Note: Descriptions are shown in the official language in which they were submitted.
Wo sl/oo364 , ~ PCr/US9O/02887
TCL-5 GENE REARRANGEMENT INVOLVED IN
T-CELL LEUKEMIA AND MELANOMA
The U.S. Government has a paid-up license in this invention
and the right in limited circumstances to require the patent owner to
license others on reasonable terms as provided for by the terms of
grant nos. CA 09485, CA 39860, CA 25875 from the National Insti-
tutes of Health.
TECHNICAL FlELD OF THE INVENTION
The invention relates to the area of cancer diagnostics and
monitoring. More particularly it relates to the diagnosis and monito~
ing of cancers associated with chromosomal translocations
t(1;14)(p32;qll). -
BACKGROIJND OF THE INVENTION
A large proportion of leukemias and lymphomas of the B- and
T-lymphocyte lineages carry characteristic chromosomal abnormali- -
ties (Yur~is, (1983), Science 221:227-235). The finding of cosistent
chromosomal translocations directly involving the MYC ar,d ABL
oncogenes in, respectively, Burkitrs lymphoma and chronic myeloid . --
leukemia (see Croce and Nowell, (1985), Blood 65:1-7; Xurzrock et al.,
(1988), N. Eng. J. Med. 319:990-998, for reviews) led to the hypothesis
that chromosomal aberrations that alter the expression of specific
genes are involved in the pathogenesis of human leukemias and -
lymphomas. In T-cell tumors, the majority of chromosomal abnor-
malities involve the T-cell receptor alpha/delta locus on chromosome
14 at band qll (Croce et al.. (1985). Science 22~:1044-1047; reviewed
in Rabbitts et al., (1988), Trends Genet. 4:300-304). The direct
involvement of this T-cell receptor locus in ~(8;14)(q24;qll).
t(l0;14)(q24;qll), t(ll:l4)(pl3;qll), t(ll:14)(plS;qll) and
t(l4;14)(qll;q32) transloca~ions observed in acute T-cell leukemias
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WO 91/00364 PCrtUS90/02887
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has been demonstrated by molecular analysis of the breakpoints
involved in these translocations (Finger et al., (1986), Science
234:982-985; Kagan et al., (198~), Proc. Natl. Acad. Sci., USA,
84:4543-4546; Kagan, et al., (1989), Proc. Natl. Acad. Sci., USA, in
press; Boehm et al., (1988) EMBO J. ~:385-394; Boehm et al., (1988),
EMBO J. ~:2011-2017; Mengel-Gaw et al., (1988), Proc. Natl. Acad.
Sci. USA 85:9171-91~5; Russo et al., (1988), Proc. Natl. Acad. Sci.,
USA 86:602-606). Human chromosome 1 band p32 aberrations have
been detected in acute T-cell leukemia (Mathieu-Mahul et al., (1986),
T.C.R. Acad. Sci. 302:525-528; Raimondi et al., (198~) Blood,
69:131-134, human cutaneous malignant melanomas (Balaban et al.,
(1986) Cancer Genet. Cytogenet. 19:113-122) and human
neuroblastomas (Gilbert et al., (1982) Cancer Genet. Cytogenet.,
~:33-42). However the oncogene or oncogenes involved have not been
identified nor the chromosomal breakpoints located. Thus the only
way to identify these aberrations is by cytogenetics, which is costly
and technically difficult.
Thus there is a need in the art for molecular methods of iden-
tifying certain chromosomal abnormalities involving human chrom~
some 1 band p32 so that diagnosis and therapy of the diseases associ-
ated with the abnormalities can be perf ormed routinely and
inexpensively.
SUMMARY OF THE INVENlION
It is an object of the invention to provide a method of diagnos-
ing or assessing a neoplasm in a human.
It is another object of the invention to provide nucleic acid
probes for diagnosing or assessing a neoplasm.
It is yet another object of the invention to provide nucleic acid
primers for amplifying translocation junctions resulting from
t(1;14)(p32;qll) translocations.
These and other objects of the invention are provided by one
or more of the embodiments described below. In one embodiment of
the invention a method of diagnosing or assessing a neoplasm in a
human is provided comprising isolating DNA of a human from cells
suspected of being neoplastic; determining whether a TCL-5 gene on
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WO 91/00364 ,~ r~ ~ j PCI'/US90/02887
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said DNA has been rearranged, a rearranged TCL-5 gene indicating a
neoplasm.
In another embodiment of the invention a method of diagnos-
ing or assessing a neoplasm in a human is provided comprising isolat-
ing from the human test cells suspected of being neoplastic; deter-
mining quantity of expression products of TCL-5 genes in said test
cells; comparing the quantity of TCL-5 gene expression products in
said test cells with the quantity in control cells, said control cells
isolated from healthy cells of the human or from a healthy human, an
altered quantity of TCL-5 gene expression products in said test cells
relative to said control cells indicating a neoplasm. ~ -
In yet another embodiment of the invention a nucleic acid
probe is provided which is derived from chromosome 1 sequences
located between a translocation junction of a lp chromosome of a
t(l;l4~(p32;qll) translocation and a restriction enzyme site, wherein
there are no other sites for said enzyme between said site and the
translocation junction.
In still another embodiment of the invention a pair of nucleic
acid primers are provided for amplifying translocation junctions of a
lp+ chromosome of a t(1;14)~p32;qll) translocation wherein one
primer of the pair is derived from chromosome 1 sequences and one
primer is derived from chromosome 14 sequences.
The present invention thus provides the art with methods,
probes and primers for diagnosis and monitoring certain neoplasms
which render such diagnosis and monitoring routine and inexpensive.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a representative trypsin-Giemsa banded
karyotyp~ of the 730-3 ceLl line: 47, XY, +17, t(1;13)(p32;q34),
t(l;l4)(p32;qll). The lp+ and 14q~ chromosomes resulting from the
t(l;l4) translocation involving TCL-5 are designated; the other abno~
malities are indicated by arrows.
Figure 2 shows analysis of the t(1:14) chromosomal breakpoint -:
on the lp+ chromosome in leukemic cell lines. Panel A shows hybrid-
ization of DNA from t(1;14) cell lines with the TCR and pJK3.0S
probe. DNA (10 ug) was isolated from cells of placenta (lanes 1 and
.
WO 9l/00364 PCr/US90/02887
,_ t," ~?c~
4), 730-3 (lanes 2 and 5) and DU.528 (lanes 3 and 6), and was digested
with BamHI (lanes 1-3) or HindIII (lanes 4-6). Panels B-D shows
restriction maps of regions surrounding the lp chromosomal
breakpoint in DU.528. Panel B shows the normal configuration of the
TCR delta locus on chromosome 14. Panel C shows the rearranged
TCL-5 locus on the lp+ chromosome. Panel D shows the complemen-
tary germline TCL-5 locus on chromosome 1. Black bars indicate
chromosome 14 DNA sequences; white bars indicate chromosome 1
sequences. Probes are shown as hatched boxes. Abbreviations used
are: E, EcoRI; B, BamHI; H, Hindm.
Figure 3 shows that sequences juxtaposed to the TCR delta
locus in the translocation chromosome of t(1;14) map to chromosome
1. DNA (10 ug/lane) from the following sources was digested with an
excess of restriction enzyme Hind III, electrophoresed, transferred to
a nitrocellulose filter, and hybridized with the p528H5.1 probe (see
figure 2): mouse LMTK cell line (lane l); M44c12S5 hybrid retaining
the 14q chromosome from the translocation t(8;14)(q24;q32) (lane 2);
GL5 hybrid retaining chromosome 4, 13, 14, 18, 20, 21, X and partial
chromosomes 17 and 22 (lane 3); 401AD5EF3 hybrid retaining partial
8 and 22 and chromosomes 19, 21, and X (lane 4); 52-63c17-17 hybrid
retaining the 14q+ chromosome from the translocation
t(14;X)(q32;ql3) (lane 5); GM7299 hybrid retaining chromosomes 1, 6
and X (lane 6); human placenta (lane 7); PB5 hybrid retaining partial
chromosomes 1, 2, 3, 5 and 17 (lane 8).
Figure 4 depicts the 14q chromosomal reciprocal breakpoint.
Panel A shows a Southern filter hybridized with the p528B0.7 probe
(see Figure 2) containing a germline chromosome 1 insert encompass-
ing the t(1;14) breakpoint. DNA (10 ug/lane) from mouse cell line
(lane 1), human placenta (lane 2), DU.528 cell line (lane 3), hybrid
~26c122 (lane 4~ and hybrid 726c124 (lane 5) was digested with EcoRI,
electrophoresed, and transferred to a nitrocellulose filter. Panel B
shows restriction maps of the chromosome 14 TCR delta locus (black
bar) and the chromosome 1 TCL-5 locus (white bar) that recombined
to form the 14q~ chromosome of the t(l;l4) translocation (black and
white bar). E, EcoRI; B, BamHI; H, EIindm.
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Figure 5 compares the nucleotide sequence at the t(1;14)
translocation junctions and the cDrresponding germline sequences.
Panel A shows the lp+ chromosome breakpoint sequence. Panel B
shows the 14q chromosomal reciprocal breakpoint sequence.
Germline chromosome 14 D-delta1 and D-delta2 sequences are shown
as previously published (Takihra, et al., (1988) Proc. Natl. Acad. Sci.
USA 85:6097-6101). Brackets indicate heptamer signal sequences of
the D-delta segments. The sequences shaded in gray are thought to
represent N regions.
Figure 6 shows the expression of a TCL-5 transcript in ~rarious
T-cell lines and a B cell line. Samples (10 ug) of total cellular RNA
from five cell lines were electrophoresed in 1% agarose containing
2.2 M formaldehyde and then transferred to nitrocellulose. Duplicate
filters were prepared and hybridized with the TCL-5 528B0.'1 probe
(panel A) or with the human ribosomal pA probe (Erickson et al.,
(1981) Gene 16:1-9) (panel B). Filters were washed with 15 mM
NaCl/1 mM sodium phosphate, pH 7.4/0.1 mM EDTA/0.1% NaDodSO4
at 42C prior to autoradiography. Lane 1, 730-3; lane 2, TALL-101
(Valtieri et ~l., (1987) J. Immunol. 138:4042-4050); lane 3, Peer (Hata
et al., (1987) Science 238:678-681); lane 4, Jurkat (Sangster et al.,
(1986) J. Exp. Med. 163:1491-1508); lane 5, 697 (Findley, Jr. et al.,
(1982) Blood 60:1305-1309).
Figure 7 demonstrates the there is a rearrangement of the
TCL-5 locus in WM8 melanoma DNA. 10 ug of GM60~ DNA used as a
germline control (lanes A) or WM8 DNA (lanes B and C) were digested
with the enzymes indicated, fractionated and hybridized with the
p528B4.4 probe.
Figure 8 depicts two models of the t(1;14) translocation event.
D deltal and D delta2 segments are shown by the stippled and filled
boxes, respectively. Chromosome 1 and 14 are shown by a single and
a double line, respectively. In Panel A the translocation and D
delta1-D delta2 attempted joining occur simultaneously. In Panel B
the D delta1-D delta2 joining precedes the transloca~ion. The trian-
gle represents the breakpoint site on chromosome 1.
,. - . . . . . , . ,. - . . .
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-- 6 --
DETAILED DESCRIPlION
It is a discovery of the present invention that there is an
oncogene on chromosome 1 band p32 which is rearranged in the
course of the neoplastic process. It is a further finding that the rear-
rangement leads to increased production of expression products of
the oncogene. The oncogene is called TCL-5.
Using nucleic acid sequences of the TCL-5 oncogene, which
are provided by the present invention, neoplasms can be diagnosed,
assessed or monitored. For example, nucleic acid probes comprising
portions of the TCL-5 gene can be used to hybridize to DNA isolated
from cel~s suspected of being neoplastic. The DNA can be digested
with restriction endonucleases to form discrete fragments. Detection
of hybridized fragments in the isolated DNA of different sizes than
those detected by the probe with control DNA samples indicates a
TCL-5 rearrangement and neoplasia. The rearrangement may be
associated with a t(1;14)(p32;qll) translocation, although other rear-
rangements are possible which would be detected by the methods of
the present invention, such as deletions, insertions and inversions.
The diagnosis of neoplasms generally provides a positive deter-
mination that a malignant state exists. Further, by determining that
a TCL-5 rearrangement has occurred a classification of the neoplasm
can be made to a class of neoplasms involving such rearrangements.
The members of a class of neoplasms may respond similarly to thera-
pies, which is useful for choosing a course of treatment.
A neoplasm may be assessed or monitored using the methods of
the present invention. This generally provides a quantitative mea-
sure of the amount of neoplastic cel1s remaining in a patient. For
example, during the course of anti-neoplastic therapy, e.g., cnemo-
therapy or radiation therapy, the method of the present invention
can be used to monitor the success of such therapy. This may involve
quantitating the amoun~ of rearranged TCL-5 genes detectable as
compared to before therapy. Alternatively, this could involve quanti-
tating the amount and type of TCL-5 gene products and comparing
these with levelc before therapy.
.
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WO 91/0~364 ~ . PCr/US90/02887
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In another application of the methods of the present invention,
TCL-5 rearrangements are determined in a human whose neoplasm
has been in remission. If any TCL-5 rearrangements are detected,
they provide a measure or indication of minimal residual disease, i.e.
the recurrence of detectable neoplastic cells. An indication of mini-
mal residual disease provides the clinician with notice to resume
anti-neoplastic therapy.
According to the present invention, DNA of a human ~s iso-
lated from cells which are suspected of being neoplastic. Such a sus-
picion may be based on morphological, cytological, or other means
relied on by clinicians. It is then determined whether the DNA iso-
lated from the cells contains rearranged TCL-5 genes. While rear-
rangements involving t(1;14)(p32;qll) translocations are specifically
contemplated, other rearrangements involving TCL-5 could occur,
such as insertions, deletions or inversions, which would lead to the
neoplastic state.
TCL-5 rearrangements have been found to occur in both leuke-
mia and melanoma cells. However, it is not beyond the scope of the
invention that other neoplasms would be associated with TCL-5 rear-
rangements. For example, neuroblastomas may be associated with
TCL-5 rearrangements, as chromosome 1 band p32 has been shown to
be rearranged in neuroblastoma cells. It is possible that a TCL-5
rearrangement is one of a number of genetic changes that occur du~
ing, and contribute to the oncogenic process. If the human is sus-
pected of having leukemia, then T-lymphocytes are the preferred cell
source for isolating DNA. If the human is suspected of having mela-
noma, then melanocytes are the preferred cell source for isolating
DNA. However, it is possible that in some cases a TCL-5 rearrange-
ment may be inherited, in which case all somatic ceUs would carry
the rearrangement. People carrying such inherited rearrangements
may be prone to develop neoplasms.
According to one method of the present invention a TCL-5
rearrangement is determined by hybridizing a nucleic acid probe
derived from chromosome 1 sequences. The probe sequences are
those retained on the lp+ chromosome after a t(l;l4)(p32;qll)
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o 91/00364 Pcr/usso/o2887
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translocation. The sequences are close enough to the translocation
junction (where chromosome 1 sequences are joined to chromosome
14 sequences) that they lie between the translocation junction and a
first restriction enzyme site; there are no other sites for the restric-
tion enzyme between the translocation junction and the first site.
Such probes hybridize to the TCL-5 RNA transcripts (expression
products) and thus can be used to detect and quantify transcripts.
Suitable probes are shown in Figure 2 and include p528B4.4, p528H5.1
and p528BO.7. Probes p528B4.4, p528H5.1 and p528BO.7, have been
deposited at the American Type Culture Collection (ATCC) in
Rockville, MD, as accession nos. Preferred probes are
those whose sequences lie between the translocation junction and the
first EcoRI site. These include those probes named above.
In another method of the present invention the step of deter-
mining is performed by amplification of particular sequences by the
polymerase chain reaction technique, as is well known in the art.
See, Mullis, Science, ~1988), vol. 239, pp. 487-491; Scharf, Science,
~1988), vol. 233, pp. 1076-1078. The sequences preferably amplified
are those which include the translocation junction of a lp+ chromo-
some resulting from a t(1;14)(p32;qll) chromosome. Preferably the
primers for such amplification are single stranded nucleic acids, one
of which comprises chromosome 14 sequences and one of which com-
prises chromosome 1 sequences. Both sets of sequences are retained
on the lp+ chromosome. The chromosome 14 sequences are prefera-
bly derived from the TCR-delta locus encoding the T cell antigen
receptor. Most preferably these comprise sequences derived from
D-delta-2 or J~elta-l segments of the locus. A suitable probe of this
kind has been deposited at the ATCC as accession no. 67996, and is
known as plOlJ-delta-E5Ø Preferred chromosome 1 primers are
derived from sequences on probe p528BO.7, which spans the
translocation breakpoint on chromosome 1.
As mentioned above, gene expression products, including both
proteins and ribonucleic acids, in cel~s suspected of being neoplastic
can be quantified and compared to the amount found in control cells
Control cells are isolated from healthy cells of the same human
.
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WO 91/00364 .J . ~ 'j PCI /US90/02887
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supplying the test cells or from a healthy human. Placental cells may
also be used. Methods of quantifying protein and nucleic acids are
well known in the art. For example, radiolabeled nucleic acid probes
can be used and the amount hybridizing can be determined by
autoradiography and densitometry. Other means of quantifying may
also be used. Gene products which have been detected in cells carry-
ing t(1;14)(p32;qll) translocations are about 5.~ kb, about 2.2 kb, and
about 2.6 kb RNA transcripts. In cells carrying the translocation, the
two smaller transcripts are much more abundant than the larger
transcript. In cells without the translocation only the larger tran-
script is detected. The protein products of these transcripts can be
detected by antibodies which recognize epitopes on the proteins.
Antibodies recognizing epitopes unique to specific protein products
can be used to distinguish the populations of TCL-5 proteins. Meth-
ods for quantitating protein products are known in the art and may be
used as are convenient. One convenient method is the ELISA tech-
nique, which employs a colorimetric assay wherein the color corre-
lates with the amount of antigen present. Means of obtaining anti-
bodies for specific protein products are also routine and well known
in the art.
Nucleic acid probes and primers discussed above which can be
used in the practice of the method of the present invention are also
provided. They can be isolated from biological sources such as the
deposited cell lines or plasmids mentioned above, or they can be syn-
thesized according to known sequences, for example those shown in
Figure 5. Methods for preparing and labeling probes are well known
in the art. Methods for preparing primers are also well known.
The following examples do not limit the scope of the inven-
tion, which is described by the preceding description of the invention
and the claims appended below.
EXAMP~-~S
ExamPle 1
This example describes the identification of the
t(1;14)(p32;qll) chromosomal breakpoint in leukemic cells.
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The t(1;14) chromosomal translocation has been described in
the leukemic cells of a 16 year-old boy with acute T lymphoblastic
leukemia (Hershfield et al., (1984) Proc. Natl. Acad. Sci. USA
81:253-257). Two cell lines, DU.528 (Kurtzberg et al., (1985) J. Exp.
Med. 162:1561-1578) and 730-3 (Kurtzberg, unpublished results),
derived from the leukemic cells prior to and after chemotherapy,
respectively, showed an apparently identical karyotype. Our descrip-
tion of the 730-3 karyotype is 47, XY, +17, t(1;13)(p32;q34),
t(1;14)(p32;qll) (See Fig. 1). This interpretation, with translocations
involving the p32 region of both number 1 chromosomes, differs
somewhat from the initial report (Kurtzberg et al., (1985) J. Exp.
Med. 162:1561-1578).
On chromosome 14 at band qll the T-cell receptor (TCR) con-
stant delta chain gene lies within the TCR alpha locus approximately
85 kb upstream of the constant alpha locus region (Isobe et al., (1988)
Proc. Natl. Acad. Sci. USA 85:3933-3937); Takihara et al., (1988) Proc.
Natl. Acad. Sci. USA 85:6097-6101). Direct involvement of the TCR
delta locus in the t(l;14) translocation was suggested by the results of
the hybridization of the TCR delta pJK3.0S probe to a Southern filter
with bound DU.528 and 730-3 DNAs (Fig. 2A). This probe, which con-
tains a 3.0 kb SstI fragment located within the TCR delta locus as
illustrated in Figure 2B, hybridizes to a 10.5 kb BamHI rearranged
fragment in both DU.528 and 730-3 Bam HI-digested DNAs (Fig. 2A,
lanes 2,3). Since the pJK3.0S insert has an internal Hindm site, the
probe detects two germline restriction fragments of 3.4 kb and 6.2 kb
in Hindm-digested placental DNA (Fig. 2A9 lane 4). In contrast to the
expected germline Ilindm pattern, pJK3.0S detects a 3.4 kb germline
restriction fragment and a 9.8 kb rearranged restriction fragment in
Hindm-digested DNAs of DU.528 and 730-3 cells (Fig. 2A). Thus, we
concluded that the t(1;14) breakpoint probably occurred within the
9.8 kb Hindm fragment detected by the pJK3.0S probe (Fig. 2A). The
lack of pJK3.0S hybridization to both a 19 kb BamHI germline frag-
ment and a 6.2 kb HindIII germline fragment originating from the
DU.528 (and 730-3) non-translocated ~hromosome 14 may be due to
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_ 1 1 _
the deletion of this delta allele following functions V alpha-J alpha
rearrangement.
We have cloned the t(l;14) chromosomal breakpoint from a
DU.528 genomic library constructed in the lambda phage vector
EMBL3A (Fishauf et al., (1983) J. Mol. Biol. 170:82~-842). One class
of rearranged clones from the phage library of DU.528 was obtained
and a representative map of the overlapping phage clones is shown in
Fig. 2C. The size of the rearranged DU.528 fragments (Fig. 2A) are
in agreement with the calculated values for the appropriate restric-
tion fragments in the rearranged clone (Fig. 2C). In order to deter-
mine the point of divergence from the TCR delta sequences in the
rearranged clone, the chromosome 14 p528E5.0 probe, containing a
5.0 kb EcoRI fragment as illustrated in Fig. 2B, was used to isolate
and map the complementary germline TCR delta locus from a human
placental library (Fig. 2B). A repeat-free 5.1 kb Hindm fragment
located close to the breakpoint, where the restriction enzyme sites
had diverged from the normal delta locus restriction sites (Fig. 2C),
was cloned into pUC19 (pS28H5.1) to be used in determining its chro-
mosomal origin. Probe p528H5.1 was hybridized to a Southern filter
with bound DNA from rodent x human hybrid cells containing either
human chromosome 1 or human chromosome 14. The presence in the
hybrids of the 5.1 kb Hindm fragment correlates only with the pres-
ence of human chromosome 1 (Fig. 3), demonstrating that we have
cloned the t(1;14) breakpoint on the DU.528 lp+ chromosome
(lqter-> lp32::14qll-~ 14qter). The pS28H5.1 probe was also used to
screen a human placental library in order to clone the normal chro-
mosome 1 counterpart of the breakpoint region (Fig. 2D).
Procedures for molecular cloning, Southern blot analysis, RNA
isolation and Northern blot analysis were performed as described as
described previously (Finger et al., (1988) Proc. Natl. Acad. Sci USA,
85:9158-9162).
The various probes cloned in this study contain inserts ligated
in pUC19. The details of each probe are described in the text and are
illustrated in figures when relevant. Al! probes were radiolabeled by
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Wo 91/00364 PC~/US90/02887
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nick translation using alpha-32P dNTPs to a specific activity of
approximately 5 x 108 cpm/ug.
Isolation, propagation and characterization of parental cells
and somatic cell hybrids used in this study have been described else-
where (Croce et al., 1985, Blood 65:1-7); Durst et al., 1987, Proc.
Natl. Acad. Sci. USA 84:1070-1074); Haluska et al., 1988, Proc. Natl.
Acad. Sci. USA 85:2215-2218). The presence of specific human chr~
mosomes or regions of chromosomes has been confirmed by DNA
hybridization using probes for genes assigned to specific human chro-
mosome regions. GM7299 is a somatic cell hybrid obtained from the
NIGMS Human Genetic Mutant cell repository (Camden, NJ).
Example 2
This example demonstrates the isolation of the reciprocal
translocation joining segment.
To complete analysis of the structural aspects of the t(1;14)
translocation, we wanted to analyze the joining segment between
chromosomes 1 and 14 on the derivative 14q~ chromosome
(14pter->14qll::1p32'1 lpter). The p528B0.? probe contains a germline
0.7 kb BamHI inse* from chromosome 1 that includes ~he DU.528 lp
translocation breakpoint (as illustrated in Fig. 2D). Upon hybridiza-
tion with the pS28B0.~ probe, an EcoRI digest of DU.528 DNA showed
a germline fragment of 17.5 kb and rearranged fragments of 8 kb and
14 kb (Fig. 4A, lane 3). The 17.5 kb EcoRI fragment (Fig. 4A, lanes
2,3) corresponds to the germline chromosome 1 fragment detected in
both the DU.528 and human control DNAs. The 14 kb EcoRI rear-
ranged fragment (Fig. 4A, lane 3) corresponds to the EcoRI segment
encompassing the breakpoint on the DU.528 chromosome lp+, a con-
clusion based on restriction analysis using the p528EB2.7 probe. The
pS28EB2.7 probe spans the DU.528 chromosome lp+ breakpoint, as
depicted in Fig. 2C, and detects the same size 14 kb rearranged frag-
ment in DU.528 EcoRI-digested DNA.
The 8 kb EcoRI rearranged fragment (Fig. 4A, lane 3) could
correspond to the translocation junction on the derivative chromo-
some 14q in DU.528. We therefore rescreened the DU.528 genomic
library with the p528B0.7 probe, our objective being to obtain clones
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corresponding to the 14q chromosome. Af ter the initial library
screening, phage clones that hybridized to the p528B0.7 probe but not
to the p528EB2.7 probe (a probe that hybridizes to DU.528 phage
clones with inserts covering the chromosome lp+ junction or the nor-
mal chromosome 1 counterpart) were isolated and characterized in
detail. The rationale for the differential screening process was to
isolate only the clones potentially containing the translocation junc-
tion on the reciprocal 14q chromosome. A comparison of the
restriction may representing the above isolated clones with the
restriction maps representing the chromosome 1 and chromosome 14
normal counterparts presented in Fig. 4B clearly demonstrates that
we have also cloned the chromosome 14q reciprocal breakpoint of
the t(1;14) translocation in DU.528.
8y subcloning mouse-human hybrids isolated af ter fusion of
mouse and 730-3 cells, we obtained hybrid lines 726c122 and 726c124
which retained the 8 kb EcoRI rearranged fragment corresponding to
the 14q (14pter-~ 14qll::1p32-> lpter) chromosomal junction region
(Fig. 4A, lanes 4,5). The two hybrids a~so retained the L-MYC locus
which has been mapped to chromosome region lp32 (Nau et al., 1985,
Nature 318:69-73) but did not retain the nerve growth factor beta
locus assigned to chromosome region lp22 or lpl3 (Munke et al.,
1984, Somat. Cell. Genet. 10:589-599); Garson et al., 1987, Nucl.
Acids Res. 15:4761-4770). Thus, analysis of the somatic cell hybrids is
consistent with the TCL-5 locus residing on the short arm of chromo-
some 1 centromeric to the L-MYC locus and telomeric to the nerve
growth f actor beta locus.
ExamDle 3
This example shows that the chromosome 14 sequences
involved in the t(l;14)(p32;qll) translocation are in the D-delta locus.
The nucleotide sequence of the translocation junction is a~so shown.
After fine genomic mapping of regions of interest, DNA frag-
ments were subcloned in M13mpl8 or M13mpl9 and their sequences
were determined by using the Sanger dideoxy method (Sanger et al.,
197?. Proc. Natl. Acad. Sci USA 7~:5463-5467) with Sequenase (United
States Biochemical, Cleveland).
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The nucleotide sequences of the translocation junction sites
and the normal counterparts are presented in Fig. 5. Except for the
loss of one nucleotide at the breakpoint, the chromosome 1 sequences
at the junction on the lp+ chromosome and the 14q chromosome are
continuous in relation to the germline chromosome 1, thus demon-
strating the reciprocity of ~he translocation in relation to chromo-
some 1. Examination of the sequence from chromosome 14 at the
translocation junction identified features which may have influenced
the translocation process. The joining on the lp+ chromosome
occurred at the D-delta2 segment (Fig. 5A). An unrearranged
D-delta2 segment normally possesses the 3~ and 5~ heptamer and
nonamer recombination signal sequences used during D segment rear-
rangements (Takihara et al., 1988, Proc. Natl. Acad. Sci. USA
85:609~-6101). Strikingly, the t(1;14) breakpoint on the lp chromo-
some occurred in D-delta2 precisely at a site where normal D seg-
ment rearrangement occurs, i.e., at the site immediately 3~ to the 5'
signal sequences, such that these sequences are lost during the
recombination process (Sakano et al., 1981, Nature 290:562-565).
The chromosome 14 sequences from the reciprocal chromo-
some 14q side of the breakpoint demonstrate that the junction site is
at the D-delta1 segment of the TCR delta locus (Fig. 5B). Analogous
to normal D segment rearrangement (Sakano et al., 1981, Nature
290:562-565), the reciprocal breakpoint has occurred precisely 5~ to
the 3' D-delta1 signal sequences. Extra nucleotides present at both
the junction sites on chromosomes lp+ and 14q are highlighted by
the stippled boxes in Fig. 5 and are not derived from the normal
germline sequences. Such additional nucleotides at breakpoints in B-
and T-cell translocations may represent N regions (Tsujimoto et al.,
1985, Science 229:1390-1393; 1985, Nature 315:340-343; Finger et al.,
1986, Science 234:982-985; Haluska, et al., 1986, Nature 324:15~-161),
stretches of extra nucleotides that are presumably added by the
enzyme terminal transferase (Desiderio et al., 1984, Nature,
311:~52-~55) at the pre-B and pre-T cell stage of differentiation.
Because of the involvement of the D~elta coding segments and their
accompanying recombinational signals, the present results suggest
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that the recombination process mediating this chromosomal
translocation is catalyzed by the saMe recombinase system that nor-
mally carries out the joining of T-cell receptor delta gene segments.
Example 4
This example demonstrates that there is a gene located at the
translocation junction on chromosome 1.
Frequently, chromosomal loci aberrantly juxtaposed to
immunoglobulin super f amily loci by chromosomal rearrangements
are inappropriately transcriptionally active, a f eature which is
thought to reflect the transcription of a gene contributing directly to
the malignant process (reviewed in Finger et al., 1989, ~Involvement
of the Immunoglobulin Loci in B-cell Neoplasms, in ImmunoFlobulin
Genes (F. Alt, T. Honjo and T.H. Rabbitts, eds.) Academic Press, Ltd.
London, pp. 221-230). We therefore sought evidence for the presence
of a gene located in the region of chromosome 1 adjacent to the
t(1;14) translocation breakpoint by performing Northern blot analysis
using purified insert from the p528B0.~ probe (528B0.7), a fragment
from normal chromosome 1 which overlaps the breakpoint.
Total cellular RNAs used for Northern blot analyses were from
730-3, a T-cell ALL cell line (TALL-101), a gamma/delta-producing
T-cell line (Peer), an alpha/beta-producing T-cell line (Jurkat), and a
pre-B-cell leukemia cell line (697). Hybridization of a filter retaining
these RNAs to a ribosomal probe indicates that there are similar
amounts of 28S ribosomal RNA in sample lanes (Figure 6B). The same
RNA preparations on a duplicate filter show varying sizes and
amounts of transcripts hybridizing to the chromosome 1 probe,
528B0.7 (Figure 6A). The 528B0.7 probe detects a 5.4 kb RNA tran-
script and possibly a low level of 2.2 kb and 2.6 kb RNA transcripts in
the T-cell lines Jurkat and TALL-101. The gene represented by these
transcripts, located at the translocation junction on chromosome 1, is
designated TCL-5 (T-cell leukemia/lymphoma-5). A barely detectable
level of a 5.4 kb TCL-5 RNA transcript in the 730-3 and Peer T-cell
lines and the 697 pre-~ cell line may be due to either low level
expression of TCL-5 or to nonspecific cross-hybridization to 28S
ribosomal RNA. In the 730-3 cell line, however, the 528B0.7 probe
.
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detects a high level of expression of the 2.2 kb and 2.6 kb TCL-5 RNA
transcripts, suggesting that an alteration of TCL-5 gene expression in
730-3 cells is directly involved in the neoplastic process in this
human T-cell leukemia.
Example 5
This example demonstrates that the TCL-5 locus is rearranged
in a melanoma cell line with a lp32 chromosomal abnormality.
A number of human cutaneous malignant melanomas (CMM)
have been shown to carry non-random chromosome lp deletions or
rearrangements which often appear to involve the proximal segment
lpl2- lp22 (Balaban et al., 1986, Cancer Genet. Cytogenet.,
19:113-122). Approximately 8-12% of all CMMs occur in individuals
with a family history of the disease (Greene and Fraumeni, 1979, ~The
Hereditary Variant of Malignant Melanoma,~ in Human Mali~nant
Melanoma, Grune and Stratton, NY, pp. 139-166). Genetic studies of
familial CMM kindreds indicate a highly penetrant, autosomal, domi-
nant mode of inheritance for a dysplastic nevus syndrome/CMM sus-
ceptibility gene with provisional linkage to the Rh blood locus on
chromosome region lp34-36 (Greene et al., 1983, Proc. Natl. Acad.
Sci. USA 80:6071-6075).
We have examined the possible involvement of the TCL-5 locus
in chromosome 1 aberrations in several melanoma cell lines. The
p528B4.4 probe, which contains a 4.4 BamHI fragment derived from ~ -
chrom~some 1 adjacent to the breakpoint on the lp+ chromosome as -
illustrated in Fig. 2D, detected a genomic rearrangement in DNA
from the primary melanoma cell line WM8. Two DNA preparations
from the WM8 cell line yielded identical results (Fig. 7, lanes B,C).
The WM8 cell line carries a del(1)(p32) and numerous other chromo-
somal aberrations (M. Herlyn and P.C. Nowell, unpublished results).
Genomic rearrangements detected in WM8 DNA compared to human
control DNA using several different restriction enzymes allowed us to ~ ;
exclude restriction fragment length polymorphisms as an explanation
of the new rearranged fragments. The lower intensity of the rear-
ranged restriction fragments relative to the germline restriction
fragments may be due to a low copy number of the del(1)(p32) -
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chromosome relative to the other chromosomes 1 observed in WM8,
which is a hypotetraploid cell line.
