Note: Descriptions are shown in the official language in which they were submitted.
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METHOD AND MARKER FOR IDENTIFICATION
OF PRE-MALIGNANCY AND MALIGNANCY
AND THERAPEUTIC INTERVENTION
s
BACKGROUND OF THE INVENTION
TECHNICAL FIELD
Io The present invention relates to methods and markers for identification of
pre-malignancy and malignancy states utilizing extrachromosomal and
intrachromosomal gene amplification. Further the present invention relates to
the identification of specific genes which undergo extrachromosomal gene
amplification and therapeutic interventions relating to their utility as
therapeutic
is targets.
BACKGROUND ART
The diagnosis of malignant conditions is approached from multiple
2o directions as for example tissue biopsies, serum levels of specific markers
(PSA
for prostate as an example), mammography and the like. However, most of
these methods do not identify pre-malignant cells where early diagnosis can
significantly increase treatment potential. Further the identification of a
malignant condition does not necessarily identity an underlying genetic
2s abnormality which can be corrected utilizing gene therapy or suggest other
points of therapeutic intervention.
Chronic lymphocytic leukemia (CLL), is the commonest leukemia, making
up 30% of all cases (O'Brien, et al. 1995), However, the cause of this disease
is
3o unknown. The leukemia primarily effects elderly males and is characterized
by
the accumulation of morphologically mature-appearing B1-lymphocytes in
peripheral blood, marrow, spleen and lymph nodes (O'Brien, et al., 1995).
Prognosis in CLL is approximately assessed by Rai staging (Table I) and
patient
survival varies from 2 years (Rai III and IV) to >10 years (Rai 0) (Rai, et
al.,
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1975). However, with each stage there is considerable variation in survival
and
patients can be further stratified according to the lymphocyte doubling time
(Montserrat, et al., 1986). Patients with a short lyriiphocyte doubling time
(<12
months) have a poorer survival rate than those with a longer doubling time
s (Montserrat, et al., 1986). At the present time, this disease is incurable
but
remissions can be obtained with alkylating agents, e.g., chlorambucil, or
nucleoside analogs, e.g., fludarabine, but relapse and the eventual
development
of drug resistance is usually observed (O'Brien, et al., 1995).
io The normal~cellular counterpart of the CLL cell is in the mantle zone of
the lymphoid follicle, and, like CLL cells, these lymphocytes are CD5+ B cells
and have high levels of bcl-2 (Schena, et al., 1992). It is presumed that a
small
fraction of CLL cells are proliferating stem cells, possibly located in the
lymphoid
tissue or marrow, but the majority of cells are non-proliferating and
accumulate
is most likely through defects in apoptosis.
The term genomic instability summarizes a variety of genomic alterations
which include the loss or gain of chromosomes as well as genetic changes at
the level of single genes, such as rearrangements, translocations,
amplifications,
2o deletions and point mutations, and has been considered to be a major
driving
force of multistep carcinogenesis (Nowell, 1976; Pienta et al., 1989; Temin,
1998; Solomon et al., 1991). Genomic integrity is maintained by checkpoint
mechanisms; when cells suffer damage imposed by exposure to genotoxic
drugs or microtubule toxins, the cell cycle is halted until the damage is
repaired
2s or apoptosis is initiated (for reviews, see Hartwell, 1992; Weinert and
Lydall,
1993; Hartwell and Kastan, 1994).
Gene amplification represents one form of genomic instability in
mammalian cells, although it can also occur as part of a normal developmental
3o program in insects, amphibia, and lower organisms (Santelli, et al., 1991;
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Delikadis, et. al., 1989; Start, et. al., 1984). With one published exception
(Prody, et al., 1989), gene amplification has not been observed in normal
diploid
cells (Lucke-Huhle, et al., 1989; Wright, et al., 1990; Tlsty, et al., 1990)
and its
presence indicates that these cells are genomically unstable, immortalized,
s transformed and/or tumorigenic. In mammalian cells lines and tumors, gene
amplification has been described after drug selection (Stark, et al., 1993;
Huang,
et al., 1994; Huang, et al., 1994; Shah, et al., 1986), DNA damage (Lucke-
Huhle, et al., 1989; Lucke-Huhle, et al., 1990; Yalkinoglu, et al., 1991) and
as a
result of c-Myc overexpression (Mai, et al., 1994; Denis, et al., 1991 ).
io Spontaneous gene amplification has also been reported (Johnston, et al.,
1983).
Gene amplification can occur in the presence of wildtype p53, but is
facilitated
by its absence (Yin, et al., 1992; Livingstone, et al., 1992); thus, gene
amplification can involve both p53-dependent and -independent pathways (Van
Der Bliek, et al., 1986; Zhou, et al., 1996). Gene amplifications often
involves
Is oncogenes, and more than 90% of these cases in patients involve c-myc where
the degree of amplification correlates with the aggressiveness of tumor growth
and poor prognosis (Schwab, et al., 1990).
c-Myc is a key regulator of growth, proliferation, differentiation, and
2o development. Deregulation of the c-Myc oncoprotein has been reported in
apoptosis, transformation, and in malignancies of lymphoid and non-lymphoid
origin (Marcu, et al., 1992; Cole, et al., 1986). c-Myc plays a role in the
modulation (Benevisty, et al., 1992; Bello-Fernandez, et al., 1993; Gaubatz,
et
al., 1994; Jansen-Durr, et al., 1993; Daksis, et al., 1994; Philipp, et al.,
1994;
2s Galaktinov, et al., 1996) and initiation of transcription (Roy, et al.,
1993, Li, et al.,
1994; Mai, et al., 1995). It is a short-lived nuclear oncoprotein (Cole, et
al.,
1986), which is strictly regulated during the cell cycle of normal diploid
cells
(Cole, et al., 1986; Heikkila, et al., 1987; Karn, et al., 1989). Increased
half life
of the protein is associated with immortalization and transformation (Marcu,
et
3o al., 1992; Cole, et al., 1986). The deregulation of c-Myc is a common
feature in
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many tumors (Marcu, et al., 1992; Cole, et al., 1986), where it frequently is
translocated (Stanton, et al., 1983; Potter, et al., 1992; Mai, et al., 1995;
Marcu,
et al., 1992; Cole, et al., 1986)' and/or amplified and overexpressed (Marcu,
et
al., 1992; Cole, et al., 1986; Feo,~''::et al., 1994; Alitalo, et al., 1985).
In addition,
s the c-myc gene is often the site of proviral insertion (Marcu, et al, 1982;
Cole, et
al., 1986). Chromosomal aberrations involving c-myc are associated with a poor
prognosis (Yokota, et al., 1986).
An amplified gene sequence is termed an "amplicon" and can be
to chromosomal ("homogeneously staining regions", HSR) or extrachromosomal
("extrachromosomal elements", Ees). Extrachromosomal submicroscopic
amplicons that replicate are termed "episomes" (250- 5,000 kb), and these can
increase in size to be visible by light microscopy, at which point they are
termed
"double minutes" (>5,000 kb) (Stark, et al., 1993; Hahn, et al., 1993). A
variety
is of mechanisms are involved in the production of gene amplification, and it
appears likely that different mechanisms can be involved for different genes
in
the same cell or for the same gene in different cell types (Stark, et al.,
1993;
Stark, et al., 1989). The "replication models" predict that a localized
replication
even can allow an isolated part of the chromosome to repeatedly replicate,
i.e.,
20 onion-skin, double roiling circle or chromosome-spiral models, and these
amplified areas can remain intrachromosomal or be released
extrachromosomally. The second major group of mechanisms are the
"segregation-driven" mechanisms, i.e., deletion-plus-episome and sister
chromatid exchange models. The deletion-plus-episome theory predicts that
as deletion of a portion of chromosome produces Ees which can proliferate and
be
subsequently incorporated into random sites on a variety of chromosomes
(Carroll, et al., 1988; Windle, et al., 1991 ).
A model of cyclin D2 gene amplification in CLL (Figure 4A) is an
so illustration of the dynamic nature of cyclin D2 gene amplification and is
based on
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the data summarized in Figures 1-4. Cyclin D2 (on chromosome 12q13) can be
amplified on chromosome 12 and thus give rise to an HSR. In addition, or
alternatively, cyclin D2 can be found on extrachromosomal elements (Ees). The
latter can be directly generated from the original locus. During this process,
it is
s possible (but not obligatory) that one allele of cyclin D2 is detected. The
extrachromosomal elements can re-integrate into chromosome 12q13 or into
random loci on chromosome 12 or on other chromosomes. Alternatively, or
additionally, Ees can remain extrachromosomally, but they can only be
maintained as extrachromosomal structures if they contain replication origins.
to According to the EM studies, the Ees in CLL cells appear to propagate by
replication (see Figures 4A and 4B). The dosage of cyclin D2 can also be
increased due to the duplication of chromosome 12. Trisomy 12 is a frequently
acquired aberration that occurs in a fraction of CLL patients (Crossen, et
al.,
1997; Dohner, et al., 1997).
Hamkalo et al, 1985 first showed using electron microscopy that the
dihydrofolate reductase (DHFR) containing Ees in methorexate-resistant murine
3T3 cells are in circles, and numerous loops can be organized together in a
rosette like structure. Similar findings have been observed by others in a
variety
of cell lines (Esnault, et al, 1994; Nonet, et al., 1993; Sen, et al., 1994;
Schneider, et al., 1992; Cohen, et al., 1996; Cohen, et al., 1997). Esnault et
al
(Esnault, et al., 1994) isolated 630 kb Ees from a methorexate-resistant cell
line
and demonstrated that each of these Ees contained on DHFR gene.
Transfection of the Ees into the methotrexate-sensitive parent cell line can
Zs confer methotrexate resistance. The adenosine deaminase containing Ees in
cells grown in 2'-deoxycoformycin (Nonet, et al., 1993), c-myc containing Ees
in
HL-60 cells (Sen, et al., 1994) and N-myc containing Ees from neuroblastoma
cells (Schneider, et al., 1992) have been isolated and cloned. In general, the
genes remain intact, can be in a high-to-tail or head-to-head configuration
(perhaps depending on the duration of time the Ees have existed) and appear to
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contain additional genomic loci to the studied gene. It has been suggested
that
these Ees can replicate and different sized molecules can develop through
intra-
or inter-molecular recombination (Esnault, et al., 1994). In addition, the
number
of Ees can increase in replicating cells, if they are unequally segregated to
each
s daughter cell and provide the cell with a survival advantage (Stark, et al.,
1993;
Hahn, et al., 1993).
CLL has been studied by comparative genomic hybridization, which
detects areas of chromosomal gene amplification or deletions (Bentz, et al.,
io 1995). Abnormalities are detected in 70% of patients and one-third of these
will
have amplifications of all or part of chromosome 12 (Bentz, et al., 1995).
Amplifications at 12q23-24, 12q13-22 and 12q13-15 have been observed by
FISH in one patient with CLL (Merup, et al., 1997). To date, extrachromosomal
gene amplification has only rarely been observed in CLL, and one patient has
Is been described with extrachromosomal amplification of c-myc (Wang, et al.,
1991 ). Recently, it was demonstrated that amplification of the cyclin D2 gene
is
a constant finding in all CLL cells, and appears to be extrachromosomal and
cyclin D2 sequences are also randomly integrated into multiple chromosomes
(Figure 2). The cause for the amplification is unknown, ~ although by an
2o extrapolation from the present murine studies, it can occur following
prolonged
m-Myc overexpression in the stem cells. As c-Myc mRNA is not increased in
peripheral blood CLL cells (Greil, et al., 1991 ), the increase can occur in
the CLL
stem cells in lymphoid tissue or marrow; the cyclin D2 amplification can then
persist in the circulating non-proliferating CLL cell, and, if they have
replicative
2s capacity, can actually increase in size and number in these cells.
Cyclin D2 is one of three D cyclins which can have an integral role in the
cell cycle. The D cyclins increase during G1 and bind to cyclin dependent
kinase-4 (CDK4) or CDK6 with the resulting phosphorylation of the
3o retinoblastoma (Rb) protein and the release of the E2F transcription
factors
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(Sherr, 1994; Hirama, et al., 1995). These factors can then induce the
transcription of a variety of genes, e.g., c-myc, DHFR and myb, which area
involved in DNA synthesis. It is likely that the three D cyclins have
epuivalent
activities, although their predominant expression depends on the cell type,
with
s cyclins D2 and D3 being primarily present in lymphoid tissue.
While there are some protocols available for providing prognosis but
within each stage there is considerable variation in survival. Therefore a
better
protocol for providing a prognosis and for making decisions on therapeutic
1o strategies is needed.
SUMMARY OF THE INVENTION
According to the present invention, there is provided a method for
is identifying pre-malignancy, malignancy, and degree of premalignancy and
malignancy of a cell by detecting extrachromosomal and intrachromosomal gene
amplification. Also provided is a marker for the identification of pre-
malignancy,
malignancy, and degree of premalignancy and malignancy of a cell containing
extrachromosomal and intrachromosomal gene amplification of a gene. A
2o diagnostic tool for the diagnosis and prognosis or cervical cancer
containing
extrachromosomal and intrachromosomal gene amplification of a gene.
DESCRIPTION OF THE DRAWINGS
2s Other advantages of the present invention are readily appreciated as the
same becomes better understood by reference to the following detailed
description when considered in connection with the accompanying drawings
wherein:
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Figure 1 shows extrachromosomal cyclin D2 gene amplification in
interphase CLL cells; A and B show control B cells from a healthy donor; C and
D show CD5+/CD19+ cells from a CLL patient; A and C show cyclin C signals
(green), this gene is present as single copygene in both normal B cells and in
s CLL cells; B and D show cyclin D2 signals (red); B shows a single copy
cyclin D2
signals in normal B cells, amplified cyclin D2 signals in CLL patient and also
shows hybridization efficiency was 30% and >80% for cyclin C and cyclin D2
respectively;
io Figure 2 shows extrachromosomal cyclin D2 gene amplification in CLL
metaphase and interphase; A shows cyclin D2 signals (red); B shows
chromosome 12 paint (green); C shows overlay of cyclin D2 hybridization
signals
(red) as obtained by FISH and chromosome 12 signals (green) as obtained by
chromosome painting;
is
Figure 3A shows a histogram of fluorescence in situ hybridization (FISH)
analysis, and a plot of cyclin D2 signals per 100 cells per CLL stage as
detected
by FISH and measured by IPLab 3.1 software; as CLL disease stage increases,.
the number of individual cyclin D2 signals increases per cell;
Figure 3B shows a histogram of fluorescence in situ hybridization (FISH)
analysis wherein total cyclin D2 signal per 100 cells as detected by FISH and
measured using IPLab 3.1 software (Signal Analytics), and as CLL disease
stage increases the fluorescence intensity of cyclin D2 signals increases;
Figure 4 shows the amplification of cyclin D2 in CLL cells: Working model
(A) and electron microscopy (EM) data (B); .
Figure 4A shows a model of cyclin D2 gene amplification in CLL; the
3o scheme of cyclin D2 amplification is based on the results obtained in CLL
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patients; cyclin D2 (chromosome 12p13) amplification generates
extrachromosomal elements (Ees) or homogeneously staining regions (HSRs)
on chromosome 12; HSRs can result from cyclin D2 gene amplification at the
original locus, however, 12p13-HSRs can also be generated through the re-
s integration of cyclin D2 containing Ees into the original locus; the
generation of
Ees can lead to the Joss of one cyclin D2 allele, Ees can re-integrate into
random chromosomes; the data also suggest the amplification-independent
increase of cyclin D2 gene dosage by the duplication of chromosome 12;
Trisomy 12 is an acquired event and has been observed in a fraction of
patients
io (see text), further the insert in this scheme shows the putative
replicative cycle of
the cyclin D2 containing Ees;
Figure 4B shows an electron microscopic (EM) analysis of
extrachromosomal DNA molecules, this demonstrates the replication
is intermediates of Ees as schematically shown in Figure 4A, arrow/arrowheads
point to replication intermediates,; the earliest stage of the replicative
cycle of
Ees is the initiation of a replication bubble (stage 1, small arrowhead), this
bubble then grows (stage 2, big arrowhead) and separates from the original
bubble which can at this time have initiated another replication cycle (stage
3,
ao arrow); extrachromosomal DNA (1 p/ml) was dissolved in 30 mM
triethanolamine
buffer 10 ng of the sample was placed onto a formvar/carbon grid followed by
the addition of 2% uranyl acetate, the analysis was carried out with Philips
EM
420 TEM microscope; Scale bar: 0.1 p;
2s Figure 5 snows a Southern analysis of DNA samples isolated from 19
CLL patients and control; the DNA was digested with EcoRl and separated on a
0.8% agarose gel, following blotting onto Hybond-N membrane, the filter was
hybridized with 600bp Ncol - fragment of human cyclin D2 cDNA, the signal
intensity was normalized with the (i-actin hybridization signals. The
amplification
3o factors obtained were 1.5 to 2.5;
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Figure 6 shows a Northern analysis of total RNA isolated from 16 CLL
patients and control (normal B); the RNA (25ug) was separated on a 1.0%
agarose gel, blotted onto Hybond-N membrane and hybridizations were
s performed with a human cyclin D2 cDNA (as described in Figure 5),
hybridizations were normalized with ~i-actin; all CLL patients express cyclin
D2,
but not normal B cells; T1165 is a positive control showing cyclin D2 mRNA
levels in a mouse plasmacytoma, normal T and human cord blood express a
cyclin D2 transcript;
io
Figure 7 shows a Western analysis of CLL patients (Rai stages 0 - IV)
and normal B lymphocytes; 100ug protein were loaded per lane onto a 10%
SDS-polyacrylamide gel, after blotting onto a Hybond C-super membrane, the
membrane was probed with cyclin D2 antibody (Santa Cruz, rabbit polyclonal
is IgG) and detected with peroxidase labeled anti-rabbit antibody (Amersham)
using the ECL detection kit (Amersham), the 35 kDa cyclin D2 protein is shown
by an arrow; control B cells and cord blood do not show detectable levels of
cyclin D2 protein; all CLL patients show cyclin D2 protein, the level
increases as
the disease progresses, stage III and IV patients show also cyclin D2 protein
2o degradation;
Figure 8 shows cyclin expression in mouse B-lymphocytic tumors;
Poly(A)+RNAs (5 ~,g) from a series of mouse B-lymphocytic cell lines
(Mushinski
et al., 1988) are arranged from left-to-right in increasing degree of
maturation,
2s HAFTL-1 3g4 and HAFTL-1 are 2 related clones of pro-B lymphocytes, the
former having more myeloid characteristics than B-cell characteristics; NFS
112,
NFS 5 and BALB 1437 are pre-B cell lines; NFS 25, WEHI 231, BAL 17 and
BAL 1131 are mature B-cell lines, SJL 4 is a plasmablastic line; and TEPC
1119, TEPC 1194, TEPC 1197 and SRPC 24 are plasmacytoma lines; the blot
3o was hybridized first with cyclin D2 cDNA and then sequentially with the
other
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hybridization probes indicated along the left margin, following stripping,
sizes of
major hybridizing bands are indicated on the right;
Figure 9 shows EMS motifs in mouse cyclin D2 5' flank, exon 1 is shown
s as a box, with the open reding frame shown in black, the mouse 5'flank is
indicated by a line to the left, positions of E-box motifs E1, E2, E3, and E4
and y
are indicated; sequences of the oligonucleotides that surround E1, E2, E3 and
y
and which were used for mobility-shift experiments are shown, the portion of
the
human 5' flank that has been sequenced is indicated by a line of double
io thickness, a dot-matrix plot of identical bases in the mouse and human 5'
flanks
is also included; H indicates the positions of the two CACGTG motifs present
in
the first 1624 5' of exon 1 in the human cyclin D2 gene (Brooks et al., 1996);
Figure 10 shows mobility "super-shift" analysis using E1, E2, and E3
is olignucleotides, 5 mg whole cell extracts of plasmacytoma MOPC 265 cells
were
incubated with the indicated antibodies or pre-immune serum (Pre) followed by
the 32P-end-labeled oligonucleotides E1-E3 (Figure 8); 1 mg of non-specific
competitor DNA was used per reaction, the arrow points to the specific complex
that does not form with bacterial protein alone; "Supershifted" bands appear
at
2o the top of the anti-MYC and anti-MAX lanes, non-specific high-molecular
weight
bands appear in all lanes that use oligonucleotide E2, the antibody
concentrations are indicated in Materials and Methods;
Figure 11 shows Southern blot analyses of mouse and human cell lines
2s hybridized with murine and human cyclin D2 cDNA probes, as indicated (panel
A), mouse lines: WEHI 231 B-cell lymphoma (low MYC) and MOPC 460D
plasmacytoma (high MYC); Human lines: GL30/92T primary fibroblasts (low
MYC), T47D brest carcinoma cells (high MYC), and COL0320HSR colorectal
carcinoma cells (very high MYC); digests were carried out with the enzymes
3o indicated. 10 mg DNA were loaded per lane, equal amounts of DNA were
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loaded as confirmed by rehybridization of the filters with the mouse
ribonucleotide reductase subunit 1 (RNR1) or the human cyclin C genes (panel
B); filled arrowheads point to amplified cyclin D2 bands; empty arrowheads
indicate lost bands that suggest another form of genomic instability in this
tumor;
s
Figure 12 shows fluorescent in situ hybridization (FISH) studies with a
cyclin D2 probe and detection with FITC-labeled anti-digoxigenin antibody; A.
COL0320HSR metaphase chromosomes stained with PI (human cyclin D2
cDNA hybridization is seen as green fluorescing spots or dark green dots); B.
io MOPC 460 metaphase chromosomes stained with PI (mouse cyclin D2 genomic
DNA hybridization is seen as green fluorescing spots or dark green dots); the
arrows point to paired dark green dots that indicate the position of the
cyclin D2
locus, single dots seen elsewhere in the spread are interpreted as ECEs that
randomly reintegrated on other chromosomes; C and D. Metaphase
is chromosomes from mouse pre-B lymphoma cells that bear the 4HT-activatable
pBabePuroMyc-ERTM expression vector were hybridized with the 5.4-kb mouse
genomic clone of cyclin D2 on a DAPI background; Cells in panel C were not
stimulated with 4HT; those in panel D were grown in 100 nM 4HT for 3 days,
arrows point to single-copy cyclin D2 in C and to extrachromosomal elements in
2o panels D; E - H. Metaphase chromosomes from y2 fibroblasts hybridized with
the 5.40kb mouse genomic clone of cyclin D2 on a DAPI background; the
image in panel E shows a negative control of FISH analysis of 4HT-treated (3
days) y2 chromosomes from cells that have not received the MYC expression
vector; Panels F, G and H show metaphase and interphase chromosomes from
2s cells bearing stable integration of the 4HT-activatable pBabePuroMyc-ERTIVI
expression vector; Cells in panel F were not stimulated with 4HT; those in
panels G and H were grown in 100 nM 4HT for 3 days;
Figure 13 shows cyclin D2 expression after MYC activation; A. Northern
3o blots of total RNA (15mg) from bulk cultures of mouse pre-B cells stably
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transduced with v-Abl (A-MuLV) or with v-Abl plus murine bcl-2 plus
pBabePurorMycERTM, both cell lines were treated with 4HT for 0, 1, 4 and 6
days, as indicated, each blot was hybridized first with cyclin D2 cDNA and
then
sequentially with the other hybridization probes indicated along the"right
margin
s following stripping, sizes of major hybridizing bands are indicated between
panels, ethidium-bromide-stained 28S ribosomal RNA bands are shown as
loading controls; B. Western blots of 10 ~.g protein per lane, isolated from
pre-B
cells, with and without pBabePuroMycERTM, after different periods of
stimulation with 4-HT, antibody specificity and size of detected protein bands
are
to indicated between panels, actin probing of duplicate blots is shown as
loading
control; C. Northern analysis of total RNA (conditions as in A) from mouse
fibroblasts (y2 cells) with and without stable integration of pBabePuroMyc-
ERTM, after different numbers of days of stimulation with 4-HT, GAPDH
hybridization is included as a loading control;
Is
Figure 14 shows DHFR gene amplification in fully developed
plasmacytoma. Arrows point to amplified DHFR sequences as detected by
FISH. Nuclei are counterstained with DAPI (4', 6'-diamidino-2-phenylindole);
2o Figure 15 shows c-Myc deregulation and the initiation of genomic
instability.;
Figure 16 shows genomic instability in p53-~- mice; Cytogenetic analysis
of bone marrow, spleen, thumus-derived cells as well as of fibroblasts
(passage
2s 0) isolated from five p53-~- and five parental p53+'+ (C57B1/6) mice, and
of p53-~-
and p53+~+ fetal liver hematopoietic cells of 16 day old embryos;
Figure 17 shows an abnormal amplification of centrosomes in p53-~- mice;
(a) Number of centrosomes detected by immunostaining in bone marrow,
so spleen, and thymic cells isolated from p53+~+ and p53-~- mice. N1: one
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centromere; N2: two centromeres; N>_3: three or more centromeres (see text);
(b) Representative picture of normal and aberrant centrosome numbers, the
. picture illustrates the normal number of centrosomes as found iy.all organs
of
p53+~+ mice (top panel) as well as aberrant numbers of centrosomes as
s observed in all organs p53-~- mice in vivo (bottom panel);
Figure 18 shows genomic instability in p53-~- mice; Representative
images of FISH analyses of DHFR and c-myc gene copies in p53+~+ and p53-~-
mice; (a) Single copy DHFR signals (green) overlaid on DAPI staining in
io thymocytes of a parental p53-~- C57BL/6 mouse; (b) Amplified signals of
DHFR
(green) overlaid on DAPI staining in P53-~- splenocytes; (c) Amplified signals
of
DHFR (pink) and c-myc (green) overlaid on DAPI staining in p53-~- thymocytes;
(d) Amplified signals of DHFR (red and c-myc (green) overlaid on DAPI staining
in p53-~- bone marrow cells; (e) Metaphase plate with extrachromosomal
Is elements, indicative of early stages of gene amplification (Wahl, 1989),
hybridizing with c-myc and DHFR probes, the most intense hybridization signals
are pointed at by arrows, note that there are many tiny hybridization signals
as
well, filled arrow point to DHFR signals (pink) and open arrows to c-myc
signals
(green);
Figure 19 shows representative images showing c-Myc expression levels
and gene amplification within the same cells in vivo using CPFA analyses
(Materials and methods); (a) p53-~- fibroblasts (passage 0) were immunostained
with anti-c-Myc antibody, two nuclei are shown that overexpress c-Myc
fourfold;
2s (a') The same cells show DHFR (red) and c-myc (green) amplification by FISH
analysis, the nuclei are stained with DAPI; (a") This image allows one to
visualize all FISH hybridization signals obtained for c-myc (green) and DHFR
(red) in (a') in the absence of DAPI staining; (b) p53-~- fetal liver-derived
hematopoietic cells were immunostained with anti-c-Myc antibody, note a four-
° to fivefold c-Myc overexpression in the nuclei; (b') shows the
overlay image of c-
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Myc staining (red) and DAPI staining of the nuclei (blue) shown in (b); (b")
FISH
analysis with a CAD probe (green) was performed on the same cells, the nuclei
are counterstained with propidium iodide; and
s Figure 20 shows apoptotic p53'~' cells exhibit atypical chromosome
morphology, gene amplification, elevated c-Myc protein levels and abnormally
amplified centrosomes, metaphase plates were prepared and evaluated as
described (Mai, 1994; Mai 35 al., 1995, /1996); (a) shows a p53'~' thymus-
derived Giemsa-stained aneuploid metaphase plate; (b) p53'j- spleen-derived
to chromosomes with atypical morphology, such chromosomes were present in all
organs examined; (c) TUNEL assay was performed on p53-~' bone marrow-
derived morphologically atypical chromosomes, extensive chromatid
fragmentation was observed, as shown by a strong positive TUNEL reaction
(yellowish green); DNA was counterstained with propidium iodide, intact DNA
is stretches can be identified in orange, and the fragmented chromatids can be
recognized by their yellowish green staining; (d) FISH analysis of bone marrow-
derived chromosomes with atypical morphology, chromosomes were
counterstained with DAPI, both c-myc (green) and DHFR (red) were amplified;
(e) Spleen-derived interphase cells with chromatin condensation typical of
2o apoptosis (arrows) and DNA fragmentation as determined by the TUNEL assay
(dUTP-fluorescein incorporated by TdT) (yellowish green arrows), DNA was
counterstained with PI, the number of apoptotic cells in P53'~' mice ranges
between 2 and 11 % in thymus, spleen, fibroblasts and bone marrow; (f)
Representative image showing apoptosis in p53-~- thymocytes p53'~' thymocytes
2s were immunostained for c-Myc (red) along with TUNEL assay and DAPI staining
(blue), the cell indicated by an arrow shows chromatin condensation yellowish
green staining; (g) shows a p53'~- bone marrow cell displaying a typical
apoptotic phenotype immunostained with anti-tubulin, the centrosomes are
indicated by arrows.
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DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method of identifying pre-malignant and
malignant cells in cells and tissues. For example the present invention allows
s identification of micrometastasis, plasmacytomas, cervical cancer, head and
neck cancer and CLL and other B-cell leukemias. More specifically, the method
includes detecting the presence of extrachromosomal gene amplification in a
cell. Additionally, a marker is disclosed which is used in the above method
for
identifying pre-malignancy and malignancy states in a cell by determining if
to extrachromosomal gene amplification of the marker is present.
The method of the present invention uses as a marker the presence of
extrachromosomal gene amplification as the marker. Further, the present
invention has unexpectedly determined that the specific genes that are
amplified
is extrachromosomally are involved in initiation of tumorigenesis and
therefore
provide therapeutic targets. Therapeutic treatment including gene therapy as
for
example utilizing suicide genes targeted to the extrachromosomal elements or
antisense therapy targeted to the identified genes can be utilized.
?o By extrachromosomal, it is meant a factor which exists, at least for a
time,
independent of the chromosome. Accordingly, the extrachromosomal factor is
not a part of the chromosome, however these factors can be considered a
genetic unit fully equal to those in the chromosomes.
2s Standard molecular biology techniques known in the art and not
specifically described are generally followed as in Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York
(1989, 1992), and in Ausubel et al., Current Protocols in Molecular Biology,
John
Wiley and Sons, Baltimore, Maryland (1989). Additionally, standard methods in
3o immunology known in the art and not specifically described are generally
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followed as in Stites et al. (eds), Basic and Clinical Immunology (8t~'
Edition),
Appleton & Lange, Norwalk, CT (1994) and Mishell and Shiigi (eds), Selected
Methods in Cellular Immunology, W.H. Freeman and Co., New York (1980).
s Cloning techniques are provided by the present invention.
Immunoassays are also provided by the present invention. In general, ELISAs
are the preferred immunoassays employed to assess a specimen. Both
polyclonal and moloclonal antibodies can be used in the assays. The specific
assay to be used can be determined by one skilled in the art.
io
Antibody production is provided by the present invention. Antibodies can
be prepared against the immunogen, or any portion thereof, for example a
synthetic peptide based on the sequence. As stated above, antibodies are used
in assays and are therefore used in determining if the appropriate enzyme has
is been isolated. Antibodies can also be used for removing enzymes from red
cell
suspensions after enzymatic conversion. Immunogens can be used to produce
antibodies by standard antibody production technology well known to those
skilled in the art as described generally in Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Springs Harbor Laboratory, Cold Spring Harbor, NY,
20 1988 and Borrebaeck, Antibody Engineering - A Practical Guide, W.H. Freeman
and Co., 1992. Antibody fragments can also be prepared from the antibodies
and include Fab, F(ab')2, and Fv by methods known to those skilled in the art.
In an embodiment, the present invention provides for the identification of
2s the cyclin D2 gene extrachromosomal amplification. It has been determined
that
all leukemia cells from patients with CLL have this abnormality and that the
extent of amplification increases with duration and stage of disease (see
Examples). The overexpression of cyclin D2 is involved in the pathogenesis of
the disease. Therefore control of cyclin D2 expression, with for example
so antisense, can provide a point of therapeutic intervention. Additional
genes
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have been identified such as DHFR, c-MYC, immunoglobulin genes, anti-
apoptosis genes, drug-resistance genes, that are amplified and play a role in
the
pathogenesis of other cancers such as plasmacytomas.
s Referring specifically to the genes relating to CLL, the CLL cells are non-
proliferating, Delmer et al, 1995 has recently made the intriguing observation
that cyclin D2 mRNA is elevated in this disease. In contrast, cyclin D1 and D3
are not increased in CLL (Delmer, et al, 1995). Whether the increase in cyclin
D2 mRNA is related to amplification of the cyclin D2 gene was examined.
to Twenty-four patients with CLL have been studied and their clinical and
laboratory details are shown in Tables 1 and 2. Cyclin D2 gene copy number
was studied by FISH and Southern blot analysis. Using FISH, amplification of
the cyclin D2 gene was observed in all examined patients, whereas this was not
observed in normal B cells. As shown in Figure 1, multiple cyclin D2 signals
is were detected suggesting the presence of extrachromosomal elements (Ees),
which was confirmed in CLL-metaphases (Figure 2). CLL metaphases,
concomitantly painted with chromosome 12 and probed with cyclin D2 by FISH,
demonstrate that cyclin D2 can be lost from its original site on one allele
and can
be integrated into loci on other chromosomes, including new locations on
2o chromosome 12. Additionally, chromosome 12 derived Ees are observed alone,
as well as in conjunction with cyclin D2. This suggests that additional genes
on
chromosome 12, apart from cyclin D2, undergo amplification.
Importantly, al! of the leukemia cells from patients with Rai stage 0
2s disease showed cyclin D2 amplification indicating that this is an early
genomic
change in CLL. The degree of amplification, as reflected by the number and
size of the cyclin D2 signals, increased with the duration and stage of
disease
(Figures 3A and 3B). When examined by electron microscopy (EM), the Ees
were found to be in a circular conformation and also showed replication
3o intermediates (Figures 4A and B).
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Southern blotting showed an increase in cyclin D2 hybridization signals in
50% of the patients (Figure 5). As Ees are not detected with a Southern Blot,
this indicates that part of the amplification is related to trisomy 12 and/or
s amplification of the cyclin D2 locus on 12p.13 (Inaba, et al., 1992). Cyclin
D2
mRNA and proteins levels were increased in most patients, with the highest
levels being seen in patients with advanced or prolonged disease (Figures 6
and
7).
io These findings suggest that cyclin D2, and/or other genes on
chromosome 12, can play an important role in the initiation of CLL. In
addition,
cyclin D2 overexpression provides the cells with a survival advantage, as the
degree of cyclin D2 amplification increases with disease duration and stage.
This is supported by the observation that trisomy 12 is a common acquired
is abnormality in CLL, which will increases cyclin D2 expression. Evidence
also
suggests a role for cyclin D2 in CLL cell survival, as flavopiridol, an
inhibitor of
CDK2 and CDK4, is highly cytotoxic to CLL cells in vitro (Byrd, et al., 1997).
Overexpression of cyclin D2 is involved in the initiation of CLL, either
2o through its effects on differentiation or apoptosis. For example, the
murine
32Dc13 non-leukemic myeloid cell line grows in blastic phase in vitro in the
presence of IL3 and undergoes apoptosis when IL3 is withdrawn (Ando, et al.
1993). However, following transfection of D cyclins these cells can survive
longer on withdrawal of IL3. In the same cells, differentiation to mature
Zs neutrophils occurs when the cells are incubated with G-CSF. However, when
these cells are transfected with cyclin D2, differentiation with G-CSF is
prevented (Kato, et al., 1993). In addition, overexpression of cyclin D2 can
cause tumor progression and transformation through its effects on genomic
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instability, as has been observed with cyclin D1 (Zhou, et al., 1996). When
cyclin D1 was overexpressed in a rat epithelial cell line, these cells showed
a
marked increase in amplification of the CAD gene when cells were exposed to
the drug, PALA (Zhou, et al., 1996). Thus, overexpression of cyclin D2 leads
to
s the accumulation of specific genetic changes, e.g., 13q14 del or 17 del,
which
are associated with disease progression and transformation.
Although the levels of cyclin D2 protein are increased in CLL, it has not
yet been established that the protein is functional. The SDKs are inhibited by
a
to variety of proteins and it has recently been shown that one of these,
p27k'p~, is
increased in CLL (Vrhovac, et al., 1997). p27~"p~ can inactivate several
cyclin/CDK complexes, including cyclin D2/CDK4 (Hirama, et al, 1995; Muller,
et
al., 1997; Blain, et al., 1997). In proliferating cells, upregulation of
p27kip1 can
induce G1 arrest (Kawamata, et al., 1998) and apoptosis (Wang, et al., 1997),
is but whether the level of p27kip1 in CLL cells is sufficient to abrogate
cyclin D2
activity requires further study. Interestingly, p27kip1 is located at 12p.12.3
(Hoglund, et al., 1996), close to cyclin D2 at 12p13 (Inaba, et al., 1992),
and can
be coamplified.
2o The extrachromosomal gene amplification is an early event during the
induction of murine malignancies and that cyclin D2 gene amplification occurs
in
a variety of murine B cell malignancies, particularly those with
overexpression of
c-Myc. More recently, the unique observation was made that extrachromosomal
amplification of the cyclin D2 gene occurs in CLL, and that all leukemia cells
2s from patients with newly diagnosed and early stage disease have this
abnormality. As the amplification is also associated with an increase in
cyclin
D2 mRNA and protein, these findings suggest that overexpression of cyclin D2
plays a role in the pathogenesis of this disease. The extent of amplification
increases with duration and stage of disease, suggesting that the cyclin D2
so provides a survival advantage. The cyclin D2 gene is located on chromosome
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12p13 and this also explains why trisomy 12 is a relatively common abnormality
in CLL, particularly in patients with advanced disease.
However, studies have also demonstrated that there is extrachromosomal
s amplification of other genes apart from cyclin D2 and other genes can be
coamplified with cyclin D2.
Inducible transfectants have been generated that allow the experimental
overexpression of the c-Myc oncoprotein (Mai, 1994). The inducible
to overexpression of c-Myc is followed by the enhanced binding of the c-
Myc/Max
heterodimer (Mai, 1994; Mai, et al., 1996). Furthermore, the DHFR gene is
amplified following the inducible overexpression of c-Myc and the increase in
the
c-Myc/Max heterodimer formation at the DHFR E-box motifs (Mai, 1994; Mai, et
al., 1996). c-Myc overexpression thus affects the genomic stability of the
DHFR
is locus.
The amplification of the DHFR gene occurs within three cell doublings
and increases 1.8- to 4.2-fold during this time period. The amplification is
locus-
specific, since other loci are unaffected irrespective of c-Myc overexpression
2o coincides with the elevated expression of the DHFR enzyme (Luecke-Huhle, et
al., 1996). The amplification of the DHFR locus is transient if c-Myc
overexpression is induced for a single time (Mai, et al., 1996). Constitutive
c-
Myc overexpression is associated with both the amplification and the
rearrangement of the DHFR gene (Mai, et al., 1996). In agreement with these
2s findings, it was observed that the prolonged induction of c-Myc in
inducible lines
is accompanied with ongoing amplification and rearrangements of the DHFR
gene (Mai, et al., 1996). Moreover, the prolonged induction of c-Myc
overexpression induces a significant increase in the formation of telomere-
centromere-fusions and of extrachromosomal elements (Mai, et al., 1996).
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Recent studies have also shown that DHFR gene amplification occurs in
association with c-Myc upregulation in p53 deficient mice in vivo (Fukasawa,
et
al., 1997). However, using p53-deficient mice as an experimental model does
not resolve the question as to whether c-Myc overexpression precedes the
s amplification of the DHFR gene. The present work was initiated to determine
whether the locus-specific amplification of the DHFR gene occurred as a result
of induced c-Myc overexpression in vivo.
To this end, an animal model of c-Myc-dependent neoplasia was
to examined, the mouse plasmacytoma. Plasmacytomagenesis is the neoplastic
development of mouse B lineage cells (Potter et al., 1992). Four criteria
generally define a plasmacytoma (PCT) cell: i) It has a well developed Golgi
and is rich in endoplasmic reticulum; ii) It predominantly secretes IgG and
IgA.
IgM and IgD were reported in a few cases; iii) It commonly displays a
is translocation between c-Myc (chromosome 15) and immunoglobulin (1g) loci
(chromosomes 12, 6, 16), and iv) c-Myc is constitutively expressed due to the
juxtaposition of myc and Ig loci. PCTs can be experimentally induced in the
mouse, with a strain specific predisposition (Potter, et al., 1992). Balb/c
and
NZB mice strains are susceptible to PCTgenesis, whereas DBAl2, C57BL/6 and
2o C3H mice are not (<5% develop paraffin oil induced PCTs). Susceptibility
loci
are located on chromosome 4 and 1, respectively (Mock, et al., 1993; Potter,
et
al., 1994). The experimental induction of PCTs is achieved with paraffin oils
and
pristane or plastic implants; in rare cases, plasmacytomas can arise
spontaneously (Potter, et al., 1992; Silva, et al., 1997).
2s
To examine whether c-Myc-dependent DHFR gene amplification occurred
in vivo, PCT-susceptible Balb/c mice were analyzed following the i.p.
injection of
pristane that leads to the induction of PCTs in these mice (Potter, et al.,
1992).
The c-Myc overexpression associated amplification of the DHFR gene in
3o pristane-injected Balb/c mice is shown. This amplification does not involve
the
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polyploidization of the DHFR carrying chromosome 13 and is predominantly
observed on extrachromosomal elements.
The focus of the experimental work herein has been on MYC-dependent
s genomic instability. The c-Myc overexpression is associated with the non-
random amplification and rearrangement of the dihydrofolate reductase (Dhfr,
ref. Mai 1994 and Mai et al., 1996) gene and the gene encoding the R2 subunit
of ribonucleotide reductase, RNR2, but not the RNR1 gene (T.). Kuschak et al.,
submitted).
~o
In the present study, evidence is shown of MYC-dependent amplification
of the cyclin D2 focus and attendant increased cyclin D2 gene products. These
findings link c-Myc overexpression and cell cycle regulation for the first
time at
the level of genomic instability of this G1 cyclin. Based on these findings, a
is model of MYC-dependent genomic instability and neoplasia is established.
Constitutive Myc expression is a key element in the induction of mouse
plasmacytomas and human Burkitt lymphomas. The mechanism through which
Myc over-expression contributes to this and other forms of carcinogenesis
2o remains elusive. One consequence of Myc overexpression is a shortening of
the G1 phase of cell cycle, these elements that regulated this phase were
studied and cloned cDNAs for the 3 mouse D cyclins. Northern blots of RNAs
from B-cell tumors showed that plasri-~acytomas have not only abundant c-Myc
transcript but also high levels of cyclin D2 transcripts. In the genomic clone
a 4
2s CACGTG "EMS (E-box Myc Site) motifs" was found upstream of the cyclin D2
coding region. These EMS motifs bind Myc/Max heterodimers, suggesting that
constitutive Myc expression can cause cyclin D2 overexpression. Cyclin D2
DNA and mRNA were examined in established mouse and human tumors that
expressed high levels of Myc. Also examined was the substantial amplification
30 of the cyclin D2 gene by Southern blotting and by FISH.
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To follow the Myc/cyclin D2 connection in vitro, Myc activity was
upregulated in two cell lines using an expression vector making a Myc-ER
chimera that is activated by 4-hydroxytamoxifen (4HT). In mouse pre-B cells
s and fibroblasts that bear Myc-ERTM, cyclin D2 mRNA rose after three to four
days of-4HT activation of Myc. Simultaneously, evidence of amplification : in
the
form of extrachromosomal elements was seen by FISH. Thus, one important
action of Myc seems to be the induction of gene amplification, a form of
genomic
instability. The associated increase in cyclin D2 gene products, apparently
due
to to the amplification, rather than upregulated transcription, can contribute
to
uncontrolled growth.
The dihydrofolate reductase (DHFR) gene is a target of c-Myc in genomic
instability. The induced overexpression of c-Myc in cell lines is followed by
the
is amplification and rearrangement of the DHFR gene. Furthermore, the
constitutive upregulation of c-Myc protein coincides with genomic instability
of
the DHFR gene in lymphoid, non-lymphoid and in tumor lines. The amplification
of the DHFR gene is locus-specific and independent of species origins. The
question has been addressed whether inducible deregulation of c-Myc is
2o followed by DHFR gene amplification in vivo. Therefore, the DHFR gene is a
target of c-Myc-dependent neoplasia in vivo and plays a role in genomic
instability during the initiation of neoplastic transformation.
The present invention also provides that the DHFR gene is a marker or
2s indicator of pre-malignant and malignancy states. More specifically, the
DHFR
gene is amplified in cervical cancer. It has additionally been established
that the
degree of DHFR gene amplification can be used as a measurement of the
degree of premalignancy and malignancy. Thus, based on the amount of DHFR
gene amplification found in a lesion, it can be determined the stage of the
so cancer. Specifically, there is a direct correlation between the amount of
DHFR
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gene amplification and the tumor stage, such that as DHFR gene amplification
increases the corresponding tumor stage also increases. Therefore, DHFR
amplification can be used as a sensitive biomarker for all stages of cervical
cancer.
s
It has also been established that the loss of p53 tumor suppressor
functions results in genetic instability, characteristically associated with
changes
in chromosome ploidy and gene amplification. In vivo, cells from various
organs
of four to six-week old p53-nullizygous (p53-~-) mice display aneuploidy and
to frequent gene amplification as well as evidence of apoptosis. Regardless of
tissue types, many p53-~- cells contain multiple centrosomes and abnormally
formed mitotic spindles. Thus, chromosome instability in vivo is associated
with
abnormal centrosome amplification. Moreover, a significant increase in the
number of cells overexpressing c-Myc in p53-~- mice is observed. Consistent
is with previous studies showing that c-Myc overexpression is associated with
gene
amplification in vitro, many of the p53-~- cells exhibited, in the same cell,
c-Myc
overexpression and amplified c-myc, dihydrofolate reductase (DHFR), and
carbamoyl-phosphate syntehtase-asparate transcarbamoyl-dihydroorotase
(CAD) genes. Furthermore, apoptosis was frequently observed in cells isolated
zo from p53-~- mice. The apoptotic cells contained abnormally amplified
centrosomes, displayed aneuploidy, high levels of c-Myc expression, as well as
gene amplification. These results indicate that a high number of aberrant
cells is
eliminated by p53-independent pathways in vitro.
2s In another embodiment of the present invention there is provided a kit for
identifying pre-malignancy and malignant states of a cell. The kit contains a
device for detecting extrachromosomal gene amplification. More specifically,
the
kit detects extrachromosomal gene amplification of genes such as DHFR, C-
Myc, immunoglubulin genes, anti-apoptosis genes and drug-resistance genes.
3o The process for detecting the gene amplification uses a combined protein
and
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FISH analysis. Such analysis includes a detecting gene amplification using
quantitative fluorescence immunohistochemistry. The kit can also include items
for therapeutic intervention of cells having such extrachromosomal gene
amplification Such therapeutic intervention includes therapeutically targeting
s the genes which have extrachromosomal amplification. Such therapy includes
gene therapy such as suicide genes which are fiargeted toward
extrachromosomal elements or antisense therapy targeted to the identified
gene.
io The methods used with and the utility of the present invention can be
shown by the following non-limiting examples and accompanying figures and
incorporated by reference in their entirety.
The above discussion provides a factual basis for the use of markers for
is the identification of pre-malignancy and malignancy of cells. The methods
used
with and the utility of the present invention can be shown by the following
non-
limiting examples and accompanying figures.
EXAMPLES
GENERAL METHODS:
General methods in molecular biology: Standard molecular biology
techniques known in the art and not specifically described are generally
followed
2s as in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs
Harbor Laboratory, New York (1989, 1992), and in Ausubel et al., Current
Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland
(1989). Polymerase chain reaction (PCR) is carried out generally as in PCR
Protocols: A Guide To Methods And Applications, Academic Press, San Diego,
so CA (1990). Reactions and manipulations involving other nucleic acid
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techniques, unless stated otherwise, are performed as generally described in
Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, and methodology as set forth in United States patents
4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057 and incorporated
s herein by reference. In-situ (In-cell) PCR in combination with Flow
Cytometry
can be used for detection of cells containing specific DNA and mRNA
sequences (Testoni et al, 1996, Blood 87:3822.)
General methods in immunology: Standard methods in immunology
io known in the art and not specifically described are generally followed as
in Stites
et al.(eds), Basic and Clinical Immunology (8th Edition), Appleton & Lange,
Norwalk, CT (1994) and Mishell and Shiigi (eds), Selected Methods in Cellular
Immunology, W.H. Freeman and Co., New York (1980).
~s Immunoassays In general, ELISAs are the preferred immunoassays
employed to assess a specimen. ELISA assays are well known to those skilled
in the art. Both polyclonal and monoclonal antibodies can be used in the
assays. Where appropriate other immunoassays, such as radioimmunoassays
(RIA) can be used as are known to those in the art. Available immunoassays
2o are extensively described in the patent and scientific literature. See, for
example, United States patents 3,791,932; 3,839,153; 3,850,752; 3,850,578;
3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345;
4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521 as well ~ as
Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor,
2s New York, 1989.
Trans eq nic and Knockout Methods
The present invention can provide for transgenic gene and polymorphic
3o gene animal and cellular (cell lines) models as well as for knockout
models.
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These models are constructed using standard methods known in the art and as
set forth in United States Patents 5,487,992, 5,464,764, 5,387,742, 5,360,735,
5,347,075, 5,298,422, 5,288,846, 5,221,778, 5,175,385, 5,175,384,5,175,383,
4,736,866 as well as Burke and Olson (1991), Capecchi (1989), Davies et al.
s (1992), Dickinson et al. (1993), Duff and Lincoln (1995), Huxley et al.
(1991),
Jakobovits et al. (1993), Lamb et al. (1993), Pearson and Choi (1993),
Rothstein
(1991), Schedl et al. (1993), Strauss et al. (1993). Further, patent
applications
WO 94/23049, WO 93/14200, WO 94/06908, WO 94/28123 also provide
information.
to
For gene therapy: By gene therapy as used herein refers to the transfer
of genetic material (e.g DNA or RNA) of interest into a host to treat or
prevent a
genetic or acquired disease or condition phenotype. The genetic material of
interest encodes a product (e.g. a protein, polypeptide, peptide or functional
Is RNA, antisense) whose production in vivo is desired. For example, the
genetic
material of interest can encode a hormone, receptor, enzyme, polypeptide or
peptide or antisense sequence of therapeutic value. For a review see, in
general, the text "Gene Therapy" (Advances in Pharmacology 40, Academic
Press, 1997).
Two basic approaches to gene therapy have evolved: (1 ) ex vivo and (2)
in vivo gene therapy. In ex vivo gene therapy cells are removed from a
patient,
and while being cultured are treated in vitro. Generally, a functional genetic
sequence is introduced into the cell via an appropriate gene delivery
2s vehicle/method (transfection, transduction, homologous recombination, etc.)
and
an expression system as needed and then the modified cells are expanded in
culture and returned to the host/patient. These genetically reimplanted cells
have been shown to have the transfected genetic sequence expressed in situ.
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In in vivo gene therapy, target cells are not removed from the subject
rather the genetic sequence to be transferred is introduced into the cells of
the
recipient organism in situ, that is within the recipient. Alternatively, if
the host
gene is defective, the gene is repaired in situ (Culver, 1998). These
genetically
s altered cells have been shown to express the transfected gene sequence in
situ.
The gene expression vehicle is capable of delivery/transfer of
heterologous nucleic acid into a host cell. The expression vehicle includes
elements to control targeting, expression and transcription of the nucleic
acid in
Io a cell selective manner as is known in the art. It is noted that often the
5'UTR
and/or 3'UTR of the gene can be replaced by the 5'UTR and/or 3'UTR of the
expression vehicle. Therefore as used herein the expression vehicle can, as
needed, not include the 5'UTR and/or 3'UTR of the gene of interest and only
include the specific amino acid coding region of the gene of interest.
is
The expression vehicle can include a promotor for controlling transcription
of the heterologous material and can be either a constitutive or inducible
promotor to allow selective transcription. Enhancers that can be required to
obtain necessary transcription levels can optionally be included. Enhancers
are
2o generally any non-translated DNA sequence which works contiguously with the
coding sequence (in cis) to change the basal transcription level dictated by
the
promoter. ,The expression vehicle can also include a selection gene as
described herein below.
2s Vectors can be introduced into cells or tissues by any one of a variety of
known methods within the art. Such methods can be found generally described
in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs
Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols
in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989), Chang
3o et al., Somatic Gene Therapy, CRC Press, Ann Arbor, MI (1995), Vega et al.,
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Gene Targeting, CRC Press, Ann Arbor, MI (1995), Vectors: A Survey of
Molecular Cloning Vectors and Their Uses, Butterworths, Boston MA (1988) and
Gilboa et al (1986) and include, for example, stable or transient
transfection,
lipofection, electroporation and infection with recombinant viral vectors. In
s addition, see United States patent 4,866,042 for vectors involving the
central
nervous system and also United States patents 5,464,764 and 5,487,992 for
positive-negative selection methods.
Introduction of nucleic acids by infection offers several advantages over
io the other listed methods. Higher efficiency can be obtained due to their
infectious nature. Moreover, viruses are very specialized and typically infect
and
propagate in specific cell types. Thus, their natural specificity can be used
to
target the vectors to specific cell types in vivo or within a tissue or mixed
culture
of cells. Viral vectors can also be modified with specific receptors or
ligands to
Is alter target specificity through receptor mediated events.
A specific example of DNA viral vector for introducing and expressing
recombinant sequences is the adenovirus derived vector Adenop53TK. This
vector expresses a herpes virus thymidine kinase (TK) gene for either positive
or
2o negative selection and an expression cassette for desired recombinant
sequences. This vector can be used to infect cells that have an adenovirus
receptor which includes most cancers of epithelial origin as well as others.
This
vector as well as others that exhibit similar desired functions can be used to
treat
a mixed population of cells and can include, for example, an in vitro or ex
vivo
2s culture of cells, a tissue or a human subject.
Additional features can be added to the vector to ensure its safety and/or
enhance its therapeutic efficacy. Such features include, for example, markers
that can be used to negatively select against cells infected with the
recombinant
3o virus. An example of such a negative selection marker is the TK gene
described
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above that confers sensitivity to the antibiotic gancyclovir. Negative
selection is
therefore a means by which infection can be controlled because it provides
inducible suicide through the addition of antibiotic. Such protection ensures
that
if, for example, mutations arise that produce altered forms of the viral
vector or
s recombinant sequence, cellular transformation will not occur.
Features that limit expression to particular cell types can also be included.
Such features include, for example, promoter and regulatory elements that are
specific for the desired cell type.
io
In addition, recombinant viral vectors are useful for in vivo expression of a
desired nucleic acid because they offer advantages such as lateral infection
and
targeting specificity. Lateral infection is inherent in the life cycle of, for
example,
retrovirus and is the process by which a single infected cell produces many
is progeny virions that bud off and infect neighboring cells. The result is
that a
large area becomes rapidly infected, most of which was not initially infected
by
the original viral particles. This is in contrast to vertical-type of
infection in which
the infectious agent spreads only through daughter progeny. Viral vectors can
also be produced that are unable to spread laterally. This characteristic can
be
ao useful if the desired purpose is to introduce a specified gene into only a
localized
number of targeted cells.
As described above, viruses are very specialized infectious agents that
have evolved, in many cases, to elude host defense mechanisms. Typically,
Zs viruses infect and propagate in specific cell types. The targeting
specificity of
viral vectors utilizes its natural specificity to specifically target
predetermined cell
types and thereby introduce a recombinant gene into the infected cell. The
vector to be used in the methods of the invention will depend on desired cell
type to be targeted and are known to those skilled in the art. For example, if
3o breast cancer is to be treated then a vector specific for such epithelial
cells can
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be used. Likewise, if diseases or pathological conditions of the hematopoietic
system are to be treated, then a viral vector that is specific for blood cells
and
their precursors, preferably for the specific type of hematopoietic cell, can
be
used.
s
Retroviral vectors can be constructed to function either as infectious
particles or to undergo only a single initial round of infection. In the
former case,
the genome of the virus is modified so that it maintains all the necessary
genes,
regulatory sequences and packaging signals to synthesize new viral proteins
io and RNA. Once these molecules are synthesized, the host cell packages the
RNA into new viral particles which are capable of undergoing further rounds of
infection. The vector's genome is also engineered to encode and express the
desired recombinant gene. In the case of non-infectious viral vectors, the
vector
genome is usually mutated to destroy the viral packaging signal that is
required
Is to encapsulate the RNA into viral particles. Without such a signal, any
particles
that are formed will not contain a genome and therefore cannot proceed through
subsequent rounds of infection. The specific type of vector will depend upon
the
intended application. The actual vectors are also known and readily available
within the art or can be constructed by one skilled in the art using well-
known
Zo methodology.
The recombinant vector can be administered in several ways. If viral
vectors are used, for example, the procedure can take advantage of their
target
specificity and consequently, do not have to be administered locally at the
as diseased site. However, local administration can provide a quicker and more
effective treatment, administration can also be performed by, for example,
intravenous or subcutaneous injection into the subject. Injection of the viral
vectors into a spinal fluid can also be used as a mode of administration,
especially in the case of neuro-degenerative diseases. Following injection,
the
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viral vectors will circulate until they recognize host cells with the
appropriate
target specificity for infection.
An alternate mode of administration can be by direct inoculation locally at
s the site of the disease or pathological condition or by inoculation into the
vascular system supplying the site with nutrients or into the spinal fluid. ~
Local
administration is advantageous because there is no dilution effect and,
therefore, a smaller dose is required to achieve expression in a majority of
the
targeted cells. Additionally, local inoculation can alleviate the targeting
lo requirement required with other forms of administration since a vector can
be
used that infects all cells in the inoculated area. If expression is desired
in only
a specific subset of cells within the inoculated area, then promoter and
regulatory elements that are specific for the desired subset can be used to
accomplish this goal. Such non-targeting vectors can be, for example, viral
Is vectors, viral genome, plasmids, phagemids and the like. Transfection
vehicles
such as liposomes can also be used to introduce the non-viral vectors
described
above into recipient cells within the inoculated area. Such transfection
vehicles
are known by one skilled within the art.
2o Antisense Therapy: Many reviews have covered the main aspects of
antisense (AS) technology and its enormous therapeutic potential (Wright and
Anazodo, 1995). There are reviews on the chemical (Crooke, 1995; Uhlmann et
al, 1990), cellular (Wagner, 1994) and therapeutic (Hanania, et al, 1995;
Scanlon, et al, 1995; Gewirtz, 1993) aspects of this rapidly developing
2s technology. Within a relatively short time, ample information has
accumulated
about the in vitro use of AS nucleotide sequences in cultured primary cells
and
cell lines as well as for in vivo administration of such nucleotide sequences
for
suppressing specific processes and changing body functions in a transient
manner. Further, enough experience is now available in vitro and in vivo in
3o animal models and human clinical trials to predict human efficacy.
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Antisense intervention in the expression of specific genes can be
achieved by the use of synthetic AS oligonucleotide sequences (for recent
reports see Lefebvre-d'Hellencourt et al, 1995; Agrawal, 1996; Lev-Lehman et
s al, 1997). AS oligonucleotide sequences can be short sequences of DNA,
typically 15-30 mer but can be as small as 7 mer (Wagner et al, 1996),
designed
to complement a target mRNA of interest and form an RNA:AS duplex. This
duplex formation can prevent processing, splicing, transport or translation of
the
relevant mRNA. Moreover, certain AS nucleotide sequences can elicit cellular
to RNase H activity when hybridized with their target mRNA, resulting in mRNA
degradation (Calabretta et al, 1996). In that case, RNase H will cleave the
RNA
component of the duplex and can potentially release the AS to further
hybridize
with additional molecules of the target RNA. An additional mode of action
results from the interaction of AS with genomic DNA to form a triple helix
which
is can be transcriptionally inactive.
Phosphorothioate antisense oligonucleotides do not normally show
significant toxicity at concentrations that are effective and exhibit
sufficient
pharmacodynamic half-lives in animals (Agarwal et al., 1996) and are nuclease
ao resistant. Antisense induced loss-of-function phenotypes related with
cellular
development were shown for the glial fibrillary acidic protein (GFAP), for the
establishment of tectal plate formation in chick (Galileo et al., 1991 ) and
for the
N-myc protein, responsible for the maintenance of cellular heterogeneity in
neuroectodermal cultures (ephithelial vs. neuroblastic cells, which differ in
their
2s colony forming abilities, tumorigenicity and adherence) (Rosolen et al.,
1990;
Whitesell et al, 1991 ). Antisense oligonucleotide inhibition of basic
fibroblast
growth factor (bFgF), having mitogenic and angiogenic properties, suppressed
80% of growth in glioma cells (Morrison, 1991 ) in a saturable and specific
manner. Being hydrophobic, antisense oligonucleotides interact well with
3o phosphofipid membranes (Akhter et al., 1991). Following their interaction
with
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the cellular plasma membrane, they are actively (or passively) transported
into
living cells (Loke et al., 1989), in a saturable mechanism predicted to
involve
specific receptors (Yakubov et al., 1989).
s Instead of an antisense sequence as discussed herein above, ribozymes
can be utilized. This is particularly necessary in cases where antisense
therapy
is limited by stoichiometric considerations (Sarver et al., 1990, Gene
Regulation
and Aids, pp. 305-325). Ribozymes can then be used that will target the same
sequence. Ribozymes are RNA molecules that possess RNA catalytic ability
io (see Cech for review) that cleave a specific site in a target RNA. The
number of
RNA molecules that are cleaved by a ribozyme is greater than the number
predicted by stochiochemistry. (Hampel and Tritz, 1989; Uhlenbeck, 1987).
Ribozymes catalyze the phosphodiester bond cleavage of RNA. Several
is ribozyme structural families have been identified including Group I
introns,
RNase P, the hepatitis delta virus ribozyme, hammerhead ribozymes and the
hairpin ribozyme originally derived from the negative strand of the tobacco
ringspot virus satellite RNA (sTRSV) (Sullivan, 1994; U.S. Patent No.
5,225,347,
columns 4-5). The latter two families are derived from viroids and virusoids,
in
2o which the ribozyme is believed to separate monomers from oiigomers created
during rolling circle replication (Symons, 1989 and 1992). Hammerhead and
hairpin ribozyme motifs are most commonly adapted for trans-cleavage of
mRNAs for gene therapy (Sullivan, 1994). The ribozyme type utilized in the
present invention is selected as is known in the art. Hairpin ribozymes are
now
2~ in clinical trial and are the preferred type. In general the ribozyme is
from 30-
100 nucleotides in length.
The present invention provides pharmaceutical compositions for the
delivery of the oligonucleotides of the present invention. The pharmaceutical
3o compositions contain active ingredients as described herein and a
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pharmaceutically suitable carrier or diluent. The compositions can be
administered orally, subcutaneously or parenterally including intravenous,
intraarterial, intramuscular, intraperitoneally, and intranasal administration
as
well as intrathecal and infusion techniques as required by the cells being
treated.
s For delivery within the CNS intrathecal delivery can be used with for
example an
Ommaya reservoir or other methods known in the art. The pharmaceutically
acceptable carriers, diluents, adjuvants and vehicles as well as implant
carriers
generally refer to inert, non-toxic solid or liquid fillers, diluents or
encapsulating
material not reacting with the active ingredients of the invention. Implants
of the
~o compounds are also useful. In general the pharmaceutical compositions are
sterile.
Modifications or analogues of nucleotides can be introduced to improve
the therapeutic properties of the nucleotides. Improved properties include
is increased nuclease resistance and/or increased ability to permeate cell
membranes.
Nuclease resistance, where needed, is provided by any method known in
the art that does not interfere with biological activity of the antisense
20 oligodeoxynucleotides and/or ribozymes as needed for the method of use and
delivery flyer et al., 1990; Eckstein, 1985; Spitzer and Eckstein, 1988; Woolf
et
al., 1990; Shaw et al., 1991 ). Modifications that can be made to
oligonucleotides in order to enhance nuclease resistance include modifying the
phophorous or oxygen heteroatom in the phosphate backbone. These include
2s preparing methyl phosphonates, phosphorothioates, phosphorodithioates and
morpholino oligomers. In one embodiment it is provided by having
phosphorothioate bonds linking between the four to six 3=-terminus nucleotide
bases. Alternatively, phosphorothioate bonds link all the nucleotide bases.
Other modifications known in the art can be used where the biological activity
is
3~ retained, but the stability to nucleases is substantially increased.
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The present invention also includes all analogues of, or modifications to,
an oligonucleotide of the invention that does not substantially affect the
function
of the oligonucleotide. The nucleotides can be selected from naturally
occurring
s or synthetic modified bases. Naturally occurring bases include adenine,
guanine, cytosine, thymine and uracil. Modified bases of the oligonucleotides
include xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other
alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza cytosine and 6-aza
thymine,
psuedo uracil, 4-thiuracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-
to thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-
halo
guanines, 8-amino guanine, 8-thiol guanine, 8-thioalkyl guanines, 8-hydroxyl
guanine and other substituted guanines, other aza and deaza adenines, other
aza and deaza guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.
Is In addition, analogues of nucleotides can be prepared wherein the
structure of the nucleotide is fundamentally altered and that are better
suited as
therapeutic or experimental reagents. An example of a nucleotide analogue is a
peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate
backbone in DNA (or RNAO is replaced with a polyamide backbone which is
2o similar to that found in peptides. PNA analogues have been shown to be
resistant to degradation by enzymes and to have extended lives in vivo and in
vitro. Further, PNAs have been shown to bind stronger to a complementary
DNA sepuence than a DNA molecule. This observation is attributed to the lack
of charge repulsion between the PNA strand and the DNA strand. Other
2s modifications that can be made to oligonucleotides include polymer
backbones,
cyclic backbones, or acyclic backbones.
The active ingredients include oiigonucleotides that are nuclease resistant
needed for the practice of the invention or a fragment thereof shown to have
the
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same effect targeted against the appropriate sequences) and/or ribozymes.
Combinations of the active ingredients can be used.
The antisense oligonucleotides (and/or ribozymes) of the present
s invention can be synthesized by any method known in the art for ribonucleic
or
deoxyribonucleic nucleotides. For example, an Applied Biosystems 380B DNA
synthesizer can be used. When fragments are used, two or more such
sequences can be synthesized and linked together for use in the present
invention.
io
The nucleotide sequences of the present invention can be delivered
either directly or with viral or non-viral vectors. When delivered directly
the
sequences are generally rendered nuclease resistant. Alternatively the
sequences can be incorporated into expression cassettes or constructs such
Is that the sequence is expressed in the cell. Generally the construct
contains the
proper regulatory sequence or promotor to allow the sequence to be expressed
in the targeted cell.
Delivery of gene productsltherapeutics (compound): The compound
ao of the present invention is administered and dosed in accordance with good
medical practice, taking into account the clinical condition of the individual
patient, the site and method of administration, scheduling of administration,
patient age, sex, body weight and other factors known to medical
practitioners.
The pharmaceutically "effective amount" for purposes herein is thus determined
2s by such considerations as are known in the art. The amount must be
effective
to achieve improvement including but not limited to improved survival rate or
more rapid recovery, or improvement or elimination of symptoms and other
indicators as are selected as appropriate measures by those skilled in the
art.
3o Isolation of EEs. 1-5x107 cells are lysed in 0.6%SDS/O.OIM EDTA (pH
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7.5) for 20 minutes at room temperature. The lysate is then brought to a final
concentration of 1 M NaC1 and left overnight at 40C. The insoluble
NaC1/SDS/chromatin fraction is pelleted by centrifugation at 17,000 rpm for 30
minutes at 4°C. The supernatant is the so-called Hirt extract and is
collected. It
s contains the bulk of the EBs, but it can also contain contamination with
small
linear fragments of genomic DNA (Ref. 2) and apoptotic DNA fragments.
Fixation of EEs. The Hirt extract is mixed with an equal volume of freshly
prepared methanol:acetic acid (3:1). This mixture can be stored at 4°C
for
Io months.
Dropping of EEs onto slides and their fixation. The following protocol
allows the fixation of the EEs onto glass slides, and it guarantees that the
EEs
are well-spread, but contained within a small area. Briefly: 40 p1 of fixed
Ees
is (Wit extract in fixative) are dropped onto precooled slides (20 seconds on
dry
ice), and the slides are immediately moved onto a slide warmer (37°C).
When
almost dry, the slides are dipped into 50% acetic acid and then dried to
completion on the slide warmer (370C). The area onto which the EEs were
dropped is marked with a diamond pen.
Analysis of the EEs sample under the fluorescent microscope. 4'6'
diamidino-2-phenylindole (DAPI) (Ipg/ml in PBS) is used to stain both the DNA
in
the, EEs and any genomic DNA contaminants. Anti-bleach (Ref. ~5.) is added to
preserve the fluorescence of the sample and as a mount for the cover slip.
2s Under a 63x oil immersion objective and a UV filter, the sample is examined
using a fluorescent microscope. DAPI-stained EEs are visible as distinct dots,
while genomic DNA appears as DAPI-stained fibres (Fig. 1).
FISH analysis of EEs (FISH-EEs). FISH is carried out according to
3o previously published protocols (Refs. 4. 5 .~. Briefly: the slides are
treated with
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RNAse and pepsin as described for metaphase chromosomes and interphase
cells. DNA probes are labeled with haptens by random priming as described,
and hybridizations are performed in 50% formamide/2 x SSC/50 mM phosphate
pH 7/10% dextran sulfate overnight at 37°C in a humidified incubator.
Post-
s hybridization washes are carried out as follows: 3 x 5 minutes at
42°C in 50%
formamide/2 x SSC; 5 x 2 minutes at room temperature in 2 x SSC. Prior to the
use of antibodies, the slides are blocked in 100% serum. Anti-hapten
antibodies,
conjugated with fluorescein (FITC) or Texas Red (TR), are used to visualize
the
hapten-labeled probes. The antibody incubation is carried out for 30 minutes
at
l0 37°C. The unbound antibodies are washed off at 42°C in 4 x
SSC/0.1 % Tween
20 for 3 x 5 minutes. DAPI (Ip.g/ml in PBS, 5 minutes) is used to stain the
DNA
and the slides are mounted in anti-bleach. In the examples shown in Fig. 2,
the
following probes have been used: human c-myc cDNA (Ref. 4.~1)~ human
cyclin C cDNA (Ref. ~), mouse cyclin D2 genomic DNA (Ref. j), mouse
is ribonucleotide reductase Ri and R2 (Ri and R2) cDNA (Ref. fID~ all of which
were labeled with digoxigenin. After hybridization, the annealed probe is
visualized by incubation with anti-digoxigenin-fluorescein antibody
(Boehringer
Mannheim). An additional probe, a mouse dihydrofolate reductase (DHFR)
cDNA (Ref. 4~), was labeled with biotin, detected with a monoclonal mouse anti-
2o biotin antibody (Boehringer Mannheim), and visualized by goat anti-mouse-
IgG-
Texas Red (Southern Biotechnology, Ass., Inc.).
Products used.
4-hydroxytamoxifen (4HT) Research Biochemical International
2s sheep anti-digoxigenin fluorescein Boehringer Maunheim
monoclonal mouse anti-biotin antibody Boehringer Mannheim
goat anti-mouse-IgG-Texas Red Southern Biotechnology Ass., Inc.
DAPI Sigma
microscope Zeiss Axiophot
3o CCD camera Photometrics
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IPLab Spectrum (version 3.1) Signal Analytics
Example 1
s Although peripheral blood CLL cells are non-proliferating, overexpression
of cyclin D2 mRNA has been observed in these cells (Blood 85:2870, 1995). In
the present study cyclin D2 protein levels are examined to determine whether
the increase in cyclin D2 mRNA in CLL can be attributed to gene amplification.
By Western blot analysis, cyclin D2 protein was undetectable in normal B or
cord
io blood CD5+/CD19+ cells, whereas the protein was detectable in 12 of 14 CLL
patients, with the highest levels being observed in patients with advanced Rai
staging. In addition, degradation of the protein was observed, with the extent
of
protein turnover being greatest in cells with the highest levels of cyclin D2.
Immunohistochemistry showed sub-populations of cells with elevated cyclin D2.
Is Cyclin D2 mRNA levels were increased in 12 of 14 CLL patients, when
compared with normal B cells. CLL cells have been examined by dispersed cell
assay (DCA) (12 patients) and fluorescent in situ hybridization (FISH) (5
patients) to asses gene copy number. An increase in cyclin D2 hybridization
signals was detected with both techniques and multiple cyclin D2 signals were
20 observed by FISH in all patients studied. This was not observed with a
control
gene (cyclin C). Ongoing studies are assessing whether the multiple signals
are
related to chromosomal or extrachromosomal gene amplification or to the
presence of trisomy 12. Thus, cyclin D2 is overexpressed at the RNA and
protein levels in most patients with CLL and preliminary studies indicate that
this
2s can be related to gene amplification.
The cyclin D2 gene is amplified in all CLL cells, primarily as a result of the
presence of multiple cyclin D2 containing Ees. These Ees can contain other
genes, apart from cyclin D2, and can independently increase in size, divide
and
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become incorporated back into chromosomes. The cyclin D2 mRNA can be
derived from all these sites of amplified cyclin D2.
Overexpression of cyclin D2 in CD19+lCD5+ cells can cause a block at a
s specific window of B cell differentiation, and cells can accumulate in this
window
through defects in apoptosis. Cyclin D2 overexpression in these cells can then
cause genomic instability leading to specific genomic changes which lead to
tumor progression and transformation.
io To analyse the size, structures and replicative/transcriptional potentials
of
the cyclin D2 containing extrachromosomal elements (Ees) in CLL cells tests
were conducted to determine whether they contain other genes in addition to
cyclin D2.
is Tests were conducted to assess the functional activity of cyclin D2 in CLL
cells and to determine whether overexpression of cyclin D2 in normal B cells
leads to enhanced cellular proliferation, changes in genome stability and
enhanced cell survival.
2o Samples are obtained by Dr. Johnston from CLL patients followed at the
Manitoba Cancer Foundation. The patients are staged using the Rai
classification (Table 3) and their initial doubling time calculated, as
previously
described (Montserrat, et al., 1986). Patients are only studied if they had no
treatment or have been off all chemotherapy for >1 month.
2s
Isolation of CLL and mouse 8 cells. The leukemia cells are isolated from
marrow and peripheral blood using a Ficoll-Hypaque gradient (Johnston, et al.,
1997). Monocytes are removed using anti-CD33 antibodies coupled to magnetic
beads (Dynabeads), and T cells depleted by sheep red cell resetting (Johnston,
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et al, 1997). Similarly, anti-B220 antibodies coupled to Dynabeads are used to
isolate mouse spleen-derived B lymphocytes.
Combined ProteinlFISH Analysis (CPFA). This assay is used to
s quantitate protein levels and determine gene amplification within the same
cells
(Fukasawa, et al., 1997.). In this study, the assay is used to demonstrate the
cell surface markers characteristic for human CLL cells, namely CD5+ and
CD19+ (O'Brien, et al., 1995), and examine the gene copy number of cyclin D2
within the same cells. Using this assay, one can assure that no normal B cells
to (CD5-/CD19+) or T cells CD5+/CD19-) interfere with the analysis.
Cytogenetics and FISH. CLL metaphases are induced by the phorbol
ester TPA (1.6x10-7m) (Juliusson, et al., 1993) and metaphase spreads are
prepared and evaluated as described (Mai, et al., 1986; Fukasawa, et al.,
1997),
is following the separation of normal T and B cells from CLL cells. Gene copy
numbers (amplified vs. Single copy gene, e.g. cyclin C) are quantitated using
IPLab Spectrum software (version 3.1) (Signal Analytics, USA). Chromosome
painting (CedarLane) is performed to determine whether cyclin D2 and/or
additional sequences from chromosome 12 are amplified or rearranged and
2o whether these structures are found on extrachromosomal elements (Ees) (76;
Figure 2).
Isolation and analysis of Ees. Ees are isolated according to the method
of Hirt et al., 1967. To examine their size and replicative potential, they
are
as analyzed by one dimensional and neutral-neutral two dimensional gel
electrophoresis respectively (Cohen, et al., 1996; Cohn, et al., 1997).
Additionally, they are studied by electron microscopy to examine the presence
of
replication intermediates (Figure 4).
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Cloning of Ees. Cloning of the Ees into the pcDNA3 vector
(INVITROGEN) are performed following isolation of the Ees (see above).
Sequencing of the Ees are carried out following PCR amplification (De Cremoux,
et al., 1997) of the Ees using Sp6 and T7 (INVITROGEN).
s
In situ hybridization of pre-mRNA. In situ hybridization of pre-mRNA is
used to localize the pre-mRNA transcript to chromosomes, Ees or both.
Nascent RNA (pre-mRNA) in situ hybridization and FISH on interphase cells are
carried out as previously described (Lawrence, et al., 1989; Wijgerde, et al.,
io 1996; Ashe, et al., 1997). Briefly, 24-50 nucleotide long sequences of the
intron-
exon boundaries of cyclin D2 are hybridized onto interphase CLL cells. The
RNA in situ analysis are followed by FISH as described (Mai, et al., 1996;
Fukasawa, et al., 1997) with a cyclin D2 probe (Lukas, et al., 1995) (Figure
3)
and chromosome 12 painting (Figure 3).
is
Proliferation, differentiation, apoptosis. Cellular proliferation are
determined after pulse-labeling with bromodeoxyuridine (BrdU) (Boechringer
Mannheim), apoptosis measured by the TUNEL assay (Fukasawa, et al., 1997)
and differentiation of CLL cells measured by IgM production and morphology
.ao (Larsson, et al., 1991 ). Cell cycle analysis is carried out by flow
cytometry using
Hoechst-staining of total DNA combined with BrdU-incorporation into
replicating
DNA (Kubbies, et al., 1985; Schindler, et al., 1987).
Abrogation of cyclin D2 protein expression. Cyclin D2 antisense
2s oligonucleotides are synthesized by standard phosphoramidite chemistry and
purified by high-performance liquid chromotography. The oligonucleotides (20
mer) are designed according to the OLIGO primer analysis software (version
3.4), which determines the potential dimer formation and self complementary
properties as well as the melting temperature and duplex formation (Anazodo,
et
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al., 1996) (see letter). Control oligonucleotides are cyclin D2 sense, single
base
mutated and scrambled.
Gel retardation analysis. Whole cell extracts and gel retardation studies
s are carried out as described earlier (Mai, et al., 1994). E2F sites from the
DHFR
promoter are used in gel retardation studies to determine E2F complex
formation (Fry, et al., 1997).
Transfection studies on 8 cells. Transfection studies on B cells are
to carried out by electroporation (Zheng, et al., 1996) and
immunoprecipitation and
Western blotting performed as described (Harlow, et al., 1988).
Amplification of cyclin D2 is a constant feature of CLL, and the size of
these amplicons and whether they are primarily extrachromosomal or are
Is reincorporated into specific chromosomes are assessed. In addition, the
structure is examined and the replicative/transcriptional potential of the
cyclin D2
containing Ees and determine if other genes are also present in these Ees.
Ees from CLL cells are isolated and separated on agarose gels to assess
2o molecular weight ranges. Using this approach, the preliminary studies have
indicated a size range from 5 to 25 kb. Gels are then blotted and filters
hybridized with a human cyclin D2 cDNA (Lukas, et al., 1995) to assess which
DNA bands contain cyclin D2 and to obtain an approximate size distribution of
these bands. If additional bands are observed that do not hybridize with the
2s cyclin D2 cDNA, this will suggest the presence of Ees that contains other
genes.
To confirm this: a) bands which do not hybridize with cyclin D2 are isolated,
the
DNA cloned and sequenced, and b) DNA purified from the same bands are
labeled for FISH and hybridized onto metaphases and interphases from CLL
cells. DNA isolated from the same bands are hybridized onto normal B cell
3o metaphases and the loci involved in the formation of these Ees identified
by
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classical cytogenetics and FISH. As the Ees in CLL cells are very small, they
are more easily cloned and sequenced than the much larger Ees usually
observed in tissue culture.
s Ees are digested with a rarely cutting restriction enzyme, e.g., Not I or
Xho I, and ligated into pcDNA3, and will then be transformed into E. coli
(DH5~). A cloning vector, rather than a cosmid or YAC, has been chosen
based on the size of the Ees. Resulting bacterial colonies are replica plated
and
probed for cyclin D2 to identify colonies with cyclin D2 carrying sequences.
The
io remaining colonies will harbour non-cyclin D2 carrying sequences. Initially
the
sequence DNA is derived from colonies that. hybridize with cyclin D2 cDNA and
determine whether the cyclin D2 gene is rearranged, e.g., inverted. If the
sequences of more than one cyclin D2 gene is rearranged, e.g., inverted. The
number and position of the restriction enzyme sites can be able to determine
i~ whether they are in a head-to-head or head-to-tail orientation.
Additionally,
assessments are made regarding whether other genes, e.g., p27kip1, are
present within the cyclin D2-containing Ees and also sequence the non-cyclin
D2 containing Ees.
2o The EM studies have suggested that the Ees in CLL can replicate, and to
confirm this the Ees are subjected to two dimensional (2D) gel
electropheresis.
If replication of cyclin D2-containing Ees is occurring, then replication arcs
are
visible after specific hybridization for cyclin D2. Similarly, the same blots
can be
hybridized with non-cyclin D2-containing DNA, purified from non-cyclin D2
2s hybridizing bands (see 1 a), to determine if these Ees are also
replicating.
Cyclin D2 mRNA is detectable in CLL (Figure 6). To determine whether
the cyclin D2 mRNA is being transcribed from the Ees, nascent pre-mRNA is
carried-out in in situ hybridization followed by FISH (Lawrence, et al., 1989;
3o Wijgerde, et. al., 1996; Ashe, et al., 1997). 24-50 nucleotide long
sequences
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derived from cyclin D2 exon/intron boundaries (Jun, et al., 1997) are used to
detect the nascent pre-mRNA. The published exon/intron sequences are for the
mouse cyclin D2 gene (Jun, et al., 1997). However, the human cyclin D2
exon/intron boundaries are similar, since the 5' and exon sequences from the
s mouse and human cyclin D2 genes are homologous (Jun, et al., 1997; Brooks,
et al., 1996). If problems are encountered in the hybridization efficiency
using
these sequence motifs, PCR is used to amplify the human exon/intron
boundaries. In in situ hybridization, control samples are processed after
RNAseH digestion or in the presence of actinomycin D, which blocks de novo
io transcription. There is no risk of hybridization of short oligonucleotides
to
genomic DNA during the in situ RNA hybridization (Lawrence, et al., 1989). To
confirm this, FISH analyzes are done with these oligonucleotides in
interphases
and metaphases of CLL cells. The RNA in situ analysis are followed by FISH
with a cyclin D2 probe (Wijgerde, et al., 1996) (Figures 1 and 2) and
is chromosome 12 painting. Localization of the origin of the cyclin D2 pre-
mRNA
transcript will confirm whether cyclin D2 is being produced by the Ees and/or
chromosomal DNA.
Cyclin D2 is overexpressed at the mRNA and protein levels in CLL
20 (Figures 6 and 7) but have not yet determined whether cyclin D2 is active.
Thus
it is determine whether: a) cyclin D2 is associated with CDK4 or CDK6 and able
to phosphorylate Rb; b) abrogation of cyclin D2 protein expression in CLL
cells
influences survival, differentiation and/or cell proliferation, and c) cyclin
D2
overexpression on normal B cells effects apoptosis, differentiation and
genomic
2s stability.
If cyclin D2 is functional in CLL, it will bind CDK4 or CDK6 and this can
lead to the phosphorylation of Rb. Recent studies have shown that p27kip1, an
inhibitor of the cyclin E/CDK2, cyclin A/CDK2 and cyclin D/CDK4 complexes
30 (Hirama, et al., 1995; Blain, et al., 1997; Kawamata, et al., 1998; Wang,
et al.,
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1997; Reynisdottir, et al., 1997) can also be overexpressed in CLL (Vrhovac,
et
al., 1997). Whether cyclin D2 is active can thus depend on the relative levels
of
specific cyclin/CDK complexes, p27kip1 and/or other CDK inhibitors. In this
study the focus is on cyclin D2 activity in CLL cells; the presence of cyclin
s D2lCDK complexes is determined by co-immunoprecipitations followed by
Western blotting and examine the abilities of the complexes to phosphorylate a
GST-Rb protein in vitro (Bosc, et al., 1995) can lead to the phosphorylation
of
Rb. In parallel, the cellular levels of p27kip1 profiein are determined.
Assessments are also be made regarding whether p27kip1 is associated with
to the cyclin D2lCDK complexes and correlate this finding with the in vitro
kinase
activities, as determined above.
Although isolated cyclin D2/CDK complexes can be active, their effects in
the cell can depend on the relative amounts of the Rb protein, which has been
is shown to be ~ low or absent in 18-42% of patients (Neubauer, et al.', 1991;
Kornblau, et al., 1994). To directly assess this possibility, the dissociation
of
E2F from Rb is examined by immunoprecipitation and Western blotting and the
binding of E2F complexes to E2F sites on the promoters of target genes, e.g.,
DHFR (Sherr, 1994; Hirama, et al., 1995; Fry, et al., 1997), by gel
retardation
2o analysis (Mai, et al., 1994). Apart from the potential pathway through the
Rb/E2F pathway, it is possible that cyclin D2 directly regulate transcription
and
thus cellular function. This speculation is based on the recent report that
cyclin
D2 represses v-Myb (canter, et al., 1998).
2s To further assess the role of elevated cyclin D2 protein levels in CLL
cells, cyclin D2 antisense oligonucleotide studies are conducted with Dr. Jim
A.
Wright, who has extensive experience with this technology. CLL cells are
treated in vitro with the antisense and control olignonucleotides. The readout
to
document the successful administration of the antisense oligonucleotides can
so initially be the decreasing levels of cyclin D2 protein in the antisense
treated, but
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not control-treated cells. If successful, cellular proliferation are examined,
differentiation and apoptosis are altered by the drop in cyclin D2. To confirm
the
data that are obtained in these antisense experiments, microinjection studies
are
conducted using anti-cyclin D2 antibodies (Pharmingen).
s
The above studies show that cyclin D2 plays a role in proliferation,
differentiation or apoptosis, direct assessments of the effect of cyclin D2
overexpression on normal non-cycling B cells are conducted. It has been shown
that overexpression of cyclin D2 can prevent differentiation and apoptosis in
io myeloid cells (Ando, et al., 1993; Kato, et al,. 1993), but the effect in
lymphoid
cells is not yet known. In addition, assessments regarding whether cyclin D2
overexpression affects genomic stability are also conducted, as has been
observed in epithelial cell line with overexpression of cyclin D1 (Zhou, et
al.,
1996).
1S
Because of the difficulty in obtaining large numbers of normal human B
cells, initial examinations of normal spleen-derived mouse B cells are
conducted.
This shows that 98% of these cells are in G° and they are thus an
appropriate
model for the CLL cells. The mouse B cells are transfected or electroporated
2o with a marine cyclin D2 carrying B-cell expression vector (C~,-driven,
pMK~,m1;
kindly provided by Dr. Konrad Huppi, NIH) and the green fluorescent protein
(GFP) (pEGFP-N1, Clontech). This vector has been used to generate
transgenic mice. However, if the transfection efficiency is too low, infection
of
the B cells with Abelson marine leukemia virus with or without the cyclin D2
2s containing plasmid takes place (Rosenber, et al., 1976). B cells are
isolated and
the transfected B cells identified due to the expression of the GFP. At
different
time points the cyclin D2 levels in transfection B cells are measured by
Western
blot analysis, cell cycle analysis is also carried out and the fraction of
apoptotic
cells calculated. In parallel, genomic instability are assessed by FISH
looking
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for the typical chromosomal changes that have been identified in CLL. Control
cells are transfected with the vector alone.
This study is l~nique in that it is the first to characterize the formation,
s structure and significance of Ees in primary tumor cells from patients. The
results will provide information as to the genes present in Ees in CLL cells
and
whether these Ees replicate and transcribe. Furthermore, they will provide
insight into the role of cyclin D2 in CLL and normal B cells. Overexpression
of
cyclin D2 is shown to induce cyclin D2 transfected B cells into SCID mice to
1o determine if the cells are transformed and give rise to B-cell tumors. To
ensure
the relevance of these studies to human cells, cyclin D2 and/or non-cyclin D2
containing Ees will also be introduced into normal human B cells using an
adenoviral vector and their tumorigenicity are assessed in SCID mice (Graham,
et al., 1991 ).
is
Example 2
Mice. Balb/c mice were obtained from Dentistry (University of Manitoba)
and kept according to the international standards of Central Animal Care. All
2o experiments performed were in accordance with the approved animal protocol
(95-441 ). An age group of 406 week old Balb/c mice (20 mice each) received
i.p. injections of 0.5 ml pristane (2,6,10,14-tetramethylpentadecane (Sigma))
or
LPS (lipopolysaccharide (Sigma)) at 125 ~g/ml (20 mice each). At the time
points indicated in the text, samples were taken from the peritoneal cavity of
the
2s experimental mice. To this end, the mice were anesthesized using avertin
(2,2,2-tribrom-ethanol (Aldrich)). 1 g avertin was dissolved in 0.5 ml liquid
tertiary amyl alcohol (Aldrich). 0.5 ml of this solution was diluted with 39.5
ml
warm (37°C) phosphate buffered saline (PBS) and used to anesthesize the
mice. The average dose per mouse was dependent on the body weight of the
3o animal; a mouse of 20g received 0.35 ml. After anesthesia, peritoneal
cavity
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cells were collected with 8-10 ml of 37°C prewarmed sterile culture
medium that
did not contain fetal calf serum.
Cells directly isolated from the experimental mice were immobilized on
s microscopic slides using a cytospin centrifuge. Fluorescent
immunohistochemistry was carried out using a mouse anti-c-Myc antibody (3C7,
reef. 13) at 20 mg per slide, followed by a goat anti-mouse IgG-Texas Red
antibody (Southern Biotechnology Associates, Inc., USA) at 10p,g per slide.
The
fluorescence intensity was quantitated using the Multiprobe 1.1 E software
to (Signal Analytics, USA) (Mai, et al., 1996). One hundred to three hundred
cells
were evaluated per sariiple.
Fluorescent in situ hybridization (FISH) was used to determine gene copy
numbers on a single cell level. 'The DHFR probe used for hybridization as well
Is as the hybridization conditions and the image analysis have been described
earlier (Mai, 1994; Mai, et al., 1996). The mouse total chromosome 13 paint
was purchased from Cambio (CedarLane Laboratories Limited, Hornby, Ontario,
Canada). The evaluation of metaphase spreads and interphase nuclei was
performed using a Zeiss Axiophot microscope and a CCD camera
20 (Photometrics/Optikon). Image analysis was performed using IPLab Spectrum
H-SU2 (Signal Analytics, USA) and Gene Join (Yale University, USA) on a
Power Macintosh 8100 computer. 100-150 interphases were evaluated in three
independent experiments. Hybridization signals were measured with IPLab
Spectrum/Multiprobe (Signal Analytics, USA), using the line measurement
2s function. Relative fluorescent intensity per pixel (1 pixel=6.8~,m) was
used to
determine both single copy and amplified fluorescent signals. A signal is
classified as amplified if the ratio between the relative fluorescent
intensity per
pixel of amplified vs. The relative fluorescent intensity per pixel of single
copy
signals is >2.
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Western blot analysis. Peritoneal cavity cells were isolated one week
LPS or pristane-treatment. Cells of control, LPS or pristane-treated mice were
pooled for further analysis. 100 ~.g protein was loaded per lane and separated
on a 10% SDS-PAGE gel (Mai, et al., 1994; Mai, 1994). Western blots were
s carried out using the ECL protocol as described (Mai, et al., 1994; Mai,
1994)
and the 3C7 anti-c-Myc antibody (Evan, et al., 1985) as primary antibody at
200ng per blot. The secondary antibody was a peroxidase labeled anti-mouse
antibody (Amersham) and was used at 1:20000 dilution. Densitometry was
performed using the "box function" of the Sigma geITM gel analysis program
io (Jandei, Scientific Software, USA).
Southern blot analysis. Peritoneal cavity cells were isolated one week
after LPS or pristane-treatment. The cells of control, LPS or pristane-treated
mice (6 mice per group) were pooled for further analysis. DNA was isolated as
is described (Mai, 1994) and 10 ~,g DNA was digested with EcoRl (Boehringer
Mannheim, Canada) according to the manufacturer's protocol. Blotting, transfer
and hybridization were carried out as outlined earlier (Mai, 1994). Equal
loading
and uniform transfer of the DNA were controlled by ethidium bromide staining.
The filter was hybridized with a 1.2 kb Pstl-fragment of the hamster DHFR gene
20 (Chang, et al., 1978).
As shown recently, lymphoid and non-lymphoid cell lines amplify the
DHFR gene as a result of c-Myc overexpression (Mai, et al,. 1996). Moreover,
p53-deficient mice show DHFR gene amplification and c-Myc upregulation within
2s the same cells (Fukasawa, et al., 1997). However, in this latter system, it
was
not possible to directly assess whether c-Myc overexpression was the cause of
DHFR gene amplification. To directly determine whether DHFR gene
amplification occurred as a result of c-Myc overexpression in vivo,
plasmacytoma (PCT)-susceptible Balb/c mice was examined that were injected
3o i.p. with pristane (Potter, et al., 1992, Materials and Methods). Control
Balb/c
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mice received i.p. injections of LPS (lipopolysaccharide) that elicites the
transient activation of B cells coricomitant with a transient upregulation of
c-Myc
protein levels, but does not lead to PCT genesis which requires the
constitutive
overexpression of c-Myc (Potter, et al., 1992).
s
Cells directly derived from the peritoneal cavity, the site of PCT diagnosis
(Potter, et al., 1992), were analyzed for their c-Myc protein levels by
quantitative
fluorescent immunohistochemistry (Materials and Methods). Pristane elicited
the elevation of c-Myc protein levels. When examined on a single cell level by
io quantitative fluorescence immunohistochemistry, the induction level of c-
Myc
protein reached 4- to 10-fold three days post pristane injection and remained
elevated for the next four weeks. LPS induced a similar, but transient
upregulation of c-Myc protein levels in B lineage cells. Non-treated
peritoneal
cavity cells exhibited low c-Myc protein levels. When the upregulation of c-
Myc
is was analyzed in the total cell population of the peritoneal cavity by
Western
blots, the upregulation was visible, however less pronounced, with 2.6- and
1.4-
fold induction for LPS vs. pristane-treatments, respectively.
Next examined is the genomic stability of the DHFR gene in the above
2o groups of pristane-treated, LPS-treated and untreated Balb/c mice.
Conventional Southern blot analysis did not show chromosomal amplification of
the DHFR gene in the total genomic DNA of pristane- or LPS-treated cells
isolated from the peritoneal cavity. Similar to previous findings (Mai, et
al.,
1996), Southern analysis suggested the partial rearrangement of the DHFR
as locus. This was only observed in pristane-treated mice. Since fluorescent
in
situ hybridization (FISH) is the most sensitive technique for the detection of
genomic instability on a single cell level, being even more sensitive than the
PCR (polymerase chain reaction)-based detection of chromosomal aberrations,
and since it is therefore becoming the method of choice in clinical studies
30 (Eckschlager, et al., 1996; White, et al., 1997; Afify, et al., 1997), this
approach
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is used to evaluate genomic instability of the DHFR gene in peritoneal cavity
cells. An increase in hybridization signals of the DHFR gene was observed
after
a single i.p. injection of pristane. LPS did~not lead to an increase in
fluorescent
hybridization signals of the DHFR gene.
s
The relative fluorescent intensities of DHFR signals were measured using
IPLab Spectrum software (Materials and Methods). The mean increase in
DHFR signals in pristane-treated mice was 4.3-fold and affected 20-60% of all
peritoneal cavity cells. The distribution of the signals suggested the
presence of
to extrachromosomal elements carrying the DHFR gene.
Next, a test is run regarding whether the increase in DHFR hybridization
signals was due to the increase in chromosome 13, the carrier of the mouse
DHFR gene, or due to DHFR gene amplification. To distinguish between those
is two possibilities, a total chromosome 13-specific paint (Materials and
Methods)
is used and painted the peritoneal cavity interphase cells. In almost all
interphases analyzed (>97%), chromosome 13 was present in two copies.
Trisomy of chromosome 13 was observed din the remaining cells (<3%). The
chromosome 13 paint also stained extrachromosomal elements that hybridized
2o with DHFR confirming the data.
In the present report, pristane-treated Balb/c mice are shown to display
an increase in c-Myc protein levels and DHFR hybridization signals as
determined by FISH. In the majority of the analyzed cells (>97%), the latter
can
2s be attributed to DHFR gene amplification, with >97% of all cells exhiting
two
copies of chromosome 13. <3% of all cells exhibited three copies of
chromosome 13.
The above in vivo findings allow two conclusions: i) the DHFR gene is a
3o molecular marker of c-Myc-dependent genomic instability following pristane
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induction in plasmacytoma-susceptible mice in vivo, and ii) one can speculate
that the amplification of the DHFR gene can be functionally important in c-Myc-
induced genomic instability and neoplasia. While the first alternative is well
documented in cell lines that overexpress c-P!lyc (Denis, et al., 1991; Mai,
1994;
s Mai,et al., 1996) and now also in the c-Myc overexpression-dependent mouse
plasmacytoma, the second alternative remains hypothetical. As shown earlier,
DHFR gene amplification and DHFR enzyme overexpression coincide (Luecke-
Huhle, et al., 1996). One can therefore speculate that the amplification and
overexpression of the DHFR enzyme, which is a key enzyme of folate
to metabolism, will lead to changes in the deoxynucleotide pool sizes,
especially in
the level of deoxythymidine triphosphate (dTTP). Changes in the nucleotide
pool enhance mutation frequencies (Kunz, et al., 1994). Thus, DHFR
overexpression can account in part for the accelerated acquisition of
mutations
and of further genomic instability. In addition, cellular proliferation and
thus the
is statistically increased chance to generate a malignant clone can be
enhanced
due to DHFR overexpression. Interestingly, the potential to amplify the DHFR
gene as well as the levels of DHFR gene amplification observed correlated with
the metastatic potential in a rat tumor model (Luecke-Huhle, 1994).
2o In conclusion, the findings presented in this report confirm the previous
in
vitro data on c-Myc-dependent DHFR gene amplification in non-lymphoid and
lymphoid cell lines of mouse, hamster, rat and human (Mai, 1994; Mai, et al.,
1996) and show for the first time c-Myc-dependent DHFR gene amplification in
VIVO.
Example 3
Cell lines and tissue culture. Human brest ductal adenocarcinoma T47D
and mouse B lymphoma WEH1 231, were obtained from the American Type
3o Culture Collection, Rockviile, MD. Mouse plasmacytomas, MOPC 265 and
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MOPC 460D, the human colorectal carcinoma line, COL0320HSR, and primary
human fibroblasts, GL30192T, have been previously described (Jaffe et al.,
1969; Mai et al., 1996; Mushinski, 1988). The spectrum of mouse B-lymphocytic
cells lines has been presented in detail earlier (Mushinski et al., 1987).
Cells
s were propagated in RPMI 1640 (Biofluids, Inc., Rockville, MD) supplemented
with 10% heat-inactivated (30 minutes, 56oC) fetal bovine serum (Gibco/BRL,
Germantown, MD), 2 mM glutamine, penicillin and streptomycin. Culture media
for B-lymphoid cell lines also contained 5x10-5 M 2-mercaptoethanol. In vitro
line of mouse pre-B lymphocytes is generated by transformation of BALB/c bone
io marrow cells with A-MuLV (Rosenberg and Bltimore, 1976). These cells were
subsequently transfected with pLXSN-bcl-2, a mouse bcl-2-expressing vector
(Gurfinckel, et al. 1987), and pBabePuroMyc-ERTM, an expression vector
(Littlewood et al., 1995) with which the human MYC protein can be activated by
100 nM 4-hydroxytamoxifen (4HT, Research Biochemicals International, Natick,
Is MA). Also produced is a line of mouse fibroblasts in which MYC is
upregulated
by 4HT due to stable transfection of pBabePuroMyc-ERTM into y2 cells (Mann,
et al., 1983).
Cloning and sequencing of mouse cyclin D2 cDNA and 5' genomic flank.
2o A cDNA library of the mouse pre-B cell, 18-81, in lambda ZAP-2, was
screened
under relaxed conditions with Cyl1 (Matsushime et al., 1991), a partial cDNA
for
murine cyclin D1, from Dr. Charles Sherr. Several clones that encoded mouse
cyclin D2 were isolated, rescued as pBIueScript clones, characterized and
sequenced. A probe derived from the clone with the longest (1255 bp) insert
2s was sequenced and found to have a coding region identical to the mouse
cyclin
D2 cDNAs in the literature (Kiyokawa et al., 1992). This probe was used to
screen a partial EcoRl library of BALB/c liver DNA in EMBL-4 arms, from Drs.
Linda Byrd and Konrad Huppi. One positive clone that contained a 17.1-kb
insert was isolated, purified and digested to completion with EcoRi. Only one
of
so the 3 EcoRl fragments that were generated from 2 internal EcoRl sites, a
5.4-kb
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fragment, hybridized with the 5' end of the cyclin D2 cDNA probe, and it was
subcloned into pBIueScript for further study. Partial sequencing of this
fragment
revealed that the 3' 505 base pairs were identical to the 5' portion of the
cyclin
D2 cDNA and that of Kiyokawa et al. (1992). The 3' 194 base pairs contained
s the AUG and an open reading frame, and the adjacent 301 upstream base pairs
contained the 5' untranslated sequence of the cDNA. The remainder was
considered 5' flank in which regulatory motifs might be expected. The complete
sequence of the mouse 5' flank is being generated and are reported elsewhere.
to Electrophoretic mobility shift and supershift assays. Whole cell extracts
were prepared as described (Mai and Jalava, 1994) from WEHI 231 mouse:B-
lymphoma cells and the mouse plasmacytomas MOPC 265 and MOPC 460D.
All mobility shift reactions as well as supershifts were performed at room
temperature. If not indicated differently, 5 mg of cellular protein were
incubated
is for 5 minutes with 1 mg of non-specific competitor DNA (salmon sperm DNA)
followed by a 30-minute incubation with 0.3 ng 32P-end-labeled
oligonucleotides
E1, E2, E3, and y (Figure 9), in low salt buffer (10 mM HEPES-NaOH, pH 7.9,
60 mM KCI, 1 mM EDTA, 1 mM DTT, 1 mM protease inhibitor AEBSF
(Calbiochem, San Diego, CA), and 4% Ficoll 400. Gell electrophoresis was
2o performed on 5% non-denaturing polyacrylamide gels in 22.5 mM Tris-
borate/0.5 mM EDTA (Sambrook et al., 1986) at 8V for 4 hours. The gels were
dried and subjected to autoradiography. Supershift analyses were carried out
as
follows: 5 mg of whole cell extracts were incubated with 1 mg non-specific
competitor DNA (salmon sperm DNA) in low salt buffer (see above). Thereafter,
2s antibodies were added at the concentrations indicated below for 30 minutes
at
room temperature prior to the addition of 32P-end-labeled oligonucleotides.
Gel
electrophoresis was performed as above. All antibodies were purchased from
Santa Cruz Biotechnology Inc., Santa Cruz, CA, except the monoclonal anti-
MAX antibody, which was obtained from Dr. Achim Wenzel, Deutsches
3o Krebsforschungszentrum, Heidelberg, Germany and the anti-c-MYC polyclonal
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antibody and antiserum, which were kind gifts from Dr. U. Deutschel, Basel
Institute for Immunology, Basel, Switzerland. Purified antibodies were used at
100 ng per reaction; the polyclonal anti-c-MYC antiserum and the respective
pre-immune serum at 1 ml per reaction.
s
Assays for genomic instability and gene amplification. Gene dosage was
examined using Southern blot analysis (Southern et al., 1975) and fluorescent
in
situ hybridization (FISH) of metaphase chromosomes (Mai et al., 1995).
Evaluation of metaphase spreads and interphase nuclei was performed using a
io Zeiss Axiophot microscope and a CCD camera (Optikon/Photometrics). 100 -
500 metaphases and interphases were evaluated in each of three independent
experiments. Extrachromosomal fluorescent signals were considered specific
when they also stained with 4', 6' diamidino-2-phenylindole (DAPI) (1 mg/ml)
or
propidium iodide (P1) (1 mg/ml) (Mai et al., 1996).
is
RNA isolation and northern blotting. Total RNA or Poly(A)+RNA was
isolated from cells as previously reported (Mushinski, et al., 1937). 5 mg of
Poly(A)+RNA or 15 mg of total RNA were fractionated on a 1 % agarose gel
containing formaldehyde. The RNA was transferred to a HybondN membrane
20 (Amersham, Arlington Heights, IL) by capillary blotting and hybridized with
32P-
labeled cDNA probes as indicated in the figure legends. Radioactive labeling
was performed with the Nick Translation System (GIBCO/BRL, Germantown,
MD) according to the manufacturer's protocol. The membranes were hybridized
overnight with 3x106 dpm/ml probe, washed with 0.1 x SSC, 0.1 % SDS at 20oC
2s and exposed to X-ray film overnight. For sequential hybridization of the
same
blot with different probes, membranes were stripped with boiling water.
Probes. A 700-by Pstl-fragment of the mouse cyclin D2 cDNA was used
to probe Southern and northern blots, and the genomic clone was used as a
3o probe for FISH. A similar strategy was used to isolate cDNAs for mouse
cyclins
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D1 and D3 (Hamel and Hanley-Hyde, 1997, generous gifts from Paul Hamel,
University of Toronto). The human cyclin D2 cDNA was from Gordon Peters. It
was used as a 1.2-kb Notl-Xhol-fragment. The ribonucleotide reductase R1
(RNR1) probe was a 1.5-kb BamHl fragment of mouse RNR1 cDNA (Thelander
s and Berg, 1986). The cDNA clone pMc-myc54 (Stanton et al., 1983) for mouse
c-Myc was from Kenneth B. Marcu, from which a 0.6-kb Sst I-Hind III fragment
was used as an exons2+3 probe for the Myc sequences expressed in
pBabePuroMyc-ERTM (Littlewood et al., 1995). Mouse cyclins C and E probes
were gifts of Steven Reed. The cDNA for the "housekeeping gene"
Io glyceraldehyde phosphate dehydrogenase (GAPDH) was from Dr. Marc
Piechaczyk (Fort et al., 1993).
hI/estern blotting. Western blots were performed on lysates of pre-B cell
cultures as previously described (Mischak et al., 1993) except that protein
is concentration was determined using the BCA Protein Assay (Pierce), and 10
mg
were loaded per lane. The immunoreactive bands were recognized by the ECL
Western blotting detection system (Amersham, Arlington Heights, IL). The anti-
cyclin D2 (M-20) was purchased from Santa Cruz Biotechnology (Santa Cruz,
CA). The anti-actin (clone AC-40) was from Sigma ImmunoChemicais (St.
2o Louis, MO). The HRP-Goat anti-Rabbit IgG and HRP-Goat anti-Mouse IgG and
IgM were purchased from Axell (Westbury, NY).
Northern blot analysis of cyclin D2 expression in murine B-lymphocytic
lines with different degrees of B-cell maturation. Figure 8 shows a blot of
2s poly(A)+ RNA from a series of mouse B-cell lymphoma cell lines of
increasing
maturation from left to right (Mushinski et al., 1987). When normalized to the
GAPDH control hybridization signals, the highest level of expression of the
predominant cyclin D2 mRNA (6.5-kb) was seen in the four plasmacytomas
(lanes 11 - 14). In addition, several smaller cyclin D2 mRNAs are prominent,
3o chiefly in these four lanes. These four lanes are the B cells with the
highest Myc
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mRNA content. This blot was stripped and rehybridized with other murine cyclin
D probes. The cyclin D3 probe revealed strong 2.3-kb bands in 4 cell lines of
early B lymphocytes, but barely detectable levels in plasmacytomas. The cyclin
D1 probe showed a very strong 3.8-kb band in the myeloid-pro-B line in lane 1,
s strong bands in 2 B-cell lines, lanes 5 and 8, but very low levels in the
remaining
RNA samples. Cyclin E transcripts were virtually undetectable. This pattern of
high levels of Myc and cyclin D2 mRNA was also seen in Northern blots of RNA
from 45 additional plasmacytomas that included tumors with t(12;15) and
t(6;15)
translocations and tumors without translocations but with Myc upregulation due
to to stable integration of Myc-expressing recombinant retroviruses
(Mushinski,
1988).
The 5' flanking region of the cyclin D2 gene has several CACGTG-motifs
that bind c-MYC/MAX. Partial sequencing of the 5' flank revealed four CACGTG
is motifs, putative "EMS (E-box MYC Sites)" (E1-E4) at positions -867, -1487, -
2988 and -3201 from the translation start site, as well as a variant motif,
CAGGTG (y) at position -3475. The positions and adjacent sequence of three of
the EMS motifs and y are indicated in Figure 9. A dot-matrix display shows
bases that are identical in mouse cyclin D2 5' flank and the published 1624
2o bases upstream of the human AUG translation start site in the human cyclin
D2
gene (Brooks et al., 1996), indicating a strong similarity between the mouse
and
human 5' flanks. Note that the 5' flank of the human cyclin D2 gene also
contains at least two cacgtg motifs. These findings prompted analysis of three
of
the EMS motifs in the 5' flank of mouse cyclin D2 for their ability to bind
2s MYC/MAX (Blackwood and Eisenman 1991), and to study whether the cyclin D2
locus can be amplified, overexpressed or both, under conditions of c-Myc
overexpression.
Oligonucleotides containing four of the above-mentioned five E boxes
3o were chosen for the mobility-shift analyses: oligonucleotides E1-E3 (EMS
motifs
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1-3) and y (the variant motif). Purified MYC and MAX bound to E1-E3 but not y.
In "super-shift" experiments, the DNA-protein complex I was found to contain
both MYC and MAX in logarithmically growing plasmacytoma MOPC 265 cells
(Figure 10). Under these conditions, antibodies directed against c-Fos, Mad,
s Mxi-1 or USF, an unrelated E-box-binding protein, did not disrupt or
supershift
complex I, whereas antibodies directed against MYC and MAX did. These data
suggested that, under proliferative conditions, MYC and MAX were present in
plasmacytoma cells, bound to canonical EMS motifs, and appeared as complex
I in mobility shifts and supershifts.
io
Mouse and human tumors with c-Myc overexpression have Southern blot
evidence of amplification of the cyclin D2 locus. The stability of the cyclin
D2
locus was characterized in two mouse B-lymphoid lines, MOPC 460D, a
plasmacytoma that constitutively overexpresses c-Myc due to Myc/Ig
is chromosome translocation (Mushinski, 1988), and WEH1 231, a lymphoblastoid
tumor with low MYC protein levels (Mai et al., 1996). Southern blot analyses
showed that the cyclin D2 gene was amplified in MOPC 460D (Figure 11, panel
A, filled arrowheads), but not in WEH1 231 cells. As in the previous studies,
mouse ribonucleotide reductase R1 (RNR1), a gene that is retained as single
2o copy gene irrespective of MYC protein levels, was used as a reference gene
(Figure 11, panel B).
These analyses were extended to human cell lines: the colon carinoma
line COL0320HSR, a classic example of c-Myc gene amplification and
2s overexpression (28-fold higher MYC protein levels than GL30/92T primary
human fibroblasts); and the breast cancer line T47D, which expresses 11 times
higher c-MYC protein levels than GL30/92T (Mai et al., 1996). As in the
previous studies, human cyclin C, a gene that is retained as single copy gene
irrespective of MYC protein levels, was used as a reference gene. The cyclin C
3o gene was not amplified in any of the human cell lines (Figure 11, panel B).
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COL0320HSR and T47D displayed amplified bands of cyclin D2 gene
hybridization (Figure 11, panel A, filled arrowheads), while primary human
fibroblasts did not. In T47D, the gene for cyclin D2 is partially deleted as
indicated by missing genomic bands in the Southern blot (see open arrowheads
s in Figure 11 ). Such deletions can reflect an additional form of genomic
instability of this locus in cells that overexpress Myc.
Cyclin D2 amplification involves the generation of extrachromosomal
elements in COL0320HSR and MOPC 460D. DAPI or PI staining of metaphase
to chromosome spreads of COL0320HSR, a human adenocarcinoma line, showed
the presence of extrachromosomal elements (ECEs). Fluorescent in situ
hybridization (FISH) showed amplified signals of the cyclin D2 gene on
chromosomes and the ECEs (Figure 12). A similar analysis of the BALB/c
plasmactoma MOPC 460D also showed ECEs that contained cyclin D2
~s sequences (Figure 12). In agreement with the Southern data (Figure 11),
FISH
hybridization of COL0320HSR and MOPC 460D showed neither evidence of
amplification of the RNR1 gene nor extrachromosomal elements that hybridized
with an RNR1 probe.
ao Induced upregulation of MYC activity in mouse pre-B cells results in cyclin
D2 amplification and increased mRNA and protein after three days. A mouse
pre-B cell line derived from bone marrow cells by transformation with
Abelsonmurine leukemia virus (A-MuLV) was stably transfected with
pBabePuroMyc-ERTM, an inducible MYC expression vector that is activated by
2s 4HT. After three days of stimulation by 4HT, numerous ECEs can be staining
of
chromatin and DNA. Several of the ECEs were shown to contain extra copies of
the cyclin D2 gene by FISH analysis (Figure 12). This evidence of genomic
instability and of cyclin D2 gene amplification was not seen in the same cells
without prolonged tamoxifen stimulation (Figure 12).
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Northern blots were prepared from total RNA from the A-MuLV-
transformed pre-B cells before and after stable introduction of pBabePuroMyc-
ERTM, and after different periods of stimulation by 4HT. Figure 13 shows the
results of successive hybridizations of this blot with cyclin D2 and other
germane
s probes. Cyclin D2 message expression is clearly elevated after 4 and 6 days
of
MYC activation by 4HT when compared to the ethidium bromide-stained 28S
ribosomal RNA bands in each lane. Transcripts from the retroviral
pBabePuroMyc-ERTM in the stably transfected line, shown in the right panels,
are detected with a Myc exon 2+3 probe. Endogenous 2.4-kb c-Myc expression
to was repressed after activation of the exogenous MYC-ER by 4HT, as shown by
hybridization with Myc exon 1 probe, since exon 1 is not present in the MYC-ER-
containing expression vector. There was no detectable mRNA for cyclin D1, and
no significant change in mRNA level of cyclin D3 was seen during the treatment
with 4HT.
Western blots of lysates from the 4HT-treated cells described in the
preceding paragraph were probed with anti-cyclin D2 antibody and, as a loading
control, with anti-actin antibody. As shown in Figure 13, cyclin D2 protein
levels
gradually rose in parallel with the levels of cyclin D2 mRNA after MYC
activation
2o by 4HT in the pBabePuroMyc-ERTM-containing cells but not in the pre-B cells
that lack this vector.
Inducing upregulation of MYC activity in mouse fibroblasts also leads to
cyclin D2 amplification and increased cyclin D2 mRNA. A mouse fibroblast line,
2s derived by transfection of y2 cells with pBabePuroMyc-ERTM, was stimulated
with 4HT to induce increased MYC activity. After three days of stimulation by
4HT, numerous cyclin D2-containing ECEs are seen in metaphase chromosome
spreads and in interphase nuclei (Figure 12). This evidence of genomic
instability and of cyclin D2 gene amplification were not seen in the same
cells
so without prolonged tamoxifen stimulation (Figure 12). A control FISH study
of
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4HT-stimulated y2 cells that do not bear the MYC-ERTM expression vector
showed no cyclin D2-hybridizing ECEs (Figure 12). Northern blots were
prepared from total RNA from the fibroblasts with and without stable
integration
of pBabePuroMyc-ERTM, and after different periods of stimulation by 4HT.
s Figure 13 shows the results of successive hybridization of this blot with
cyclin D2
and other probes. As with the B cells, cyclin D2 message expression is clearly
elevated after several days of MYC activation by 4HT, when compared to the
GAPDH loading control.
io The data presented in this report indicate that the mouse cyclin D2 gene
has at least four MYC-MAX-binding EMS motifs 5' of its first exon.
Oligonucleotides that contain any of these motifs bind purified MYC and MAX
proteins, as well as MYC and MAX proteins that are present in lysates of mouse
plasmacytoma cells. The presence of at least two CACGTG motifs upstream of
Is the human cyclin D2 coding region suggests that these motifs have been
evolutionarily conserved due to some essential role in normal cell physiology.
This region of high structural homology has been recently suggested to play a
role in regulation of cyclin D2 expression, but the role of MYC in genomic
instability or cyclin D2 expression was not addressed directly (Jun et al.,
1997).
2o The details of how MYC/MAX binding to each individual EMS motif affects
cyclin
D2 transcription are the subject of another report (J. Hanley-Hyde, in
preparation).
A direct role for MYC in cyclin D2 gene amplification was first suspected
2s when a coupling was observed between Myc overexpression and amplification
of the cyclin D2 gene in established tumors. Amplification of cyclin D2 was
first
seen in Southern blots of two human cell lines, COL0320HSR and T47D, which
were known to have c-Myc amplification and overexpression. Similar evidence
of cyclin D2 amplification was also found in mouse plasmacytomas that did not
3o have c-Myc gene amplification but which did have c-Myc overexpression, due
to
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chromosomal translocations. Extracts from these cells were shown to-contain
MYC/MAX complexes that bound in vitro to three of the CACGTG-containing
oligonucleotides that is identified on the 5' of cyclin D2.
s The cyclin D2 amplification that was detected in mouse plasmacytomas is
associated with enhanced mRNA levels on RNA blots; more transcripts are
found in plasmacytomas than in other cell lines that do not have c-Myc-
activating
chromosome translocations. Such increased expression of other members of
the G1 cyclins, D1, D3 and E was not found in plasmacytomas, indicating that
io this was a special attribute of cyclin D2. Since cyclin D1 is not thought
to be
expressed in normal B lymphocytes (Sinclair et al., 1994), it can be
noteworthy
that two mouse B-cell lines exhibit substantial cyclin D1 expression: BALB
1437
and BAL 17. The mechanisms responsible for the overexpression of cyclin D1
in these lines have not been investigated.
is
To directly implicate MYC levels in the induction of cyclin D2
amplification, the effects of inducible overexpression of Myc in mouse pre-B
cells is studied using a tamoxifen-activated pBabePuroMyc-ERTM chimeric
expression vector. Since amplification of genes occurs gradually, over
2o successive replication cycles. Instead, concentration is on the state of
the locus
and its expression over several days of 4HT stimulation. 4HT had no effect on
the cyclin D2 of parent A-MuLV-transformed pre-B cells. In situ hybridization
showed no evidence of genomic instability, and mRNA expression remained
very low. In the cells with activated MYC-ERTM chimera, extrachromosomal
2s elements, also called double-minutes or polydispersed circular DNA,
episomes
and extrachromosomal DNA (Cohen et al., 1997), that hybridized with the cyclin
D2 probe, appeared after three to four days, indicating increased genomic
instability. At these same time points, blots of RNA and cell lysates isolated
from these cells began to show increased expression of cyclin D2 mRNA and
so protein. The data obtained to date do not require upregulation of either
RNA
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transcription or changes in RNA stability. Simple status-quo rates of
expression
can yield increased steady-state levels of mRNA and protein if the template
were increased, such as by the amplification that is demonstrated. Such a
mechanism is also be responsible for the high levels of cyclin D2 mRNA in
s plasmacytomas, secondary to their constitutive expression of high levels of
c-
Myc mRNA and protein. It is interesting to note that Southern blots of DNA
from
pre-B cells after 3 days of 4HT-induction did not show increased cyclin D2
hybridization signals like those that were seen in well-established tumor
cells
that have experienced high MYC levels for many generations. This can be
to connected with the nature of DNA in extrachromosomal elements that have
been amplified for a short time. This is not surprising, since it has been
well
documented in the literature that FISH analysis is a much more sensitive
technique for identification of amplification of genes than Southern blotting
(Cohen et al., 1997).
is
Such a connection between MYC expression and cyclin D2 amplification
is probably not limited to B lymphocytic tumors, because amplified cyclin D2
in
human colorectal and breast carcinomas is seen. In addition, a gradual
increase in cyclin D2 expression in mouse fibroblasts is found when MYC is
20 overexpressed and activated by 4HT treatment of cells that bear .the
pBabePuroMyc-ERTM expression vector.
This MYC-associated genomic instability can have another possible
consequence: the frequent aneuploidy seen in plasmacytomas (and other
2s cancer cells) that express high MYC levels and have been passaged for
extended periods of time in vivo or in vitro. It has been reported that tumor-
specific initiating non-random chromosome translocations become increasingly
difficult to recognize with repeated passages due to accumulations of
additional,
presumably random, chromosomal aberrations (Coleman et al., 1997).
3o However, it is important to emphasize that this MYC-associated tendency to
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amplification is locus-specific. It has been demonstrated previously for Dhfr
and
in this paper for cyclin D2, but is has been determined that high Myc
expression
produces no such amplification in the genes encoding ornithine decarboxylase,
syndecan-2, glyceraldehyde-3-phosphate-dehydrogenase and cyclin C (Mai et
s al., 1996).
It possible to construct a hypothetical model for how Myc overexpression
and the genes that are amplified in its presence might work together toward
neoplasia. It is their common role in promoting cell cycle progression and
cell
Io proliferation that produces a potent combination favoring induction,
promotion or
progression of neoplastic transformation. Overexpression of Myc has been
shown to shorten the G1 phase of the cell division cycle (Karn et al., 1989),
which favors further mutations by curtailing the period available for cells to
assess and repair DNA damage before it is duplicated in S phase. A similar
is effect can be expected from overexpression of cyclin D2, an important G1
cyclin.
High levels of such cyclins are also foreshorten G1 and rush cells prematurely
into S by titrating out cdk inhibitors such as p21 and p27. Perhaps such
changes are responsible for the transformed characteristics that are induced
by
overexpression of cyclin D1 in fibroblasts (Jiang et al., 1993). Cyclin D1
ao amplification and overexpression is a well-known step' in various cancers
(Motokura and Arnold 1993; Wang et al., 1994; Zho et al., 1995). Amplification
andlor overexpression of cyclin D2 can have similar effects. Overexpression of
cyclin D2, along with D1 and D3, has been found in mouse skin neoplasms and
has been associated with tumor progression (Zhang et al., 1997). Similar to
the
2s finding of cyclin D2 gene amplification in COL0320HSR, Leah et al. (1993)
reported the amplification of this cyclin in a subgroup of colorectal
carcinomas.
What is more, inappropriate expression of cyclin D2 also occurs as a result of
retroviral integration in retrovirus-induced rodent T-cell lymphomas (Hanna et
al.,
1993). Finally, the expression of G1 cyclins and their control of the cell
division
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cycle is known to vary between normal and transformed cells (Hamel and'
Hanley-Hyde 1997).
As mentioned above, amplification associated with Myc overexpression is
s not random, but is locus-specific. Another gene that is amplified by Myc
overexpression, Dhfr, is a key enzyme of folate metabolism, and it is
essential
for DNA synthesis. High levels of the product of this gene can contribute of
this
gene can contribute to maintenance of cell proliferation. High copy number of
Dhfr genes, e.g., following amplification, have been correlated with the
io metastatic potential of tumor cells in a rat carcinoma model (Lueke-Huhle,
1994). Similarly, the gene encoding ribonucleotide reductase R2 subunit, which
is required for dNTP (deoxynucleoside triphosphate) synthesis, is amplified as
a
result of c-Myc deregulation (T.1. Kuschak et al., submitted) Furthermore, it
has
been reported recently that the R2 protein is a malignancy determinant in
is neoplastic cells (Fan et al., 1996).
A model in which MYC promotes genomic instability fits the data reported
here and offers a potential explanation for the frequent observation that high
expression of c-Myc contributes to neoplastic development. Not only does it
zo force cells through the G1 phase of the cell cycle abnormally rapidly, but
also, if
they escape apoptosis, these cells can suffer increased genomic instability in
certain loci, which compounds the precocious cell cycling problem. This makes
it possible, and indeed likely, that such cells will accumulate additional
genomic
alterations and complete the multi-step process of neoplastic transformation.
Example 4
To determine whether the locus-specific amplification of the DHFR gene
occurred as a result of c-Myc overexpression in vivo, an animal model of c-Myc-
3o dependent neoplasia is examined, the mouse plasmacytoma (Potter, et al.,
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1992). Using plasmacytoma-susceptible Balb/c mice, it is analyzed whether
DHFR gene amplification occurred during pristane-induced
plasmacytomagenesis. Balb/c mice are examined that were injected i.p. with
pristane (Potter, et al., 1992). Control Balb/c mice received i.p. injections
of LPS
s (lipopolysaccharide) that elicites the transient activation of B cells
concomitant
with a 'transient upregulation of c-Myc protein levels, but does not lead to
PCT
genesis which requires the constitutive overexpression of c-Myc (Potter, et
al.,
1992).
io c-Myc protein levels of cells directly isolated from the peritoneal cavity
(Potter, et al., 1992) were analyzed by quantitative fluorescent
immunohistochemistry. Pristane elicited the elevation of c-Myc protein levels
(Table 4). The induction level reached 4 to 10 fold three day post pristane
injection and prevailed elevated for the next four weeks. LPS induced a
similar,
Is but transient upregulation of c-Myc protein levels in B .lineage cells
(Table 4).
Non-treated peritoneal cavity cells exhibited low c-Myc protein levels (Table
4).
Next the Balb/c mice are examined to determine whether DHFR gene
amplification in Balb/c mice was induced as a result of the above treatments.
2o Peritoneal cavity cells were analyzed by fluorescent in situ hybridization
(FISH).
The amplification of the DHFR gene was observed after a single i.p. injection
of
pristane (Table 4). Interestingly, LPS did not lead to the amplification of
the
DHFR gene (Table 4). These findings allow one to conclude that the DHFR
gene is a molecular marker of c-Myc-dependent genomic instability in vivo.
2s Moreover, these findings confirm the previous in vitro data on c-Myc-
dependent
DHFR gene amplification in non-lymphoid and lymphoid cell lines of mouse,
hamster, rat and human. Interesting, the fully developed plasmacytoma also
exhibited DHFR gene amplification.
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As shown earlier, DHFR gene amplification and DHFR enzyme
overexpression coincide (Luecke-Huhle, et al., 1996). It is not known whether
the amplification of the DHFR gene is functionally important in c-Myc-induced
genomic instability and neoplasia. Based on the fact that DHFR gene
s amplification occurs both early and late during pristane-induced
plasmacytomagenesis in Balb/c mice, and this amplification event plays a role
during the initiation of genomic instability and neoplastic transformation
(Figure
15). It has been described by others that imbalances in the nucleotide pool
enhance mutation frequencies (Kunz, et al., 1994). Consistent with these
to findings, the amplification and overexpression of DHFR enzyme, which is a
key
enzyme of the folate metabolism, can lead to changes in the nucleotide pool,
especially in the dTTP pool, which affect both DNA synthesis and mutation
frequencies. Thus DHFR amplification and overexpression can account in part
for accelerated acquisition rates of mutations and for further genomic
instability.
is Moreover, DNA synthesis and cellular proliferation and thus the
statistically
elevated probability to generate a malignant clone can be enhanced due to
DHFR amplification and overexpression. It is noteworthy that the potential to
amplify the DHFR gene as well as the levels of DHFR gene amplification
correlate with the metastatic potential in a rat tumor model (Luecke-Huhle"
20 1994).
Example 5
Here, genomic instability in vivo is studied in different organs of p53-~-
2s mice (4-6 weeks old), with age-matched p53 homozygous (p53+~+) mice as
controls. In all p53-~- tissues examined, a substantial percentage of cells
contained abnormal numbers of centrosomes and displayed aneuploidy.
Moreover, c-Myc overexpressi.on was observed in 5-15% of p53-~- cells. In
these
cells, dihydrofolate reductase (DHFR), carbamoyl-phosphate synthetase-
so aspartate transcarbamoyl-dihydroorotase (CAD) and c-myc genes exhibited
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gene amplification. Apoptosis of cells, which displayed abnormal numbers of
centrosomes, aneuploidy, gene amplification and c-Myc overexpression, was
frequently observed.
s Aneupolidy in p53~ mice. Genomic instability is examined in different
organs of clinically healthy p53-'- mice (4-6 weeks old) by cytogenetically
assessing chromosome ploidy. Age-matched parental p53+'+ mice were used. as
controls and then characterized as spleen-, thymus-, and bone marrow-derived
cells, as well as skin- and spleen-derived fibroblasts. These analyses
revealed
to aneuploidy; hyperdiploid, hypo-, hypertetraploid, and polyploid metaphase
plates
were present in all organs examined (Figure 16). The percentage of aneuploid
mitotic plates varied between individual mice; the mean frequencies were 25.6%
in the thymus, 34.8% in fibroblasts, 10% in the bone marrow, and 20% in the
spleen (Figure 16). No aneuploidy was observed in p53+'+ mice (Figure 16).
Cytogenetic studies also revealed that five percent of all metaphases
present in the p53-'- fetal liver hematopoietic cells were aneuploid (Figure
16). In
contrast, there was no evidence of aneuploidy in age-matched p53+'+ fetal
liver
hematopoietic cells.
Abnormal centrosome amplification in vivo in p53-~ mice. p53 has been
implicated in the regulation of centrosome duplication, and multiple
centrosomes
are generated in p53-'- embryonic fibroblasts (MEFs) during a single cell
cycle
(Fukasawa et al., 1996). In the present work, the chromosome instability is
2s tested and observed in p53-'- mice was associated with multiple centrosomes
per cell in vivo. Spleen-, thymus-, and bone marrow-derived cells were
immunostained with anti-y-tubulin antibody to identify centrosomes (reviewed
in
Oakley, 1992; Joshi, 1994), and the number of centrosomes per cell was
scored. The numerical distribution of centrosomes in various organs of p53+'+
so and p53-'- mice is summarized in Figure 17. More than 99% of the interphase
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p53+'+ cells contained one or two centrosomes, most likely depending on their
duplication cycle, while 20-30% of the interphase p53-'- cells contained >2
centrosomes. A typical example of normal vs. Aberrant centrosome duplication
is shown in Figure 17. Thus, as previously observed in vitro in p53-'- MEFs,
in
s vivo that mitotic p53-'- cells frequently displayed abberant spindles,
organized by
multiple copies of centrosomes. However, as reported (Fukasawa et al., 1996)
in some mitotic p53-'- cells, abnormally amplified centrosomes sequestered to
the poles to form bipolarity. These results imply that abnormal amplification
of
centrosomes occurs in vivo, and this can lead to chromosome instability in p53-
'-
io mice.
Gene amplification in p53-~ mice. To evaluate the genomic (in)stability of
single genes in p53-'- mice in vivo, fluorescent in situ hybridization (FISH)
was
performed. Then analysis is conducted on the c-myc gene, which is frequently
Is translocated and/or amplified in tumors (for review, see Marcu et al.,
1992;
Bishop, 1995), and the DHFR gene, whose genomic instability is altered
following either drug selection (Stark, 1993), growth factor (Huang and
Wright,
1994) or c-Myc overexpression (Denis et al., 1991; Mai, 1994; Mai et al.,
1996).
The CAD and the ribonucleotide reductase R1 (R1 ) genes were also examined.
ao The CAD gene encodes a trifunctional enzyme of the pyrimidine biosynthesis
and has been shown to be amplified after exposure to PALA (N-
(phosphonoacetyl)-L-aspartate) (Otto et al., 1989; Yin et al., 1992;
Livingstone et
al., 1992). R1 forms a functional ribonucleotide reductase molecule in
association with a second subunit, ribonucleotide reductase R2 (R2). The
2s genomic stability of R1 is maintained even in malignant cells (Mai et al.,
1996)
and served as a control.
In all p53-'- tissues examined, there was detected an increase in
fluorescent signals as observed by FISH for DHFR and c-myc as well as for
3o CAD in both interphases and metaphases (Figure 18 and Figure 19). In
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contrast, no increase in fluorescent signals for the above genes was detected
in
age-matched control p53+~+ mice (Figure 18). R1 was present as single copy
gene in both p53-~- and p53+'+ cells.
s An increase of fluorescent signals as detected by FISH can be due to two
forms of genomic instability, karyotypic instability (gain of chromosomes)
and/or
gene amplification. The latter can also involve the formation of
extrachromosomal elements which is frequently found in early stages of
tumorigenesis (Wahl, 1989). Extrachromosomal elements were observed
to hybridizing with either DHFR or c-myc in all organs of 4-6 week old p53-~-
mice; a
representative picture is shown in Figure 18.
c-Myc overexpression and amplification of c-myc, DHFR and CAD in the
same p53~ cells. Since c-Myc overexpression has been shown to be
is associated with locus specific gene amplification, the levels of c-Myc
protein in
p53-~- bone marrow-, thymus-, spleen-derived cells as well as fibroblasts were
examined by quantitative fluorescent immunohistochemistry (Materials and
methods). Five to fifteen percent of the p53-~- cells in all tissues examined
showed a two- to eightfold increase in c-Myc expression, while less than 1 %
of
2o p53+~+ cells expressed detectable levels of c-Myc protein. To determine
whether
c-Myc deregulation and genomic instability of single genes occurred within the
same p53-~- cell(s) in vivo, the Combined Protein/FISH Analysis (CPFA)
(Materials and methods) was developed. This assay allows the simultaneous
analysis of c-Myc protein levels and FISH hybridization signals in the
identical
2s cells) in vivo. It therefore allows one to conclude whether c-Myc
overexpression
is associated with genomic instability in the same cells) in vivo. A similar
technique has been independently established by Hessel et al. (1996).
Using CPFA, c-Myc overexpression and amplification of DHFR, c-myc,
~o and CAD genes was observed, but not of the R1 gene within the same
individual
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p53-~- bone marrow-, thymus-, spleen-derived cells as in primary fibroblasts
(Figure 19, panels a-a", b-b"). Thus, locus-specific gene amplification in p53-
~-
mice appears to be accompanied by c-Myc overexpression.
s Apoptosis of genomically altered cells in p53-~ mice. Despite the
extensive genomic alterations present in fetal and neonatal life, p53-~- mice
develop without apparent abnormalities (Donehower et al., 1992; Jacks et.al.,
1994). One possibility is that cells with deleterious genomic alterations can
be
efficiently eliminated by apoptosis. Therefore it was determined that
apoptosis
io occurred in organs and fibroblasts from p53-~- mice. Apoptosis frequently
occurred in tissues and fibroblasts of p53-~- mice, with cells displaying
chromatic
condensation characteristic of apoptosis and a positive TUNEL reaction (Figure
20). Cytogenetic and FISH analyses of p53-~- cells revealed the frequent
presence of morphologically atypical chromosomes (Figure 20) in contrast to
is aneuploid plates with the typical chromosome morphology (Figure 20).
Moreover, morphologically atypical chromosomes exhibited a strong positive
TUNEL reaction in all organs examined (Figure 20). Such morphologically
atypical chromosomes were exclusively observed in p53~~- mice. They seem to
undergo chromatid fragmentation and degradation and often showed gene
2o amplification as well (Figure 20). Apoptotic cells characteristically
displayed
multiple copies of centrosomes (Figure 20), aneuploidy, c-Myc overexpression
(Figure 20), and/or gene amplifications (Figure 20),
p53-~- mice develop a variety of tumors early in life (Donehower et al.,
2s 1992; Jacks et al., 1994), but the mechanisms underlying this tumor
predisposition have remained elusive. Here, it is shown that cells directly
isolated from p53-~- mice display extensive aneuploidy and gene amplification.
This enhanced genomic instability is coupled with the high susceptibility and
frequency of tumor development in these mice.
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Genomic instability initiates during embryonic development and increases
during life; haematopoietic cells of the fetal liver displayed 5% of
aneuploidy,
while all organs of young (4-6 week old) exhibited higher levels of aneuploidy
(Figure 16).
Abnormal amplification of centrosomes occurs in p53-~- mice in vivo, in all
cell types tested. The adverse effects of abnormal centrosome amplification
are
readily observed in cells undergoing mitosis. For instance, the formation of
aberrant mitotic spindles organized by multiple copies of centrosome was
io frequently observed in p53-~- mice. Such events are likely to impair
chromosomal segregation, and induce karyotypic instability and aneuploidy.
Consistent with in vitro studies demonstrating that c-Myc expression is
negatively regulated by p53 (Ragimov et al., 1993), it is found that in the
is absence of p53, all tissues became permissive to c-Myc overexpression.
However, only 5-15% of p53 cells expressed high levels of c-My protein,
suggesting that the additional events) can be required for c-Myc
overexpression
to occur and/or that cells overexpressing c-Myc undergo apoptosis (Figure 20;
Evan et al., 1992; Packham and Cleveland, 1995).
The absence of p53 and the deregulation of c-Myc expression seem to
cooperate during tumor development. For instance, c-Myc overexpression and
lack of p53 have synergistic effects during lymphomagenesis in E~,myc/p53~~+
and CD2-myc/p53-~- mice (Blyth et al., 1995; Hsu et al., 1995). Thus, c-Myc
can
2s play an important role in the overall tumor susceptibility of p53-~- mice
by
contributing to genomic instability, such as locus-specific gene amplification
as
well as increased proliferation rates (Karn et al., 1989).
It has been previously shown that cell lines overexpressing c-Myc display
3o DHFR amplification, independent of species and tissue origins (Mai et al.,
1996).
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Moreover, the c-myc gene was both translocated and amplified, and the protein
was overexpressed in mouse plasmacytoma cells (Mai et al., 1995). In this
study, the genomic stability of c-myc, DHFR, and CAD were tested, all of which
were amplified concomitant with c-Myc overexpression in cells isolated from
p53-
s ~- mice.
While no mutagens were used in this study, several groups have shown
earlier that the administration of the drug PALA (N-(phosphonoacetyl)-L-
aspartate) leads to the selection of drug-resistant cells with amplified CAD
genes
io (Otto et al., 1989; Yin et al., 1992; Livingstone et al. 1992), and this
occurs with
an enhanced frequency in p53-~- cells (Yin et al., 1992; Livingstone et al.,
1992)
A role for c-Myc in PALA-induced CAD amplification has not been described.
Recent work suggests that c-Myc is involved in the transcriptional regulation
of
the CAD gene (Boyd and Farnham, 1997). Since c-Myc acts both as a
is transcription and replication factor, one can propose a role for c-Myc not
only in
the transcriptional activation, but also in the replication/amplification of
the CAD
gene. Consistent with this idea, it has recently been observed the PALA-
dependent upregulation of c-Myc protein levels in p53-~- fibroblasts.
Moreover,
PALA-induced c-Myc overexpression occurred prior to CAD gene amplification
20 (SM). C-Myc overexpression can thus precede the genomic instability of the
CAD gene as it does for DHFR (Mai and Jalava, 1994; Mai et al., 1996), cyclin
D2 (Mai et al., 1997) and ribonucleotide reductase R2 (Kuschak et al., 1997).
Further investigation shows that c-Myc deregulation is a necessary and
limiting
molecular event in CAD gene amplification.
2s
Genomic instability in p53-~- embryos and young mice affects several
genetic loci, includes c-Myc overexpression, abnormal centrosome numbers and
aneuploidy. Further genomic and molecular alterations, such as changes in the
expression and/or half fife of additional oncogenes, cell cycle related genes,
so growth factor-mediated signaling, DNA repair, etc., are conceivable, but
have
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not been examined here. During the multistage process of carcinogenesis, all
of
the above events can ultimately contribute to the selection and evolution of
neoplastic cell(s).
s Despite extensive genomic alterations, p53-~- mice mature without
discernible abnormalities until tumors start to appear (Donehower et al.,
1992;
Jacks et al., 1994). As is show here, there is a high incidence of apoptosis
in all
p53-~- tissues. In addition to interphase cells exhibiting chromosome
condensation and a positive TUNEL staining, there is detected chromosomes
io with atypical morphology. These chromosomes stained in the modified TUNEL
reaction and thus shows chromatid fragmentation. It is noteworthy that these
apoptotic cells usually contained abnormally amplified centrosomes, expressed
high levels of c-Myc protein, and displayed aneuploidy and gene
amplifications.
Thus, many of the genetically abberant cells can be eliminated through p53-
is independent apoptosis. This can explain how p53-~- mice seemingly develop
normally. However, the yield of p53-~- offspring from heterozygous crosses has
been reported to be only ~60% of the expected yield (Jacks et al., 1994),
suggesting that some homozygous lethality occurs during embryogenesis.
Indeed, genomic alterations were observed in a fraction of embryonic cells
20 (Fukasawa et al., 1996, and this study). Since c-Myc has been implicated in
apoptosis (reviewed in, Amati and Land, 1994; Harrington et al., 1994; Packham
and Cleveland, 1995) and apoptosis occurs in the presence of elevated c-Myc
expression in p53-~- mice.
2s Mice, cell suspensions, cell culture. Mice were obtained from Taconic
farms (Ca, USA), with the exception of two p53+~+ parental C57B1/6 mice that
were obtained from Charles River (Quebec, Canada). Nine p53-~- and six p53+'+
mice between 4 and 6 weeks old were used to prepare single cell suspensions
of spleen, bone marrow, and thymus. Ten-day-old skin- and spleen-derived
3o primary fibroblasts (passage 0) were also obtained from these mice by
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explanting subcutaneous skin tissue pieces and spleen fragments into RPMI
1640 medium supplemented with 10% fetal calf serum, 2mM L-glutamin, and 1
mM sodiumpyruvate. Hematopoietic cells were prepared from fetal livers of 16-
day-old embryos. Ten fetal livers were pooled for further analyses.
s
y~-tubulin immunostaining. Cells isolated from mice were seeded onto the
slides. Samples were fixed in 3.7% formaldehyde in phosphate buffered saline
(PBS) for 20 minutes at room temperature (RT). Cells were then incubated in
the blocking solution (10% normal goat serum in PBS) for 1 h at RT. The
io samples were then incubated with anti-~-tubulin antibody raised against
CSREIVQQLIDEYHAATRPDYISWGTQ for 1 h at 37°C. The samples were then
washed extensively in PBS, followed by incubation with fluorescein
isothiocyanate (FITC)-conjugated goat anti-rabbit immunoglobulin G (IgG) for
30
minutes at RT. The samples were then washed extensively in Tris buffered
Is saline (TBS), followed by 4',6' diamidino-2-phenylindole (DAPI) DNA
staining (1
~,g/ml). For each cell type, >400 cells were examined.
c-Myc determination. Quantitative fluorescent immunohistochemistry was
used to determine c-Myc protein levels in single cells directly isolated from
the
ao mice. Following immunohistochemistry with 20 ng/slide of a monoclonal anti
c-
Myc antibody 3C7 (Evan et al., 1985) and a secondary antibody (anti-mouse
IgG-Texas Red; Southern Biotechnology Associates, Inc., USA) at 10 ng/slide,
images were acquired using a Zeiss Axiophot microscope, coupled to a CDD
camera (Optikon/Photometrics), and the relative fluorescence intensity per
pixel
as (one pixel = 6.8 ~,m) was analyzed on a Power Mac 8100, using IPLabSpectrum
and Multiprobe software, version 3.1 (Signal Analytics, USA). One hundred to
three hundred cells were evaluated per sample.
Cytogenetics and fluorescent in situ hybridization (FISH). Metaphase
3o spreads for cytogenetic analysis, FISH and a modified TUNEL assay (see
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below) were performed according to standard protocols (Mai, 1994; Mai et al.,
1996) using cells directly isolated from the mice. Analyses of interphase
cells by
FISH, CPFA (see below) and the TUNEL assay (Li et al., 1995) were carried out
on cytospin preparations. FISH determination of metaphase chromosomes and
s interphase cells was performed as described (Mai, 1994; Mai et al., 1996).
All
probes used have been described elsewhere (Mai et al., 1996), except the CAD
probe (a generous gift from Dr. George Stark, The Cleveland Clinic Foundation,
OH). The number of metaphase plates evaluated for cytogenetic analyses was
50 per organ and 100 per fetal liver cells. The number of metaphase plates and
io interphases evaluated for FISH analyses was 100 per sample. Using the
lPLabSpectrum and Multiprobe softwares (Signal Analytics, USA), amplified
signals were determined with the 'Line Measurement' function; relative
fluorescent intensities per pixel (one pixel = 6.8 ~.m) were measured for
single
and amplified hybridization signals. As signal is classified as amplified if
the
is ratio between the relative fluorescent intensity per pixel of amplified vs.
the
relative fluorescent intensity per pixel of single copy signals is >2 in one
hundred
interphase cells.
Combined proteinlFISH analysis (CPFA). To visualize c-Myc protein
2o expression and genomic (in)stability within the same cells,
immunohistochemical
analysis and FISH are combined. Cells immobilized on slides were fixed (3.7%
formaldehyde), permeabilized (0.2% Triton X-100), incubated with 20 ng/slide
of
the anti-c-Myc monoclonal antibody (3C7, Evan et al., 1985), followed by the
secondary Texas Red-conjugated goat anti-mouse IgG (Southern Biotechnology
2s Associates, Inc., USA) (10 ng/slide). The nuclei were counterstained with
DAPI
(1 ~,g/ml in PBS), photographed, and the positions were recorded. Thereafter,
the slides were processed for FISH analysis as described (Mai, 1994; Mai et
al.,
1996). Cells in the recorded positions were re-evaluated for their respective
gene copy numbers. A microscopic field of 104 to 105 cells was analysed, and
30 100 cells were evaluated per sample. As above, relative fluorescent
intensity
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per pixel was determined using the IPLabSpectrum software (Signal Analytics,
USA).
Apoptosis assays. The TUNEL assay was performed on interphase cells
s as described (Li et al., 1995). A modified TUNEL assay was designed to
visualize gaps and DNA fragmentation on metaphase chromosomes.
Metaphase chromosomes were prepared as described (Mai, 1994; Mai et al.,
1996). Briefly, the slides underwent fixation, RNAse and pepsin treatments,
and
postfixation. Thereafter, the apoptotic assay was performed using dUTP-
to fluorescein and the terminal deoxynucleotidyl transferase (TdT) enzyme
according to the suppliers (Boehringer Mannheim, Canada). Chromosomes
were counterstained with propidium iodide (1 ~,g/ml) and analysed as described
(Mai, 1994; Mai et al., 1996). Chromatin condensation was visualized with DAPI
(1 ~,g/ml). One hundred to three hundred cells were evaluated per sample.
is
Example 6
c-Myc-dependent locus-specific genomic instability. In this study, the
focus has been on the modulation of cellular proliferation as determined by
the
2o presence of elevated c-Myc protein levels. Furthermore, the presence of c-
Myc-
dependent locus-specific genomic (in)stability has been examined. Genomic
instability is a hallmark of pre-malignant and neoplastic cells. However,
current
knowledge about the genomic changes that occur in a cell or cell population at
the initiation of the carcinogenic process is still limited, and therefore no
exact
2s definition of "genomic instability" relevant to neoplastic transformation
has been
possible to date. In this example, the term "genomic instability" refers to c-
Myc-
dependent locus-specific genomic instability. In tissue culture as well as in
animal models, it has been shown that the deregulated expression of the c-Myc
oncoprotein mediates locus-specific genomic instability (Mai, 1994, Mai et
al.,
so 1996a, 1999, Kuschak et al., 1999; Taylor and Mai, 1998) as well as
karyotypic
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instability (Mai et al., 1996b). Locus-specific genomic instability affects a
number of genes, among them are the dihydrofolate reductase (DHFR) (Mai,
1994, Mai et al., 1996, Taylor and Mai, 1998), cyclin D2 (Mai et al., 1999)
and
ribonucleotide reductase R2 (R2) (Kuschak et al., 1999) and carbomoyl-
s phosphate synthetase-aspartate transcarbarnoyl-dihydroorotase (CAD)
(Fukasawa et al., 1997) genes. At the same time, other genes such as
syndecan-7, ribonucleotide reductase R7, ornithine decarbocylase (ODC) and
cyclin C, remain unaltered in their genomic stability (Mai et al., 1996a). c-
Myc-
dependent karyoptypic instability is reflected by the generation of
io extrachromosomal elements, centromere-telomere fusions, DNA breakage and
the presence of ring chromosomes (Mai et al., 1996b, Felsher and Bishop,
1999). These chromosomal changes are directly associated with the
tumorigenic potential of the genomically unstable cells (Felsher and Bishop,
1999).
is
Based on these findings, it was thought to examine in human cervical
cancer with well-defined stages of pre-neoplasia (dysplasia) as well as
carcinoma whether c-Myc played a role in tumorigenesis and affected a target
gene of c-Myc in genomic instability, the DHFR gene.
MATERIALS AND METHODS
Thirty-six patients referred to the Colposcopy Clinic in the University of
Manitoba's Department of Obstatrics, Gynecology and Reproductive Sciences,
2s that is located at the Health Sciences Centre (HSC), were enrolled for the
study.
This study has ethics approval from The University of Manitoba Faculty
Committee on the Use of Human Subjects in Research and a written consent
was obtained. The study protocol included: A standardized history on each
patient, collection of exfoliated cervical cells for the Pap smear and a
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colposcopically directed cervical biopsy and/or endocervical curretage (ECC),
when clinically indicated.
Following collection of the cervical cells and preparation of Pap smears,
s the cervix was treated with 5% acetic acid and a colposcopic examination was
performed using a Zeiss Colposcope OP-1 or OP-9, or a photocolposcope. The
findings were recorded in a standardized form. Colposcopically directed
biopsies and/or ECCs were performed and the cervical biopsy specimen was
divided in two parts; one part was put in 10 formalin and the other kept
fresh;
to both were sent to Pathology immediately.
The fresh tissue was then frozen in a cryostat (company) at -26°C
and
serially sectioned at 5w intervals. The first few sections were stained by
hematoxylin and eosin (H and E) and microscopically examined to make sure
is that the sections contained perpendicularly cut tissue from the
transformation (T)
zone. The following 5-6 sections were kept unstained and air dried for up to
24
hours, before being examined by fluorescent immunohistochemistry and
fluorescent in situ hybridization (FISH). The remaining of the frozen tissue
was
put in 10% formalin and sectioned and stained together with the originally
20 obtained and formalin-fixed tissue, dehydrated and paraffin embedded
overnight.
The pap smear obtained at the time of colposcopic evaluation of the
patient was fixed and stained by the classical Papaniscolaou method. It was
2s screened at the Cytology laboratory of the Department of Pathology at HSC.
The smear was reviewed and reported according to the combined Walton
classification (no abnormal cells, reactive, atypical, mold, moderate, severe
dysplasia, carcinoma in situ and invasive carcinoma) and the Bethesda system
(no abnormal cells, reactive, ASCUS, low grade squamous intraepithelial lesion
30 (LSIL), high grade squamous intraepithelial lesion (HSIL) and carcinoma).
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The histologic sections were cut from the paraffin blocks at 8~, intervals
and stained by II/E. These sections were reviewed and reported by one of the
authors (M.P.). The degree, if any, of dysplasia present was reported as mild
if
s the squamous epithelium contained enlarged, hyperchromatic, mitotically
active
cells occupying the lower third of squamous epithelium. Dysplasia was reported
as moderate, if the dysplasia involved 2/3 of the thickness of squamous
epithelium and severelCIS if it involved the total thickness of the squamous
eithelium. Any evidence of koilocytic surface changes and/or individual cell
io keratinization was noted and, in their presence, the diagnosis of HPV
infection
was suggested. Breaking of basal membrane of the surface squamous
epithelium by neoplastic tissue and extension into the underlying stroma was
taken as evidence of early (micro) or frank invasion, depending on the depth
of
invasion.
is
For the molecular and cytogenetic studies, sections were positioned on
coated slides. The slides were coated with 3-aminopropyltriethoxy-silane
(Sigma). The slides were dipped in 2% silane (in acetone), rinsed two times in
distilled water, dried at 37°C for two hours and stored at room
temperature. All
2o sections were then placed onto these slides and were analyzed in the
following
way. 5~, thick parallel sections were examined by quantitative fluorescent
immunohistochemistry as described (Fukasawa et al., 1997). The antibody used
was an anti-human Myc antibody (Oncogene Research). Images were acquired
using a Zeiss Axiophot microscope, a CCD camera (Photometrics) and analyzed
2s with IPLab Spectrum software (v3.1) (Scanalytics, Fairfax, Virginia).
Parallel
sections were processed for fluorescent in sifu hybridization (FISH) using a
digoxigenin-labeled human dihydrofolate reductase (DHFR) probe (CHB204,
obtained from ATCC). Detection of the' hybridization was carried out using a
FITC-conjugated sheep-anti-digoxigenin antibody (Roche Diagnostics). FISH
3o analysis was performed with IPLab Spectrum software.
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All images taken were matched with the respective areas of the parallel
sections of the H and E stained slides.
s Myc protein levels were assessed in the following way: elevated c-Myc
protein levels were assessed with +, ++, +++ depending on the amount of c-Myc
protein present in the nuclei ofi the cervical cells. No detectable nuclear
immunofluorescence was recorded as negative, +; less than one third of all
cells
in the section displayed elevated c-Myc protein, ++; 2/3 of all cells
displayed
to elevated c-Myc protein, +++; every cell displayed elevated c-Myc protein.
DHFR
gene copy numbers were recorded as negative, if all cells displayed single
copy
DHFR signals, as +l- when the occasional cell showed additional DHFR copies,
+ when a limited group of cells manifested DHFR gene amplification, ++ when
more than 50% displayed DHFR gene amplification, +++ when >80% of the cells
is showed DHFR gene amplification.
RESULTS
The results of the colposcopic, cytological and histopathological
2o diagnoses were tabulated and then compared with those of the fluorescent
immunohistochemistry and fluorescent in situ hybridization.
Quantitative fluorescent immunohistochemistry of biopsies derived from
36 patients enrolled in the study showed that c-Myc protein levels were
elevated
2s in 29 out of 36 patients (80.5%) (Table 1). Seven patients out of 36
(19.4%)
without significant c-Myc protein elevation were found to have either no
deectable cervical lesion (five patients out of 36 or 13.8%) or had CIN1 (one
patient of 36 or 2.7%). One patient was CIN 3 (one patient out of 36 or 2.7%).
In general, c-Myc protein levels were upregulated in early and late cervical
30 lesions. Among the biopsies that were classified as negative, five showed
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elevanted c-Myc protein (5110 or 50%). The exact test was used to analyze the
data. When negative and CIN1 (group A) was compared to C1N2, CIN3, and
carcinoma (group B), c-Myc levels were significant ([=0.098). When the exact
trend test was performed, the one tailed result for c-Myc was significant as
well
s (p=0.032). ,
DHFR gene copy numbers were examined in 29 patients (Table 2) who
were also studied by quantitative fluorescent immunohistochemistry. The
presence of DHFR gene amplification was indicative of early and late
~o malignancy (five out of 29 patients (17.2%) did not show DHFR gene
amplification, two of these had no detectable cervical lesion (6.8%), three
out
were CIN1 (10.3%)). The level of DHFR gene amplification was associated with
stage of disease. It is noteworthy that out of seven patients that were
classified
as negative, five showed DHFR gene amplification (one had low level
is amplification), the other one had high level with areas of very strong
amplification (DHFR++~+++)_ The PAP smear of this patient was CIN3.
Statistical analysis was carried out for the above results. The exact test
gave
significant values for DHFR (p=0.041 ) as did the one tailed trend test
(p<0.0001 ).
2o DISCUSSION
The data presented in this work shows that the assessment of c-Myc-
dependent DHFR gene amplification is suitable as a molecular biomarker for
cervical intraepithelial lesions. This biomarker is useful both for the
detection of
2s early cervical cancer and for tumor progression. If compared to other
biomarkers, the genomic instability of the DHFR gene is one of the earliest
markers available to date.
No studies have been performed on the genomic stability of the DHFR
so gene in cervical cancer. In contrast, a series of studies have been carried
out on
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the amplification and. expression of c-Myc. The data reported on c-Myc in
cervical cancer are controversial. When evaluating the published reports, one
has to distinguish between those that examined c-myc gene amplification and
those that described c-Myc protein elevation or both. Both molecular
alterations
s do not necessarily coincide. While indicative of genomic instability, the
amplification of a gene without its overexpression is functionally irrelevant
(see
also Kuschak et al., 1999).
c-Myc gene amplification in cervical cancer. Some studies have
Io suggested that c-Myc amplification is one of the early events in cervical
intraepithelial lesions (Aoyama et al., 1998). However, Choo et al. (1989)
reported that a Chinese group of cervical patients did not show c-Myc gene
amplification. These data are supported by a more recent study that reports on
low c-Myc amplification in invasive carcinoma, stages I and II of cervical
cancer
Is (Kersemaekers et al., 1999). In contrast, a French study found c-Myc
amplification and protein overexpression associated with early stages of
cervical
cancer and related to the risk of relapse (Riou et al., 1990). The authors
further
report on the significant overexpression of the oncoprotein in later stages
and
suggest that c-Myc can be both an early marker and a progression marker (Riou
2o et al., 1990). Bourhis et al. (1990) essentially come to similar
conclusions. In a
different study, Wu (1996) reported that c-Myc can be a valuable biomarker for
cervical cancer
c-Myc overexpression in cervical cancer. In studies performed on
2s Mexican cervical cancer patients, c-Myc protein deregulation was found in
90%
of the lesions (Ocadiz et al., 1989). Indian patients also displayed >90%
overexpression of c-Myc. A Swiss study showed elevated levels of c-Myc by
immunostaining, with the highest level of c-Myc found in high grade CIN
(Deltas
et al., 1997). These findings were contrasted by studies in Japan where the
so incidence of c-Myc protein deregulation was significantly lower (Iwasaka et
al.,
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1992), however, c-Myc protein level was suggested as a prognostic marker for
disease progression. A study carried out by Sowani et al., (1989) also
suggested that c-Myc has prognostic value in treatment decisions. In contrast,
Slagle et al. (1998) found no elevated c-Myc protein levels in normal to high
s grade cervical lesions (dysplasia). Work by Dellas et al. (1998) suggests
that c-
Myc expression is associated with proliferation in pre-cancerous lesions, but
not
with overall survival in invasive carcinoma (see also Symonds et al. 1992). In
support of the c-Myc-induced proliferative potential is the study by Helm et
al.
(1993). The authors inhibited growth of ovarian and cervical carcinomas in
situ
to by the use of a c-myc-targeted triplex forming oligonucleotide.
Trisomy of chromosome 8. Trisomy of a specific chromosome in
cancer cells is usually associated with the overexpression of genes) involved
in
transformation. An example is trisomy chromosome 12 in a subset of chronic
is lymphocytic leukemia patients (Aver et al., 1999; Liso et al., 1999; Dohner
et al.,
1999; Van Kessel et al. 1999). Interestingly, Mark et al. (1999) have recently
reported on trisomy 8 in cervical cancer. Chromosome 8 carries the c-Myc
proto-oncogene (8q24).
2o HPV and integration sites. Preferential sites of HPV integration have
been reported (Popescu and Di Paolo, 1989). Interestingly, HPV has been
shown to integrate next to the N-Myc gene (2p24) and c-Myc gene (8q24.1)
(Couturier et al., 1991). This integration can result in the deregulated
expression
of the Myc proteins. Co-amplification of HPV and c-Myc has been observed in a
2s newly established cervical carcinoma line (Gotoh et al. 1991). Recently,
Maeville et al. (1999) reported on HPV18 integration sites next to the c-Myc
locus in HeLa cells. The authors also detected 8q24 amplification by CGH and
confirmed the amplification of c-Myc by FISH. Additional chromosomes carry
HPV copies next to 8q24-derived material (Maeville et al. 1999).
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c-Myc protein levels in cervical biopsies determined by quantitative
fluorescent immunohistochemistry. This data shows that c-Myc protein levels
play a role in early and late cervical lesions. Therefore a prolonged
overexpression of c-Myc in early lesions allows for the stable amplification
of
s DHFR, which shows increased gene amplification over time and remains an
indicator of tumor progression.
c-Myc-induced DHFR gene amplification determined by FISH: The
amplification of the DHFR gene correlates best with the pathological and
to cytological evaluations. In cell lines and in a mouse model of c-Myc-
dependent
genomic instability, DHFR is amplified as a consequence of deregulated c-Myc
expression (Mai, 1994, Mai et al., 1996a, Taylor and Mai, 1998). For this
example, it is concluded that c-Myc overexpression is also associated with
enhanced copy numbers of DHFR in cervical cancer.
CONCLUSIONS
This work shows that c-Myc-dependent DHFR gene amplification as a
2o suitable marker of early and late cervical cancer.
The invention has been described in an illustrative manner, and it is to be
understood that the terminology which has been used is intended to be in the
nature of words of description rather than of limitation.
Example 7:
Recently, greater advances have been made in the characterization of
amplified genes and specifically EEs. Each of the more sophisticated
3o technologies currently used can be applied to greater potential following
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additional purification of EE samples. Recently, it was demonstrated that
functional EEs carried active histone proteins (Wiener et al., 1999). The
presence of histones on functional Ees was used to develop a method enabling
the isolation of EEs that carry genes. To facilitate the isolation of
functional EEs
s extracted from tissue culture cells or clinical samples, the Hirt protocol
(Hirt,
1967) was adapted that was originally designed to isolate polyoma virus
particles. Preparations of EEs are frequently contaminated with varying
amounts
of genomic DNA and/or apoptotic DNA fragments (Regev et al., 1998). The
contamination of EEs with genomic and apoptotic DNA was addressed by
Io Gaubatz and Flores (Gaubatz et. al., 1990), who described the use of
exonuclease III, an enzyme that removes linear DNA molecules from these
preparations. Exonuclease III also digests open and nicked circular
extrachromosomal DNA molecules, potentially eliminating some of the EEs.
Therefore, the use of this enzyme can compromise the isolation of a true and
Is representative population of potentially transcriptionally or
replicationally active
EEs from a cell.
There are several advantages to immunopurifying EEs prior to their
analysis. First, this immunoprecipitation method can be utilized as a primary
2o post-extraction purification step following Hirt isolation of EEs. This
immunopurification method allows for a number of analytical procedures to be
conducted on a pure and heterogeneous population of EEs without genomic
contaminants, derived from cultured cells, primary tissue, or tumor samples.
In
the experiments, immunopurified samples showed an enriched population of
2s EEs that carry DHFR gene sequences and are free of genomic contaminants.
Second, this purification method allows further' experimentation with a
greater
degree of resolution. In the past, classical karyotype analysis was only able
to
show that EEs were present in the karyotype, though the sequence content of
these EEs was not characterized. Recently, FISH-EEs (Kuschak et al., 1999)
3o was successfully applied to test for the presence of genes on freshly
isolated
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EEs. Using the ability to immunopurify EEs prior to performing FISH-EEs, it is
now possible to identify potentially active genes that are (co)localized on a
selected, but representative population of potentially functional EEs.
s SKY has proven invaluable in visually identifying chromosomal re-
arrangements in tumor cells and in showing the presence of EEs as well as the
chromosomal origins) of those EEs (Ariyama et al., 1998). SKY analysis of EEs
can be refined by probing immunopurified EEs specifically for histone-carrying
and potentially active genes that are amplified or deleted from whole
io chromosomes, as opposed to the SKY analysis of all extrachromosomal DNA
that is not necessarily associated with active chromatin.
CGH is a technology that is capable of detecting amplifications and
deletions in genomic DNA (Kallioniemi et al., 1992). CGH can also be used to
is examine extrachromosomal DNA. Comparative analysis of CGH on
immunopurified EEs in combination with CGH on genomic DNA can together
resolve (i) the degree of amplification found on EEs, (ii) answer mechanistic
questions about whether certain EEs are extrachromosomal amplification
products derived from HSRs or the products of a chromosomal deletion. Thus,
2o CGH can be used more precisely as a tool when only potentially active
populations of EEs are compared. The isolation of large quantities of
potentially
active EEs using the immunoprecipitation method also facilitates the use of
more traditional approaches such as Southern analysis of extrachromosomal
DNA. Finally, the immunopurification of EEs expedites the isolation of
sufficient
2s quantities of purified and potentially active EEs, allowing for the
creation of EE
libraries, which was not previously possible.
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MATERIALS AND METHODS
Cell Culture
A mouse Pre-B ABM cell line was used for the generation and isolation of
s extrachromosomal DNA purification. This is a Myc-regulatable cell line that
carries a Myc-ERTM that is activated by the addition of 100 nM 4-
hydroxytamoxifen (4-HT) (Sigma-Aldrich Canada, Winston, ON, Canada). For
simplicity, non-activated Pre-B cells are referred to as Pre-B- and 4-HT-
activated
Pre-B cells are referred to as Pre-B+ cells. The origin of Pre-B cells (Mai et
al.,
Io 1996) and their culture conditions (Kuschak et al., 1999) have been
described.
Isolation of EEs
Ees were isolated from Pre-B+ cells following activation as described in
Is (Kuschak et al, 1999). The procedure for EE isolation is based on the
protocol of
Hirt (Hirt, 1967) and modified as previously described (Kuschak et al, 1999).
The
Hirt extract contains the bulk of the EEs and the total cellular RNA, but it
can
also be contaminated with small linear fragments of genomic DNA, and
apoptotic DNA fragments which have been estimated to comprise 0.5 n
1.0°I° of
ao the total amount of EEs isolated (Regev et al., 1998). Following isolation,
all
manipulations of EEs were performed in sterile, siliconized micro-centrifuge
tubes (Fisherbrand, Fisher Scientific, Pittsburgh, PA, USA).
Determination of Apototic or Genomic DNA Contamination in Non-
2s Immunopurified Pre-B- and Pre-B+ Cell-Derived EEs
Preparations of EEs were made from Pre-B- and Pre-B+ cells in triplicate.
4',6' diamidino-2-phenylindole (DAPI) (Sigma-Aldrich Canada, Winston, ON,
Canada) (1 ~.gmL-~ in PBS) was used to counter-stain both the EEs and any
3o genomic DNA contaminants. The samples were then visualized and
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photographed using a Zeiss Axioplan2 microscope (Carl Zeiss Canada, Inc.,
Ottawa ON, Canada) under a 63x oil immersion objective and a UV filter. The
images were acquired using Northern Eclipse 5.0 software (Empix Imaging Inc.,
Mississauga, ON, Canada) and a Sony (model XC75) CCD camera. The
s percentage of contaminating DNA was assessed in each of the preparations by
manually counting and totaling EEs and signals from contaminating apoptotic or
genomic DNA (Khaira et al., 1988). It was determined that Pre-B- and Pre-B+
cell-derived EE preparations carried 10.8 ~ 2.5 % and 12.1 ~ 4.9 % apoptotic
or
genomic DNA contamination, respectively.
io
Purification of Histone-Containing Extrachromosomal DNA Molecules
The Protein G sepharose beads were pretreated and (Amersham
Pharmacia Biotech, Inc, Baie diUrfE, PQ, Canada), used for the
Is immunopurification of he EEs. 1 ~,g of Protein G sepharose beads was washed
in 5 mL Buffer A (100 mM KCI, 10 mM Trizma pH 7.4, 1 mM Na2EDTA, 1 mM
DTT, 1 mM AEBSF) by placing on a rotating platform for 10 minutes at room
temperature. KCI, Trizma base, and Na2EDTA were purchased from Sigma-
Aldrich Canada, Winston, ON, Canada. DTT was purchased from FLUKA
2o through Sigma-Aldrich Canada, Winston, ON, Canada. AEBSF was purchased
from Roche Diagnostics, Laval, PQ, Canada. The beads were centrifuged at
13,000 rpm (16,000 x g) for 10 minutes at room temperature and the
supernatant was removed and discarded. This washing procedure was
performed a total of three times. The non-specific binding sites on 40 ~,L of
2s beads were blocked by resuspending the beads in 300 p,L of Buffer B (Buffer
A
+ 4% ('"/") Bovine Serum Albumin) (FLUKA purchased through Sigma-Aldrich
Canada, Winston, ON, Canada).
Immunopurification of extrachromosomal elements was used to perform
so the isolation of the Hirt-extracted histone-bound EEs from the impurities
found in
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the Hirt-extracted EE sample. Freshly extracted EEs were dialyzed against 1x
TE buffer (10 mM Trizma base, 1 mM Na2EDTA, pH 8.0) overnight at
4°C. The
EEs were then incubated in 500 p.L Buffer B on a rotating platform for 10
minutes at room temperature. 1 ~,g of sheep polyclonal anti-core histone
antibody
s was added per p,g of EEs and incubated the mixture for 30 minutes at room
temperature on a rotating platform. The blocked beads were then added to the
antibody-treated EEs and incubated overnight on a rotating platform at room
temperature. Following this incubation, the EE/beads were centrifuged at
13,000
rpm for 10 minutes at room temperature to remove any unbound material. The
to EE/beads were washed three times with Buffer A to remove any residual
unbound material. The bound histone-containing EEs were eluted by adding 300
~,L of 100 mM glycine (Sigma-Aldrich Canada, Winston, ON, Canada) (pH 2.3)
to the beads and mixing gently by inverting the tube 5-10 times. The tube was
centrifuged at 13,000 rpm for 10 minutes at room temperature and the
is supernatant (eluate) was removed and collected. This eluate was immediately
neutralized to pH 7.2 using 1 M Trizma, pH 8Ø The elution step was repeated
once more and the two collected eluates were pooled together. The sample was
concentrated to approximately 40 ~.L by roto-evaporation.
ao Analyses of Immunopurified Extrachromosomal Elements
The following protocol allows the fixation of the EEs onto glass
microscope slides and it ensures that the EEs are contained within a small
area.
Briefly: 40 ~,L of Hirt extract (Hint, 1967) or immunopurified and
concentrated
as EEs are diluted 1:1 in a fixative solution (freshly prepared 3:1
methanol:actetic
acid) and then delivered onto pre-cooled (60 seconds on dry ice) slides (O.
Kindler, Germany). The slides are immediately moved onto a slide warmer
(37oC). The fixation procedure was completed as described previously (Kuschak
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et al., 1999). Methanol was purchased from FLUKA through Sigma-Aldrich
Canada, Winston, ON, Canada.
Non-immunopurified and immunopurified Hirt-extracted Ees were
s visualized by immunohistochemical staining (Kuschak et al, 1999). For this
procedure, a sheep anti-core histone antibody diluted 1:200 and incubated for
30 minutes at room temperature was used. This was followed by incubation with
a secondary antibody, a FITC-conjugated donkey anti-sheep IgG antibody
(Sigma-Aldrich Canada, Winston, ON, Canada) diluted 1:400 in Iamb serum
to (Gibco/BRL, Life Technologies, Inc., Burlington, ON, Canada) and incubated
for
30 minutes at room temperature. Immunofluorescent analysis of histone-bound
EEs was performed as previously described (Kuschak .et al, 1999), the only
modification being the omission of the permeabilization step. Anti-bleach
(Mai,
1994) was added to preserve the fluorescence of the sample and to function as
is a mount for the cover slip. The immunostained EEs were analyzed using a
Zeiss
Axioplan2 microscope (Carl Zeiss Canada, Inc., Ottawa ON, Canada) under a
63x oil immersion objective and a UV filter. The images were acquired using
Northern Eclipse 5.0 software (Empix Imaging Inc., Mississauga, ON, Canada)
and a Sony (model XC75) CCD camera. Adaptive thresholding tools (Northern
ao Eclipse version 5.0 from Empix Imaging Inc., Mississauga, ON, Canada) were
used to remove all dots from the DAPI-stained images that were c 5 x 5 pixels
in
size when visualized using a 63x oil immersion objective lens and a 0.63x
adapter.
2s FISH-EEs (Kuschak et al, 1999) was performed on Hirt-extracted EEs
and on immunopurified EEs using a DHFR probe (Mai, 1994). The EEs were
analyzed using a Zeiss Axiophot microscope (Carl Zeiss Canada, Inc., Ottawa
ON, Canada) under a 100x oil immersion objective, a 1x magnification adapter,
and a UV filter. The images were acquired using IPLab software (Scanalytics,
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Fairfax VA, USA) Photometrics (CH250/a) CCD camera equipped with a KAF-
1400-50 sensor chip (1317 x 1035 pixels, Kodak).
RESULTS
s
The isolation of the histone-bound population of extrachromosomal DNA
extracted from Pre-B+ cells (Hirt, 1867) was performed by immunoprecipitation.
Pre-B+ cells contain a 4-HT-responsive MycERTM construct and have been
activated with 4-HT to overexpress c-Myc (Kuschak et al, 1999). This induces
to the activation of EEs from c-Myc activated Pre-B+ cells (Kuschak et al,
1999,
Mai et al., 1999). These EEs were isolated, first by Hirt extraction (Hirt,
1967)
and then followed by immunoprecipitation of histone-bound EEs.
Binding of Anti-Core Histone Antibody to Protein G Sepharose Beads
is
Sheep anti-core histone antibody was bound to Protein G sepharose beads
according to manufacturer's instructions. Briefly, the anti-histone antibody
was
eluted from the Protein G sepharose beads by incubating in 100 mM glycine
buffer, pH 2.3. The eluate was immediately neutralized to pH 7.2 using 1 M
2o Trizma Buffer, pH 8Ø
Immunostaining shows Enrichment of Histone-Bound EEs following HIP-
EEs
2s In the antibody-purified population, there was observed an enrichment in
histone-bound EEs. In contrast, the sheep anti-core histone antibody was
amplified by the FITC-conjugated donkey anti-sheep secondary antibody, giving
a greater relative signal intensity in comparison to the weaker DAPI signal
intensity. The data show that there are fewer DAPI signals in the
immunopurified
3o sample, indicating fewer EEs in the immunopurified sample, as compared to
the
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non-immunopurified EEs. However, of the EEs isolated by immunopurification,
the majority is co-localized with bright yellow or white signals indicative of
histone protein. This demonstrates an enrichment of histone-bound EEs.
s FISH-EEs shows Enrichment of DHFR Sequences on EEs following HIP-
EEs
To assess the value of this EE immunopurification method, it was
assayed for the enrichment of an extrachromosomally amplified gene. It was
to demonstrated previously that c-Myc deregulation results in amplification..
of
DHFR in mouse, rat, hamster, and human cell lines (Mai et al., 1996). There
was
shown by the FISH-EEs method, that DHFR was present on extrachromosomal
DNA from Pre-B+ cells (Kuschak et al, 1999). It was hypothesized that the
enrichment of the Hirt-extracted EEs by the immunopurification method can
Is increase the relative ratio of DHFR-containing EEs when comparing non-
immunoprecipitated against immunoprecipitated EE sample populations.
The overlay shows an increased proportion of DHFR-containing EEs in
comparison to the non-purified EE sample. There is shown an increase in the
zo number of co-localized DAPI-FITC signals in comparison with the proportion
of
DAPI-FITC co-localized signals in the non-purified sample shown previously. In
addition, there was also observed a number of DAPI signals that did not show
co-localization with a FITC signal. These are likely histone containing EEs
that
do not contain DHFR sequences, but can contain sequences from other gene
2s families (Coller et al., 2000, Kuschak et al, 1999, Mai et al., 1996).
Overall, these data indicate enrichment in the number of EEs that carry
DHFR sequences, and presumably sequences of other c-Myc target genes,
such as ribonucleotide reductase R7 and R2 genes (Kuschak et al, 1999), cyclin
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D2 (Mai et al., 1999), and potentially other, as yet unidentified genes
(Coller et
al., 2000).
DISCUSSION
The immunopurification of the active population of extrachromosomal
DNA molecules is advantageous in studying functional EEs since it enriches for
a potentially active population of EEs, removing the non-histone-bound and
presumably inconsequential population of EEs from the extrachromosomal DNA
io population. The value of this method as a primary means of isolating
potentially
functional EEs from a large population of extrachromosomal DNA amplicons
was assessed by two methods.
It was first determined the percentages of contanimating apoptotic or
is genomic DNA in each EE preparation. Ees were prepared from non-
immunoprurified Pre-B- and Pre-B+ cells. The results indicate the Pre-B- and
Pre-B+ had 10.8 ~ 2.5 % and 12.1 ~ 4.9% genomic DNA contaminant,
respectively. Immunoprecipitation of histone-bound EEs helps eliminate these
contaminants.
Non-immunopurified and immunopurified EEs were examined by
immunostaining for histone protein and comparing the ratio of DAPI-stained
DNA molecules that co-hybridized with the signal from an anti-histone
antibody.
An enrichment of histone-bound Ees was seen in the immunopurified samples.
Zs Nearly all of the EEs in the sample of immunopurified EEs showed co-
localization with FITC, indicating that the majority of the EEs in the
purified
samples contained histone proteins. The results of the immunostaining assay
shows that this method is successful in isolating histone-bound EEs from a
large
pool of EEs that do not contain histone protein.
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Then the number of EEs that hybridize with a gene of interest, in this
case DHFR, was assessed. The experiments show that immunopurified EEs
carry a larger relative number of DHFR sequences, indicating an overall
enrichment of specific extrachromosomal amplicons. There was shown that
s although there is an increase in the proportion of EEs that hybridize with a
DHFR probe, there are also a number of EEs where no FITC signal is seen to
co-localize with DAPI .stained EEs. This is expected since previous work has
shown that a number of genes can be found on EEs from Myc-ERo-activated
mouse Pre-B+ cells. These include ribonucleotide reductase R7 and R2
io (Kuschak et al, 1999), cyclin D2 (Mai et al., 1999), as well as DHFR (Mai,
1994)
sequences. It is likely that there are others as well (Coller et al., 2000).
In conclusion, it has been shown that the present method for
immunopurification of EEs is useful as a means of studying extrachromosomal
is gene amplification phenomena as well as amplification-mediated expression
of
oncogenes, drug-resistance genes, and potentially others. The
immunopurification of EEs is a novel tool that is ideally suited as a first
step
purification of EEs for a variety of studies in cultured cells, primary cells,
and
tumor samples. These analyses and procedures include generating libraries of
2o EEs from cells, analyses of EEs by electron microscopy, fluorescent in situ
hybridization (FISH-EEs) (Khaira et al., 1988, Kuschak et al, 1999, Mai, 1994,
Mai et al., 1999), mRNA FISH (Start et al., 1984, Von Hoff, 1991), cloning,
and
Southern blotting.
2s Example 8
The activation of the c-myc gene is key to the development of all murine
plasmacytomas (PCTs), resulting in deregulated levels of endogenous c-Myc
protein expression (Cory, 1986; Ohno et al., 1979; Potter et al., 1992). In~
the
3o majority of pristane-induced mouse PCTs, the deregulation of c-myc
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transcription is achieved by chromosomal translocation that juxtaposes the c-
myclpvt-7 locus on chromosome 15 to one of the immunoglobulin (1g) loci: on
chromosome 12 (IgH), 6(IgK) or 16(IgL) (Ohno et al., 1979; Potter et al.,
1992).
More than 90% ofPCTs carry the typical T(Janz et al. 1997;Wang et al., 1971)
s translocation, whereas the variant T(corcoran et al., 1984;Wang et al., 1971
) or
T(Wang et al., 1971: Committee, 1969) translocation is present in fewer than
10% ofPCTs (Potter et al., 1992).
In a few PCTs, classical G-banding analysis could not identify any of the
io plasmacytoma-associated typical or variant translocations (Potter et al.,
1992).
Molecular and cytogenetic analysis oftwo translocation-negative PCTs, ABPC22
and RFPC 2782 (Shaughnessy et al., 1993), revealed that the overexpression of
the c-myc gene was achieved by promoter/enhancer insertion brought about by
retroviral insertion into the 5' flanking region of c-myc. Such retroviral
is deregulation of the c-myc gene is not unique. It was shown to operate in
avian
bursal lymphomas and also in MuLV induced lymphomas of T-cell origin
(Hayward et al. 1981; Corcoran et al., 1984; Graham et al., 1985). Unusual
gene
rearrangements have been described in two other translocation-negative PCTs
that lack retroviral insertion, namely in ABPC45 and DCPC21 (Fahrlander et
al.,
20 1984; Ohno et al., 1989; Ohno et al., 1991). In both cases, it has been
reported
that the c-myc-IgHjuxtapositon was achieved via complex rearrangements that
resulted in a new gene order on the myc-activated chromosome (Fahrlander et
al., 1984; Ohno et al., 1989; Ohno et al., 1991), different from that found at
the
chromosomal breakpoint of typical T (Janz et al. 1997;1 5) PCTs (Muller et
al.,
2s 1995, Janz et al. 1997). For both of these translocation-negative PCTs,
molecular analysis revealed that the 5' flanking region of c-myc is juxtaposed
either to 3' Sa or to 3' Smjt, respectively, in 5' to 3' ("head to tail")
orientation.
This is in striking contrast to the "head to head" (5' to 5') configuration of
the Ch
locus and the c-myc gene that is present in every PCT of the typical T(Janz et
3o al., 1997; Wang et al., 1971) type.
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Although fewer than 1 % of the PCTs analyzed to date belong to the
group of translocation-negative plasmacytomas, they are of interest because
they reveal a new mechanism of plasmacytomagenesis that is unrelated to viral
s LTR insertion, or to the interchromosomal recombination that has been
implicated in virtually all PCT-associated deregulation of c-myc
transcription.
Consequently, the lack of cytogenetically identifiable translocations suggests
alternate pathways by which c-Myc overexpression is achieved in this group of
tumors.
io
To examine the mechanisms) of c-Myc deregulation in translocation~
negative PCTs, the investigation was focused on DCPC21, a plasmacytoma that
had been induced by intraperitoneal implantation of a plastic diffusion
chamber
into a BALBIc female mouse (Ohno et al., 1989). Previous work by these
is authors had suggested that DCPC21 exhibited complex molecular
rearrangements leading to the IgH-myc gene juxtaposition by the insertion of
the
myc and pvt-7 loci-containing chromosome 1 5 segment into the IgH focus on
Chr 12(Ohno et al., 1991). The realization of such a complex rearrangements
requires the occurrence of a paracentric inversion, a deletion/insertion, and
2o multiple translocations both on chromosome and gene levels during the
process
of the IgH-myc illegitimate recombination (Ohno et al., 1991).
Here it is shown that the results of classical and molecular cytogenetic
analyses show that the DCPC21 plasmacytoma lacks any type of
as interchromosomal recombination that could cause the constitutive activation
of
the c-myc gene. However, chromosomal segments containing c-myc and IgH
sequences are present, either alone or jointly, on extrachromosomal elements
(EEs) in the DCPC21 plasmacytoma. It is demonstrated that the deregulated
expression of c-myc occurs on EEs, and this appears to be sufficient to
sustain
3o the malignant phenotype of the DCPC21 tumor.
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MATERIAL AND METHODS
Tumor cells. DCPC21 was induced in a female BALB/c mouse by i.p.
s implantation of a Millipore diffusion chamber (Merwin et a., 1963).
Trypsin-Giemsa Banding. Metaphase spreads were prepared without
colcemid treatment. Trypsin-Giemsa banding was performed as described
previously (Wang et al., 1971) and adapted to mouse chromosomes.
to Chromosome identification followed the recommendations of the Committee on
Standardized Genetic Nomenclature for Mice (Committee, 1969).
Molecular cytogenetics. Chromosomes were analyzed by FISH
(fluorescent in situ hybridization ) as previously published (Mai, 1996,
Fukasawa
is et al., 1997). Analysis of slides was performed using a Zeiss Axiophot
microscope, a PowerMacintosh 8100 computer, and a CCD camera
(Photometrics); the analytical software used was IPLabSpectrum Version 3.1
(Signal Analytics, USA).
2o FISH probes and detection of hybridization. The following probes were
used, c-myc (Mai, 1994), IgH (pJll; Greenberg et al., 1982) andpvt-7 (Huppi et
al,
1990). The probes were labeled by random priming with either digoxigenin- or
biotin-dUTP (Roche Diagnostics, Laval, Quebec, Canada). The detection of
hybridization signals with digoxigenin-labeled probes was carried out using a
2s fluorescein conjugated polyclonal sheep anti-digoxigenin- antibody (Roche
Diagnostics). For the detection of hybridization signals obtained with
biotinylated
probes, a monoclonal anti-biotin antibody (Roche Diagnostics) was used,
followed by a Texas Red-conjugated goat anti-mouse-IgG secondary antibody
(Southern Biotechnology Assoc., Inc., Birmingham, USA).
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FISH-EEs (FISH on purified extrachromsomal DNA molecules). The total
population of extrachromosomal elements (EEs) was purified and examined by
FISH as described in (Kuschak et al., 1999). EEs were hybridized with c-myc,
IgH and pvt 7 . The specificity of these hybridizations was confirmed by the
s absence of hybridization signals with a negative control, cyclin C (Mai et
al.,
1996; Kuschak et al., 1999) and hybridization signals obtained with a positive
control, cot i DNA.
Chromosome painting. The chromosome paints used (CedarLane,
Io Laboratories Limited, Hornby, Ontario, Canada) were a FITC-conjugated mouse
chromosome 15 and a biotinylated mouse chromosome 12-specific paint.
Hybridization of chromosome paints, alone or in combination with FISH probes,
was carried out as described in the general FISH protocol. Chromosome 12
hybridization signals were detected with a monoclonal anti-biotin antibody
is (Roche Diagnostics) at 0.5 ng/slide followed by a Texas Red conjugated goat
anti-mouse-IgG secondary antibody (Southern Biotechnology Assoc., Inc.,
Birmingham, USA) at 2.5 ng/slide. The hybridization signals of the FITC-
labeled
chromosome 15 paint were amplified using a rabbit anti-FITC antibody
(CedarLane), followed by a FITC-labeled goat anti-rabbit IgG secondary
2o antibody (Sigma). Both antibodies were used at 1 :40 dilution.
SKY. Spectral karyotyping was performed using the ASI (Applied Spectral
Imaging, CA, USA; Migdal Ha'Emek, Israel) kit for mouse spectral karyotying
and the suppliers' hybridization protocols. Analyses were carried out using
the
2s Spectra CubeTM on a Zeiss Axiophot 2 microscope and the SkyView 1.2
software on a PC (P11-3 50).
mRNA track studies. mRNA tracks studies were carried out as
described in (Szeles et al., 1999) on freshly isolated ascitic DCPC21 tumor
cells.
3o The cells were cytospun onto microscopic slides (10~ cells/slide) and fixed
in
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formaldehyde (1% in 1xPBS/SOmM MgC12). The slides were washed in 2xSSC,
dehydrated sequentially in 70%, 90% and 100% ethanol. A denatured mouse c-
myc probe, pMycEx2, a 460 by Pstl-fragment of myc exon 2 (gift from Dr. K.
Huppi, NIH), was added in 50% formamide/2xSSClSOmM phosphate buffer,
s 10% dextran sulfate for overnight hybridization at 37°C in a
humidified incubator.
As expected, subsequent RNAse treatment removed any hybridization signals,
and hybridization to chromosomes or extrachromosomal material was only
achieved after the slides had been treated with RNAse and pepsin and
denatured prior to the addition of FISH probes (see also Lawrence et al.,
1989).
io
Fluorescent immunohistochemistry. Immunohistochemistry was
performed as described (Fukasawa et al., 1997). The following antibodies were
used, a monclonal anti-c-myc antibody, 3C7 (Evan et al, 1985) at 20 ng/slide.
Visualization of this antibody was achieved with a Texas Red-conjugated
is secondary goat anti-mouse IgG antibody (Southern Biotechnology Assoc.,
Inc.,
Birmingham, USA) at 2.5 ng/slide. A sheep anti- CORE histone antibody (US
Biological) was used at 5 ng/ slide and visualized with a FITC-conjugated
donkey anti-sheep IgG antibody (Sigma) at 2.75 ng/slide, and an anti-histone
H3P antibody that were received. The anti-histone H3P used is a histone H3-
2o phophoserine monoclonal antibody from Dr. Z. Darzynkiewicz (Juan et al,
1998).
It was used at 4.0 ng/slide and visualized with a Texas Red-conjugated goat
anti-mouse IgG antibody (Southern Biotechnology Assoc., Inc., Birmingham,
USA) at 2.5 ng/slide.
2s Southern analysis. For Southern analyses, 10 ~tg DNA from primary
BALBlcRb6.15 spleen or DCPC21 tumor DNA was digested was digested
overnight with 40 units of either Hindlll or Sad restriction endonucleases
(Roche
Diagnostics) and electrophoretically separated on a 0.8% agarose gel, blotted
onto Hybond XL membrane (Amersham Pharmacia Biotech), and baked at 80°C
3o for 2 hours. Hybridizations and washes were carried out according to
standard
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procedures (Sambrook et al., 1989). The probes used were, c-myc (Mai, 1994),
pJi i (Greenberg et al., 1982) pvt i (Huppi et al., 1990; Mai et al., 1995),
JQ2
(Ohno et al., 1991).
s Electroporations. Spleen cells of BALB/cRb6. 15 mice were harvested
for extrachromosomal gene transfer studies as follows. Green fluorescent
protein (GFP, pEGFP-N1 Clontech, Mississauga, Ontario, Canada) was used as
a tracer molecule for determination of gene transfer efficiencies. Lymphocytes
isolated from one spleen were divided into three groups: electroporation of
GFP
Io plus c-mycllgH-carrying EEs (2.5 ~tg), electroporation of GFP (2.5 jig),
and
"mock" electroporation. Electroporations were carried out in OPTI-MEM solution
(Canadian Life Technologies, Burlington, Ontario, Canada) using 1 ml Gene
PuIserR cuvettes, 0.4 , cm (Bio-Rad, Hercules, CA, USA) using a Bio-Rad
electroporator, model #1652076, and a Bio-Rad Capacitance Extender, model
is #7652087. The settings used were: 960K, 240V, cap. 25 units. Subsequent to
electroporation, the cells were washed in complete medium (RPM11640 with
10% fetal calf serum (Canadian Life Technolgies, Burlington, Ontario, Canada),
2 mM Lglutamine, 5 IU/ml ofpenicillin and 5 ~g/ml streptomycin and 50 ~tM/ml
~3-mercaptoethanol and allowed to grow in complete medium in a humidified
2o incubator at 37°C and in the presence of 5%C02. 24 hours after
transfer, cells
were cytospun onto microscope slides (10~ cellslslide), and c-Myc protein
expression was determined in splenic B cells that also expressed GFP. A FITC-
conjugated anti-B220 antibody ( Pharmingen, Mississauga, Ontario, Canada)
was used to visualize splenic B cells on cytospin preparations. Fluorescent
2s immunohistochemistry of the electroporated cells was carried out as
previously
described (Fukasawa et al., 1997).
RESULTS
3o DCPC21 is a translocation-negative plasmacytoma harboring
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extrachromosomal elements
Karyotyping of DCPC21 metaphase spreads by standard G-banding
revealed that chromosomes 15, 12, 6, and 16, regularly involved in mouse PCT-
s specific translocations, were not part of reciprocal translocation events.
To
confirm the results provided by G-banding, DCPC21 metaphases were further
examined by chromosome painting, fluorescent in situ hybridization (FISH), and
spectral karyotyping (SKY). Since the most frequent translocation (>90%) in
pristane-induced mouse PCT transposes the c-myc containing segment of
io chromosome 15 into the neighborhood of the IgH gene loci on chromosome 12
(Potter et al., 1992), chromosome painting was performed to ascertain whether
chromosomes 12 and 15 are carriers of cryptic rearrangements. The painting
with chromosome 15- and 12-specific probes revealed the presence of four
copies of chromosome 15 (green) and chromosome 12 (red) in the majority of
is the DCPC21 plates analyzed. More importantly, neither chromosome 15- nor
chromosome 12-derived genetic material was found to be translocated or
inserted into any other chromosome of DCPC21 metaphases.
When either chromosome 12 paint was combined with FISH using a c-
2o myc probe or chromosome 15 paint used in combination with an IgH probe (pJi
i), it was also evident that chromosomes 12 and 15 were not involved in
reciprocal translocations. However, extrachromosomaf elements (EEs) carrying
either cmyc or IgH genes alone or c-myc and IgH genes jointly became
apparent.
The possible involvement of the IgK and IgL-carrying chromosomes 6
and 16 in Iglmyc translocation was analyzed by SKY. SKY corroborated the data
obtained by standard cytogenetics, painting and FISH, namely, that DCPC21
does not carry any plasmacytoma-associated c-myc-activating translocation.
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In addition, SKY revealed the nature and structure of the chromosomal
aberrations detected by G-banding. Noteworthy, SKY showed that the
duplicated D2 band on one of the chromosomes 15 contained only chromosome
75- derived genetic material, excluding the likelihood of an interchromosomal
s rearrangement involving chromosome 15 . The additional band on chromosome
9 was identified as derived from chromosome 16, while one copy of
chromosome was centromerically fused with one chromosome 19. A "hidden"
chromosomal aberration, undetected by classical G-banding, was the insertion
of chromosome 3-derived material into one chromosome 2. Since the
io aberrations involving chromosomes 9 and 2, as well as the fusion of
chromosomes 16 and 19, were not consistently seen in all metaphases, they are
likely chromosomal aberrations acquired during tumor progression, rather than
during tumor initiation.
is Classical cytogenetics, chromosome painting, FISH and SKY establish
that the DCPC21 plasmacytoma lacks any chromosomal aberration that could
reasonably be involved in the constitutive activation of the c-myc gene.
However,
the presence of IgH and c-myc sequences on extrachromosomal elements
(EEs) suggests that these genetic entities can be responsible for the
2o deregulation of c-Myc in this tumor.
Southern blot analysis shows rearrangements within the IgH locus and in
the 5' flanking region of c-myc.
2s Southern blot analysis was performed with normal mouse spleen DNA
and DCPC21 tumor DNA. The c-myc gene, visualized by using a mouse exon 2
-specific probe, showed no rearrangements) and exhibited identical
hybridization patterns in Hindlll- and Sacl-digests of normal spleen and
DCPC21
DNA. Similarly, pvt-7 showed no evidence of rearrangements. The stronger
3o hybridization signals of cmyc andpvt-i in DCPC21 DNA reflect both the
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duplication of the myclpvt-i-containing 1 5D2 band of one of the chromosome 15
and the additional copies of chromosome 15. In contrast to the germ line bands
observed with c-myc and pvt-f, rearrangements within the IgH sequences and in
the 5' flanking region of c-myc became apparent when using the IgHprobe (pJii)
s as well as a 5' flanking probe of the c-myc gene (JQ2).
Since none of the bands that hybridized with pJii co-hybridized with JQ2,
it can be excluded that any of the additional bands represent a cryptic
transposition of sequences detected by pJi 7 and JQ2. Furthermore, a
to transposition ofpvt 7 and c-myc within the chromosomal DNA ofDCPC21 is
unlikely, since these two genes were not involved in translocation and or
rearrangement events detectable in genomic DNA. These results suggest that
the rearranged genomic bands represent intrachromosomal rearrangements,
possibly due to the excison of c-myc and IgH sequences from the relevant
is chromosomes rather than interchromosomal recombination.
c-myc and !gH co-localize on extrachromosomal elements (EEs) and are
functional genetic units
2o Extrachromosomal c-myc and IgH hybridization signals in DCPC21
metaphases were observed. To analyze these EEs further, FISH was performed
on the total population of EEs. In the majority of the cases, c-myc and
IgHwere
found together on the large EEs (0. 1 -0.2 ~tm in diameter, as determined by
electron microsopy (EM) measurements). Noteably, c-myc and IgH were also
2s found alone on EEs of smaller sizes (0.01 ~tm in diameter). pvt-I could be
detected on some of the EEs, together with c-myc and IgH.
The co-localization of c-mycllgH on some of the EEs raised the question
whether these EEs are biologically active structures. To investigate this
3o hypothesis, it was analyzed whether these EEs were associated with active
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chromatin, could transcribe c-myc mRNA and confer c-Myc overexpression to
resting primary B cells in extrachromosomal gene transfer studies.
To determine whether the EEs contained active genes, first examined
s was: i) the presence of histones and of the transcription-associated
phosphorylated form of histone H3 (H3P) (Juan et al., 1998; Juan et al., 1999)
on the EEs and ii) carried out mRNA track studies. Using a pan-histone
antibody
that detects all histones irrespective of chromatin activation, histones were
found
on the large, but not on the small EEs. To determine whether the former were
io also transcriptionally active, the presence of H3P was examined using a
monoclonal anti-histone H3P antibody (Materials and Methods). In over 90% of
the pan-histone-containing EEs it was found that they also stained with the
monoclonal anti-histone H3P antibody, indicating that these EEs contained
active chromatin.
~s
To examine whether c-myc mRNA was produced from these EEs, mRNA
track studies were carried out. Multiple short c-myc RNA tracks were observed,
typical of episomal (extrachromosomal) gene transcription (Szeles et al.,
1999),
were generated from DCPC21,-EEs. To unequivocally demonstrate that the
2o mRNA was derived from the EEs, the identical slides were processed for FISH
after RNAse and pepsin treatment and following slide denaturation. Co-
localizing
c-myc mRNA (red signals) and c-myc-EEs DNA signals (green). All c-myc
mRNA tracks were consistently observed to be colocalized with EEs that
showed c-myc DNA by FISH. However, the number of c-myc-carrying EEs in a
~s DCPC21 cell was higher than the amount of EEs that were transcribing c-myc
mRNA.
To further examine the functional activity of DCPC2 1 EEs, purified EEs
were electroporated into normal BALB/cRb6. 15 spleen cells together with a
3o vector expressing green fluorescent protein (GFP). The latter served as
tracer
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molecule for gene transfer efficiency. The B lineage-specific marker B220 was
used to determine the lineage origin of the electroporated cells. When
purified
DCPC21 EEs were introduced into normal BALB/cRb6. 15 spleen cells they
conferred c-myc expression to GFP-expressing B220-positive B cells. However,
s within 24 hours, the DCPC-21 EEs induced cell death in the majority of the
GFP-
expressing B cells (>90%), while cells electroporated with GFP only surivived.
Cell death was associated with c-Myc overexpression and visible by . the
appearance of apoptotic bodies.
to DISCUSSION
c-mycllgH-carrying EEs represent an alternative mechanism of c-Myc
overexpression in DCPC21 plasmacytoma.
is In the present study, there was demonstrated, by classical cytogenetics,
chromosome painting, FISH and SKY, that the DCPC21 plasmacytoma lacks
any of the chromosomal translocations involved in the deregulation of the c-
myc
gene in this malignancy. The presence of extrachromosomal c-myc and IgH-
carrying EEs in this tumor raised the question whether these EEs could replace
ao the .function of chromosomal translocation in PCT-genesis. A series of
experimental approaches have confirmed that the c- mycllgH-carrying EEs are
functional genetic units capable of c-myc transcription.
The evidence that the deregulated expression of c-Myc in the DCPC21
2s plasmacytoma can be attributed to DCPC21-EEs is further supported by the
presence of H3P histone, by c-myc mRNA tracks derived from the EEs, and by
extrachromosomal gene transfer studies. The phosphorylated form of histone
H3 (H3P) is associated with active chromatin found during transcription and
replication (Juan et al., 1998, Juan et al., 1999, Wei et al., 1999). The mRNA
3o track studies followed by FISH show that the c-myc gene is transcribed in
the
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EEs and this confirms that the generation of Myc RNA initiates outside the
chromosomal location of the c-myc gene. The later was further confirmed by
extrachromosomal gene transfer experiments. The introduction of DCPC21-EEs
into primary mouse B cells was associated with c-Myc overexpression in the
s electroporated cells and was followed by cell death.
Based on the above findings, it was concluded that the deregulation of c-
Myc in plasmacytomas can occur by a mechanism alternative to chromosomal
translocation or viral insertion. This novel pathway of c-Myc deregulation
to involves the formation of extrachromosomal elements that allow c-Myc
deregulation similar in extent to that of juxtaposed Iglmyc sequences
generated
by chromosomal translocation in conventional plasmacytomas.
Models for the generation of DCPC21-EEs carrier of myclpvt-1 and IgH
is sequences.
Extrachromosomal DNA is found in all organisms analyzed to date (for
review see, Gaubatz, 1990). Normal cells seem to carry repetitive sequences on
their extrachromosomal DNA and their role in the cells as well as their
2o mechanisms of generation have been widely discussed, but are essentially
unknown. EEs can be generated transiently during normal developmental
processes (Iwasato et al., 1990; Matsuoka et al., 1990). The size of
extrachromosomal elements (EEs) varies (Brothman et al, 1987; Gaubatz, 1990;
Gaubatz et al., 1990) as does their number which increases following genotoxic
2s treatments (Cohen et al., 1996; Cohen et al., 1997; Regev et al., 1998).
Tumor
cells often harbour EEs (Wahl, 1989; Cox et al., 1965). Some of the genes
found on EEs have been studied; they include oncogenes and drug resistance
genes (Fegan et al., 1995; Wullick et al., 1993; Chen et al., 1989;
Delinassios et
al., 1983; Rowland et al., 1985; Stahl et al., 1992; Wettergren et al., 1995).
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Previous work has indicated that a T(Janz et al. ,1997; Wnag et al.,
1971) translocation-carrying plasmacytoma cell line, MOPC26S, also contains c-
myc and pvt-i genes in duplicated units on . chromosome 15 and on
extrachromosomal elements (Mai et al., 1995). However, DCPC21 -EEs
s represent the first reported case of functional c-myc-transcribing genetic
units in
a plasmacytoma that allow c-Myc expression to initiate outside the
chromosomes, i. e., the usual chromosomal translocation units. The presence of
c-mycllgH-containing EEs raises the question about the mechanisms) that
account for their formation in the DCPC21 plasmacytoma.
lo
Model 1 assumes a transposition/insertion of myclpvt-i into the IgH locus
on chromosome 12, followed by the release of the juxtaposed sequences from
chromosome 12 and their survival as EEs. This model requires one single
illegitimate recombination event between c-myc and IgH leading to their
is juxtaposition on chromosome 12 only, followed by their release from
chromosome 12. In the present study, none of the approaches applied detected
an insertion of chromosome 15-derived sequences into chromosome 12.
Model 2 proposes reciprocal chromosomal translocation between IgH
2o and c-myc genes. The juxtaposed unit is released from the reciprocally
translocated chromosomes T(Janz et al. 1997; Wang et al., 1971) and T(Wang
et al., 1971; Janz et al. 1997) in the from of independently replicating EEs.
This
model, although probable, requires additional molecular events to be
consistent
with the experimental data obtained in this work. For example, one predicts to
zs find that all EEs contained both c-myc and IgH. Since EEs of various sizes
are
found which carry both c-myc and IgH or either one alone, replication,
recombination and/or breakage of extrachromosomal DNA would have to occur.
Model 3 assumes the independent generation of myclpvt i and IgH
3o carrying EEs that recombine to generate mycllg- carrying EEs. This model
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postulates the sequence of two events, namely the recombination between c-
myc- and IgH-containing EEs that were generated concomitantly. Such
illegitimate extrachromosomal recombination events are not unlikely, since Ig-
sequences-containing EEs are generated as circular extrachromosomal
s elements during switch recombination in normal B cell development (Iwasato
et
al., 1990). Following such recombination(s), selective processes allow
survival of
those extrachromosomal units that confer a growth/survival advantage to the
DCPC21 tumor cells. Those EEs that allow deregulated c-Myc expression
presumably have favored DCPC21 tumor growth. This model is most consistent
Io with the data obtained. In the absence of any detectable chromosomal
translocation involving c-myc and Ig loci, EEs of various sizes were found by
hybridizing with c-myc and/or lgH.
Currently, the significance of EEs for tumorigenesis is poorly understood,
is although they are present in a variety of human tumors and presumably
involved
either in the initiation of the tumorigenic process or during tumor
progression
(Trent et al., 1986; Martinsson et al., 1988; Von Hoff et al, 1988). The
elimination of amplified c-myc or N-myc located on double minutes from human
and mouse tumors contributes to the reduction of tumorigenicity in vitro and
in
2o vivo. These studies provided the first indication that EEs can be involved
either
in initiation or progression of malignancy. Recently, it has been suggested
that
the amplification of the rearranged c-myc gene and its integration into novel
chromosomal sites can involve the formation of extrachromosomal elements
(Coleman et al., 1999).
2~
A large number of human neoplasia were found to belong (Von Hoff et
al., 1992; Eckhardt et al., 1994; Shimizu et al., 1994) to the translocation
negative group. Recent analyses of a series of chronic myelogenous leukemia
(CML) revealed an incongruity between the overexpression of the oncogenic
3o fusion product involved in malignant transformation of the precursor cell,
and the
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absence of cytogenetical(y detectable T(Ohno et al., 1989; Kuschak et a(.,
1999)(q34;q1 1) carrier Philadelphia (Ph) chromosome (van der Plas efial.,
1989;
Kurzrock et al., 1990; Costello et al., 1995; Selleri et al., 1990; Janssen et
al.,
1992; Estop et al., 1997). In a recent study of adult and childhood acute
s (ymphoblastic leukemia (ALL), the authors investigated the relationship
between
the T(Muller et al, 1995 ;Merwin et al., 1963) translocation and - the
overexpression of the MLL-AF4 gene implicated in the leukemogenesis of infant
ALL (Uckun et al., 1998). Nested polymerase chain reaction (NT-PCR) revealed
that in 7 out of 18 patients, the generation of the chimeric oncogenic MLL-AF4
to protein occurs without cytogenetically detectable T(Muller et al., 1995
;Merwin et
al., 1963) translocation. As in mouse PCTs and rat immunocytomas, Burkitt
lymphomas (BLs) are also carrier of Ig/myc-juxtaposed sequences resulting from
chromosomal translocation between myc and lg gene carrier chromosomes (for
review see, Klein, 1989; Klein, 1993; Klein, 1995). Notably, in one of the
is translocation-negative BLs that were analyzed, c-Myc overexpression was
found
similar to that found in translocation-carrying BLs. c-myc and IgH sequences
were found on EEs, and the c-myc gene was germ line.
In conclusion, the results provide evidence that the EEs represent
2o functional genetic units that play essential roles both in the initiation
andlor
promotion of the malignant phenotype of the translocation-negative DCPC21
plasmacytoma. The findings also show that different experimental and human
neoplasms with fusion transcripts or oncogenic activation, although
cytogenetically classified as "translocation-negative", can indeed carry
specific
2s translocation(s), however in an extrachromosomal form.
Obviously, many modifications and variations of the present invention are
possible in light of the above teachings and following Examples. It is,
therefore,
to be understood that within the scope of the described invention, the
invention
3o can be practiced otherwise than as specifically described.
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Throughout this application, various publications, including United States
patents, are referenced by author and year and patents by number. Full
citations for the publications are listed below. The disclosures of these
s publications and patents in their entireties are hereby incorporated by
reference
into this application in order to more fully describe the state of the art to
which
this invention pertains.
The invention has been described in an illustrative manner, and it is to be
Io understood that the terminology which has been used is intended to be in
the
nature of words of description rather than of limitation.
Obviously, many modifications and variations of the present invention are
possible in light of the above teachings. It is, therefore, ~to be understood
that
is within the scope of the appended claims, the invention can be practiced
otherwise than as specifically described.
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Negative Myc+ Myc++ Myc+++ Total
Negative 5 4 0 1 10
CIN1 I 1 7 0 2 11
CIN2 0 4 1 0 4
CIN3 1 4 2 3 10
Carcinoma0 0 0 1 1
Total 7 19 3 7 36
Table 1. Myc protein levels as determined by quantitative fluorescent
immunoh istochemistry.
DHFR- DHFR+/- DHFR+ DHFR++ DHFR+++ Total
Negative 2 3 0 2*** 0 7
CIN1 3 2 2 0 0 8
CIN2 0 1 1 3 0 4
CIN3 0 0 0 4 5 9
Carcinoma0 0 0 0 1 1
Total 5 6 3 9. 6 29
*Pap was CIN3
**Strong maplification in limited areas
io
Table 2. DHFR gene copy numbers in cervical ncoplasia as determined by FISH.
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