Note: Descriptions are shown in the official language in which they were submitted.
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MODULATION OF TELOMERE-INITIATED CELL SIGNALING
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001) This application is a continuation-in-part of International Application
No.
PCT/US03/11393, filed April 11, 2003, the contents of which are hereby
incorporated in their
entirety.
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0002) The present invention relates to the regulation of signaling pathways.
More specifically,
the present invention relates to the regulation of telomere-initiated
senescence, apoptosis, tanning
and other DNA damage responses.
2. Description of Related Art
[0003) The frequency of cancer in humans has increased in the developed world
as the
population has aged. For some types of cancers and stages of disease at
diagnosis, morbidity and
mortality rates have not improved significantly in recent years in spite of
extensive research:
During the progression of cancer, tumor cells become more and more independent
of negative
regulatory controls, including resistance to senescence and apoptosis, the
important aspects of
how the interaction of normal cells with their tissue-specific environment is
regulated.
[0004) Cellular senescence has been suggested to be an important defense
against cancer.
Extensive evidence implicates progressive telomere shortening or telomere
dysfunction caused
by an inability to replicate the 3' ends of chromosomes in senescence. In
germline cells and most
cancer cells, immortality is associated with maintenance of telomere length by
telomerase, an
enzyme complex that adds TTAGGG repeats to the 3' terminus of the chromosome
ends.
Telomeres, tandem repeats of TTAGGG, end in a loop structure with a 3' single-
stranded
overhang of approximately 150-300 bases tucked within the proximal telomere
duplex DNA and
stabilized by telomeric repeat binding factors (TRFs), particularly TRF2.
Ectopic expression of
a dominant-negative form of TRF2 (TRF2DN) disrupts telomere loop structure,
exposes the 3'
overhang and causes DNA damage responses, followed by senescence in primary
fibroblasts and
fibrosarcoma cells.
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[0005] Senescence can also be precipitated acutely by extensive DNA damage or
the
overexpression of certain oncogenes. Ectopic expression of the telomerase
reverse transcriptase
catalytic subunit (TERT), which enzymatically maintains or builds telomere
length, can bypass
senescence with subsequent immortalization of some human cell types, strongly
suggesting a
telomere-dependent mechanism of replicative senescence. Moreover, malignant
cells commonly
express TERT and/or contain mutations that allow the cell to bypass the
senescent response and
to proliferate indefinitely despite often having shorter telomeres than normal
senescent cells.
However, some tumor cells undergo senescence in response to various anticancer
agents,
indicating that acquisition of immortality does not necessarily imply a loss
of this basic cellular
response to DNA damage.
[0006] Senescence in human cells is largely dependent on the p53 and pRb
pathways. The
tumor suppressor p53 plays a key role in cellular stress response mechanisms
by converting a
variety of different stimuli, for example, DNA damage, deregulation of
transcription or
replication, oncogene transformation, and deregulation of microtubules caused
by some
chemotherapeutic drugs, into cell growth arrest or apoptosis. When activated,
p53 causes cell
growth arrest or a programmed, suicidal cell death, which in turn acts as an
important control
mechanism for genomic stability. In particular, p53 controls genomic stability
by eliminating
genetically damaged cells from the cell population, and one of its major
functions is to prevent
tumor formation.
[0007] An intact tumor suppressor pRb pathway is needed to prevent
tumorigenesis. In pRb~~~
tumor cells that do not contain wild-type p53, introduction of pRb induces
senescence. Although
cervical cancer cells frequently retain wild-type p53 and pRb genes, the HPV
E6 and E7 proteins
interfere with the p53 and pRb pathways, respectively. Ectopic expression-of
viral E2 protein
represses HPV E6 and E7 gene transcription and induces a rapid and prominent
senescent
response in cervical carcinoma cell lines, again affirming the important roles
of p53 and pRb in
cancer cell senescence.
[0008] Suppressing only the p53 or the pRb pathway is not sufficient for
fibroblasts to bypass
replicative senescence. Indeed, human fibroblasts either transfected with SV
40 T antigen or
transduced with combinations of adenovirus ElA+E1B or HPV E6+E7, suppressing
both the p53
and pRb pathways, have an extended life span and escape replicative
senescence.
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[0009) Double strand breaks in DNA are extremely cytotoxic to mammalian cells.
The highly
conserved MRN complex is involved in the repair of double strand breaks in
eukaryotes. The
MRN complex adheres to sites of double strand breaks immediately following
their formation.
The MRN complex also migrates to telomeres during the S-phase of the cell
cycle associates
with telomeric repeat binding factors (TRF).
[0010) The MRN complex consists of Mrel l, Rad50 and NBS (p95). Mrel 1, as
part of the
Mrel 1/p95/Rad50 complex, associates with the telomere 3' overhang DNA during
S phase of the
cell cycle. Mrel l is an exonuclease with preference for the 3' end of a DNA
strand. The activity
of Mrel 1 is believed to be dependent on interaction with Rad50, which is an
ATPase. Nbsl is
believed to be involved in the nuclear localization of the MRN complex, as
well as its assembly
at the site of a double strand break.
[0011 ] Cancers are typically treated with highly toxic therapies, such as
chemotherapy and
radiation therapy, that comparably damage all proliferative cells whether
normal or malignant.
Side effects of such treatments include severe damage to the lymphoid system,
hematopoietic
system and intestinal epithelia, as well as hair loss. Other side effects
include hair loss. There
continues to be a need for safer and more effective cancer therapies,
especially for alternative
therapies that would avoid some or all of these side effects by preferentially
targeting malignant
cells relative to normal but proliferative cells.
SUMMARY OF THE INVENTION
[0012] The present invention relates to an in vitro method of screening for a
modulator of Mrel l
comprising contacting candidate modulators with Mrel 1 in vitro in the
presence of a nucleic acid
substrate for Mrel l, and measuring the hydrolysis of the substrate. A
modulator may be
identified by altering hydrolysis of the substrate nucleic acid compared to a
control. The nucleic
acid substrate may be an oligonucleotide with at least 50% nucleotide sequence
identity with
(TTAGGG)", wherein n=1 to 20. The hydrolysis of the substrate nucleic acid may
be measured
by W absorbance, gel analysis of labeled oligos, or recovery of non-
precipitatable nucleotide
bases.
[0013] The present invention also relates to an in vitro method of screening
for an agent that
specifically binds to Mrel l, comprising contacting candidate agents with Mrel
l, and
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determining whether a candidate agent specifically binds to Mrel 1. Mrel l may
be attached to a
solid support.
[0014] The present invention also relates to a cell-based method of screening
for a modulator of
Mrel l, comprising contacting candidate modulators with a cell that expresses
Mrel 1 under
conditions in which the modulator is taken up by the cell, and measuring a
property of the cells
including, but not limited to, cellular proliferation, cellular viability,
cellular morphology, SA-b-
Gal activity and phosphorylation of p53 or p95. A modulator may be identified
by altering the
property compared to a control. The candidate modulator may be an agent that
specifically binds
to Mrel 1 as identified above. Mrel 1 may be expressed as a fragment, homolog,
analog or
variant of Mrel 1, which may have exonuclease activity.
[0015] The present invention also relates to an in vitro method of screening
for a modulator of
tankyrase comprising contacting candidate modulators with tankyrase in vitro
in the presence of
a substrate for tankyrase, and measuring the ribosylation of the substrate. A
modulator may be
identified by altering ribosylation of the substrate compared to a control.
The substrate may be a
peptide or polypeptide, which.may be TRF. The ribosylation of the substrate
may be measured
by LTV absorbance or labeling of the substrate.
[0016] The present invention also relates to an in vitro method of screening
for an agent that
specifically binds to tankyrase, comprising contacting candidate agents with
tankyrase, and
determining whether a candidate agent specifically binds to tankyrase.
Tankyrase may be
attached to a solid support.
[0017] The present invention also relates to a cell-based method of screening
for a modulator of
tankyrase, comprising contacting candidate modulators with a cell that
expresses tankyrase under
conditions in which the modulator is taken up by the cell, and measuring a
property of the cells
including, but not limited to, cellular proliferation, cellular viability,
cellular morphology, SA-b-
Gal activity and phosphorylation of p53 or p95. A modulator may be identified
by altering the
property compared to a control. The candidate modulator may be an agent that
specifically binds
to tankyrase as identified above. Tankyrase may be expressed as a fragment,
homolog, analog or
variant of tankyrase, which may have ribosylase activity.
[0018] The present invention also relates to an in vitro method of screening
for a modulator of
MRN complex formation comprising contacting candidate modulators with Mrel 1,
Rad50 and
Nbsl in vitro, and measuring the formation of the MRN complex. A modulator may
be
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identified by altering formation of the MRN complex compared to a control.
Candidate
modulators may be contacted with Mrel l, Rad50 and Nbsl in the presence of a
nucleic acid
substrate or inhibitor of Mrel 1. The nucleic acid may be an oligonucleotide
with at least 50%
nucleotide sequence identity with (TTAGGG)", wherein n=1 to 20. Formation of
the MRN
complex may be measured by centrifugation, coprecipitation or nondenaturing
electrophoresis.
[0019] The present invention also relates to a cell-based method of screening
for a modulator of
the DNA damage pathway, comprising contacting candidate modulators with a cell
that
expresses Mrel 1 and tankyrase in the presence of an oligonucleotide under
conditions in which
the modulator is taken up by the cell, and measuring a property of the cells
including, but not
limited to, cellular proliferation, cellular viability, cellular morphology,
SA-b-Gal activity and
phosphorylation of p53 or p95. A modulator may be identified by altering the
property
compared to a control. The oligonucleotide may have at least 50% nucleotide
sequence identity
with (TTAGGG)n, wherein n=1 to 20. Mrel 1 may be expressed as a fragment,
homolog, analog
or variant of Mrel 1, which may have exonuclease activity. Tankyrase may be
expressed as a
fragment,.homolog, analog or variant of tankyrase, which may have ribosylase
activity.
[0020] The cell used in the cell-based screening methods described above may
be a cancer cell.
The cell used in the cell-based screening methods described may maintain
telomeres by
telomerase reverse transcriptase or the ALT pathway. The candidate modulators
and agents
described in the in vitro and cell-based screening methods above may be
carbohydrates,
monosaccharides, oligosaccharides, polysaccharides, amino acids, peptides,
oligopeptides,
polypeptides, proteins, nucleosides, nucleotides, oligonucleotides,
polynucleotides, lipids,
retinoids, steroids, glycopeptides, glycoproteins, proteoglycans, or small
organic molecules.
[0021] The present invention also relates to the use of compositions
comprising an activator of
Mrel l, tankyrase, the DNA damage pathway or MRN complex formation. The
activator may be
used to treat cancer, inducing apoptosis, inducing cellular senescence,
inhibiting tanning,
promoting cellular differentiation or promoting immunosuppression. The
activator may be an
oligonucleotide activator of Mrel l, which may have at least 50% nucleotide
sequence identity
with (TTAGGG)", wherein n=1 to 20. From about one to about ten of the first 3'-
nucleotide
linkages may be hydrolyzable by a 3' to 5' nuclease.
[0022] The present invention also relates to the use of compositions
comprising an inhibitor of
Mrel l, tankyrase, the DNA damage pathway or MRN complex formation. The
inhibitor may be
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used to inhibit apoptosis, inhibit cellular senescence, promote growth,
promote tanning, inhibit
cellular differentiation, reduce cancer treatment side effects. The
composition may be given in
combination with chemotherapy or ionizing radiation. The inhibitor may be an
oligonucleotide
inhibitor of Mrel 1, which may have at least 50% nucleotide sequence identity
with (TTAGGG)",
wherein n=1 to 20. From about zero to about ten of the first 3'-nucleotide
linkages may be
hydrolyzable by a 3' to 5' nuclease.
[0023] The present invention also relates to a composition comprising an
oligonucleotide with at
least 50% nucleotide sequence identity with (TTAGGG)" and with at least one
nonhydrolyzable
internucleotide linkage, wherein n=1 to 20. From one to about ten of the first
3'-nucleotide
linkages may be hydrolyzable by a 3' to 5' nuclease, such as Mrel 1. The
oligonucleotide may
have at least 50% nucleotide sequence identity with TTAGGG. The
oligonucleotide may also
have the sequence GTTAGGGTTAG. The nonhydrolyzable linkage may be a
phosphorothioate.
The oligonucleotide may be a PNA.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figures lA-1H show FACS analysis of propidium iodide stained Jurkat
cells
(immortalized T lymphocytes), treated with diluent (Figures lA and lE); 40 pM
l lmer-1
pGTTAGGGTTAG (SEQ 1D NO: 2) (Figures 1B and 1F); 40 pM l lmer-2 pCTAACCCTAAC
(SEQ >D NO: 3) (Figures 1C and 11G); 40 ~.M l lmer-3 pGATCGATCGAT (SEQ )D NO:
4)
(Figures 1D and 1H). Jurkat cells were treated with the stated reagents for 48
hours before
analysis (Figures lA-1D) or 72 hours (Figures lE-1H).
[0025] Figures 2A-2F are profiles showing the results of fluorescence
activated cell sorting, for
the following additions to the cells: Figure 2A, diluent; Figure 2B, 0.4 ~,M l
lmer-1; Figure 2C,
0.4 ~.M l lmer-1-S; Figure 2D, diluent; Figure 2E, 40 ~M l lmer-l; Figure 2F,
40 ~,M l lmer-1-
S.
[0026] Figures 3A-3G are profiles showing the results of fluorescence
activated cell sorting, for
the following additions to the cells: Figure 3A, diluent; Figure 3B, 10 ~.M l
lmer-1; Figure 3C,
p.M l lmer-1 and 1 ~,M l lmer-1-S; Figure 3D, 10 p,M l lmer-1 and 5 ~,M l lmer-
1-S; Figure
3E, 10 ~,M l lmer-1 and 10 p.M l lmer-1-S; Figure 3F, 20 p,M l lmer-1-S;
Figure 3G, 10 ~,M
l lmer-1-S.
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[0027] Figure 4 is mbar graph showing the melanin content (in pg/cell) of
cells treated with
diluent, pTpT or pTspT.
[0028] Figure 5 is a bar graph showing the melanin content (in pg/cell) of
cells treated with
diluent, l lmer-1 'or l lmer-1-S.
[0029] Figure 6 is a bar graph showing the melanin content (in pg/cell) of
cells that have been
sham-treated (no irradiation, no oligonucleotides), or treated with
ultraviolet light (LTV), or
unirradiated but given pTspT, or irradiated with UV and given pTspT.
[0030] Figure 7 is a diagram of oligonucleotides of nucleotide sequence SEQ m
NO: 2 which
were synthesized with phosphorothioate linkages.
[0031] Figure 8 is a bar graph showing the results of testing the effects of
phosphorothioate
oligonucleotides 1, 2, 3 and 4 depicted in Figure 7 in causing senescence in
cultures of normal
neonatal human fibroblasts, indicated by the cells staining positive for (3-
galactosidase activity.
Oligonucleotide "11-1" indicates fibroblast cultures treated with SEQ m NO: 2
synthesized
entirely with phosphodiester linkages. "Dil" indicates fibroblast cultures
treated with diluent not
containing oligonucleotide.
[0032] Figures 9-11 demonstrate that downregulating Mrel 1 protein levels
blocks r~esporise of
T-oligos.
[0033] Figure 12 demonstrates that the p53 and pRb pathways both contribute to
T-oligo-
induced senescence in human fibroblasts. Figure 12a: Irnmunoblot analysis of
p53DD and
cdk4~4C expression. Cells were collected for protein analysis by western blot
using 30p,g total
protein and probed for total p53 and cdk4. Lanes l, 2, 3 and 4 contain protein
samples from
R2F, R2F (p53DD), R2F (cdk4~4C) and R2F (p53DD/ cdk4~4c) fibroblasts,
respectively.13-
actin was used as a loading control. Figure 12b: Contribution of p53 and pRb
pathways to T-
oligo-induced SA-(3-Gal activity. R2F fibroblasts and derived transductants
were treated with
diluent or 40p,M T-oligo for one week and then assayed for SA-[3-Gal activity.
Figure 12c:
Quantitative analysis of SA-~i-Gal positive cells. Cells expressing SA-[i-Gal
activity were
counted and presented as percentage of total cells in the cultures. Averages
and standard
deviations were calculated from 3 representative fields from each of 3
independent experiments.
[0034] Figure 13 shows that exposure of human fibrosarcoma HT-1080 cells to T-
oligo induces
senescence. Figure 13a: Exposure to T-oligo increases SA-~3-Gal activity. HT-
1080 cells were
treated for 4 days with diluent alone or 40pM T-oligo or the complementary
control oligo, then
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stained and assayed for SA-(3-Gal activity. Figure 13b: Quantitative analysis
of SA-(3-Gal
positive cells. Cells expressing SA-(3-Gal activity were counted and presented
as percentage of
total cells in the cultures. Averages and standard deviations were calculated
from 3
representative fields from each of 3 independent experiments. Figure 13c:
Effect of T-oligo on
cell proliferation. Cells were treated for 4 days as in Figure 12 and assayed
for DNA synthesis by
BrdU incorporation. Figure 13d: Quantitative analysis of BrdU incorporation.
Dark black nuclei
indicate BrdU incorporated into nuclear DNA. BrdU positive cells were
calculated and
presented as percentage of total cells in the cultures. Averages and standard
deviations were
calculated from 3 representative fields from each of 3 independent
experiments. Figure 13e:
Effect of T-oligo on pRb phosphorylation. Cells were treated as in Figure 13a
and were then
collected for protein analysis by western blot using 30pg total protein and
probed for pRb-
ser780*, ser795* and ser807/811* (pRb phosphorylated at serine 780, serine 795
and serine
807/811 respectively). Lanes D, T and C contain protein samples from cells
treated with diluent,
T-oligo and complementary oligo respectively.13-actin was used as a loading
control.
[0035] Figure 14 shows the persistent effect of T-oligo removal on the
senescent phenotype in
human fibrosarcoma HT-1080 cells. Parallel cultures were treated as described
in Figure 13a.
Cells were then washed once with PBS and refed with complete medium without
additional
treatment for 24 hours or 48 hours. Figure 14a: SA-(3-Gal activity. Cells were
stained for SA-[3-
Gal activity. Figure 14b: Cell cycle arrest. BrdU incorporation was assayed.
Figure 14c:
Phosphorylation and activation of pRb. Immunoblot analysis was performed as
described in
Figure 13 e.
[0036] Figure 15 shows the effect of prolonged exposure to T-oligo on
clonogenic capacity of
human fibrosarcoma HT-1080 cells. Cells were treated with diluent, 40pM T-
oligo or
complementary oligo for one week, and then assayed. Figure 15a: Appearance of
stained dishes.
Figure 1 Sb: Quantification of clonogenic capacity. Colonies of triplicate
cultures were counted
and plotted as percentage of diluent treated control.Figure 16 shows the
effect of T-oligo on
mean telomere length (MTL) in human fibrosarcoma HT-1080 cells. Cells were
treated as
described in Figure 13a. Lanes 1, 2, 3 contain genomic DNA from cells treated
with diluent (D),
T-oligo (T), or complementary oligo (C). Lanes 4 and 5 contain high (H)
molecular and low (L)
molecular weight standard telomeric DNA.
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[0038] Figure 17 shows that T-oligos and TRFDN initiate DNA damage responses
via the same
pathway. The graphs show densitometric readings of the western blots, with
diluent control set
at 100%. Figure 9f-. Lane 1, diluent, GFP; lane 2, diluent TRF2DN; lane 3,
3AB, GFP; lane 4,
3AB, TRF2DN; lane 5, IQ, GFP; lane 6, IQ, TRF2DN. .~
[0039] Figure 18 shows that the effect of T-oligos are not dependent on
telomerase. Figure 18a:
FACS profiles from one representative experiment of three with the percentage
and standard
deviations of cells in each phase of the cell cycle was calculated from
triplicate cultures of each
condition. Figure 18b: Western blots with an antibody specific for phospho-
p95/Nbsl. Lanes 1,
2 and 3 contained protein from cells treated with diluent, l lmer-1 or l lmer-
2, respectively.
Control cells (3 hours) were irradiated with 10 Gy of IR (+), or were sham
irradiated (-).
[0040] Figure 19 shows that downregulating tankyrase protein levels blocks the
response of T-
oligos. The upper panel shows the densitometry readings and the lower panel
shows the western
blot.
(0041] Figure 20 shows that T-oligos cause phosphorylation of p53 on serine
37. Western blot
analysis was performed on normal neonatal cells using a antibody specific for
p53 phosphoserine
37 after being treated with either diluent or 40 pM for the indicated times.
DETAILED DESCRIPTION OF THE INVENTION
[0042] The present invention is based on the discovery that Mrel 1-mediated
hydrolysis of the
3' telomere overhang sequence initiates signaling cascades important for
protective cellular
responses to DNA damage, such as senescence, tanning and apoptosis. Not being
bound by
theory, we believe that DNA damage, such as W irradiation, oxidative damage to
DNA, or
formation of carcinogen adducts to DNA, or age-associated telomere shortening
destabilizes the
telomere loop, exposing the 3' overhang sequence comprising repeats of TTAGGG.
Telomere-
associated proteins then attach to the overhang in a sequence-dependent manner
and serve as an
"anchor" for the Mrel 1/p95/Rad50 complex. Mrel 1 then begins to hydrolyze the
telomere
overhang from the 3' end, which leads to activation of the Rad50 ATPase.
Activation of Rad50
leads to activation of tankyrase by phosphorylation, conformational change of
some kind or other
mechanism, which then activates ATM and possibly other kinases such as ATR.
ATM then
phosphorylates p95 and other DNA damage response effectors such as p53,
ultimately leading to
the biologic endpoints of cell cycle arrest, gene induction, apoptosis and/or
senescence.
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[0043] Based on the role of Mrel 1 and tankyrase in the proposed signaling
pathway, activators
of Mrel l, tankyrase, the DNA damage pathway or MRN complex formation are
expected to
activate the DNA damage response pathway regardless of the presence of DNA
damage or
telomere loop disruption. This is illustrated in the Examples herein showing
that telomere
homolog oligonucleotides (T-oligos) serve as a substrate for Mrel l thereby
leading to apoptosis,
senescence or growth arrest in the absence of DNA damage or telomere loop
disruption.
[0044] Similarly, inhibitors of Mrel 1, tankyrase, the DNA damage pathway or
MRN complex
formation are expected to inhibit the signal transduction pathway, even in the
presence of DNA
damage or telomere loop disruption. This is illustrated in the Examples herein
showing that
apoptosis and growth arrest are inhibited under conditions that cause DNA
damage and telomere
loop disruption by the following: (i) non-hydrolyzable T-oligos, which act as
an antagonist of
Mrel l, (ii) RNAi-mediated reduction in Mrel l protein levels; and (iii) RNAi-
mediated
reduction in tankyrase protein levels.
[0045] Before the present products, compositions and methods are disclosed and
described, it is
to be understood that the terminology used herein is for the purpose of
describing particular
embodiments only and is not intended to be limiting. It must be noted that, as
used .in the
specification and the appended claims, the singular forms "a," "an" and "the"
include plural
referents unless the context clearly dictates otherwise.
[0046] Throughout this application, where patents or publications are
referenced, the disclosures
of these publications 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.
1. Definitions
[0047] As used herein, the term "activator" means anything that activates a
protein or increases
the activity of a protein.
[0048] As used herein, the term "administer" when used to describe the dosage
of a modulator
means a single dose or multiple doses of the agent.
[0049] As used herein, the term "analog", when used in the context of a
peptide or polypeptide,
means a peptide or polypeptide comprising one or more non-standard amino acids
or other
structural variations from the conventional set of amino acids; and, when used
in the context of
an oligonucleotide, means an oligonucleotide comprising one or more
internucleotide linkages
other than phosphodiester internucleotide linkages.
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[0050] As used herein, the term "antibody" means an antibody of classes IgG,
IgM, IgA, IgD or
IgE, or fragments or derivatives thereof, including Fab, F(ab')Z, Fd, and
single chain antibodies,
diabodies, bispecific antibodies, bifunctional antibodies and derivatives
thereof. The antibody
may be a monoclonal antibody, polyclonal antibody, affinity purified antibody,
or mixtures
thereof which exhibits sufficient binding specificity to a desired epitope or
a sequence derived
therefrom. The antibody may also be a chimeric antibody. The antibody may be
derivatized by
the attachment of one or more chemical, peptide, or polypeptide moieties known
in the art. The
antibody may be conjugated with a chemical moiety.
[0051] As used herein, "apoptosis" refers to a form of cell death that
includes, but is not limited
to, progressive contraction of cell volume with the preservation of the
integrity of cytoplasmic
organelles; condensation of chromatin (i.e., nuclear condensation), as viewed
by light or electron
microscopy; and/or DNA cleavage into nucleosome-sized fragments, as determined
by
centrifuged sedimentation assays. Cell death occurs when the membrane
integrity of the cell is
lost (e.g., membrane blebbing) with engulfment of intact cell fragments
("apoptotic bodies") by
phagocytic cells.
[0052] .As used herein, the term "cancer treatment" means any treatment for
cancer known in. the
art including, but not limited to, chemotherapy and radiationtherapy.
[0053] As used herein, the term "combination with" when used to describe
administration of a
modulator and an additional treatment means that the modulator may be
administered prior to,
together with, or after the additional treatment, or a combination thereof.
[0054] As used herein, the term "derivative", when used in the context of a
peptide or
polypeptide, means a peptide or polypeptide different other than in primary
structure (amino
acids and amino acid analogs); and, when used in the context of an
oligonucleotide, means an
oligonucleotide different other than in the nucleotide sequence. By way of
illustration,
derivatives of a peptide or polypeptide may differ by being glycosylated, one
form of post-
translational modification. For example, peptides or polypeptides may exhibit
glycosylation
patterns due to expression in heterologous systems. If at least one biological
activity is retained,
then these peptides or polypeptides are derivatives according to the
invention. Other derivatives
include, but are not limited to, fusion peptides or fusion polypeptides having
a covalently
modified N- or C-terminus, PEGylated peptides or polypeptides, peptides or
polypeptides
associated with lipid moieties, alkylated peptides or polypeptides, peptides
or polypeptides
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linked via an amino acid side-chain functional group to other peptides,
polypeptides or
chemicals, and additional modifications as would be understood in the art.
[0055] As used herein, the term "fragment", when used in the context of a
peptide or
polypeptide, means any peptide or polypeptide fragment, preferably from about
5 to about 300
amino acids in length, more preferably from about 8 to about 50 amino acids in
length; and,
when used in the context of an oligonucleotide, means any oligonucleotide
fragment, preferably
from about 2 to about 250 nucleotides, more preferably from about 2 to about
20 nucleotides in
length. Representative examples of peptide or polypeptide fragments are 8, 9,
10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49 or SO amino acids in length. Representative
examples of
oligonucleotide fragments are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19 or 20
nucleotides in length.
[0056) As used herein, the term "homolog", when used in the context of a
peptide or
polypeptide, means a peptide or polypeptide sharing a common evolutionary
ancestor or having
at least 50% identity thereto; and, when used in the context of an
oligonucleotide, means an
oligonucleotide sharing a common evolutionary ancestor or having at least 50%
identity thereto.
[0057] As used herein, the term "inhibit' when referring to the activity of a
protein, means
preventing, suppressing, repressing, or eliminating the activity of the
enzyme.
[0058] As used herein, the term "treat" or "treating" when referring to
protection of a mammal
from a condition, means preventing, suppressing, repressing, or eliminating
the condition.
Preventing the condition involves administering a composition of the present
invention to a
mammal prior to onset of the condition. Suppressing the condition involves
administering a
composition of the present invention to a mammal after induction of the
condition but before its
clinical appearance. Repressing the condition involves administering a
composition of the
present invention to a mammal after clinical appearance of the condition such
that the condition
is reduced or prevented from worsening. Elimination the condition involves
administering a
composition of the present invention to a mammal after clinical appearance of
the condition such
that the mammal no longer suffers the condition.
[0059] As used herein, the term "variant", when used in the context of a
peptide or polypeptide,
means a peptide or polypeptide that differs in amino acid sequence by the
insertion, deletion, or
conservative substitution of amino acids, but retain at least one biological
activity; and, when
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used in the context of an oligonucleotide, means an oligonucleotide that
differs in nucleotide
sequence by the insertion, deletion, or substitution of nucleotides, but
retain at least one
biological activity. For purposes of the present invention, "biological
activity" includes, but is
not limited to, the ability to be bound by a specific antibody.
2. Modulators
a. Modulator of Mrell
[0060] The present invention relates to a modulator of Mrel 1 activity. The
modulator may
induce or increase Mrel l activity. The modulator may also inhibit or reduce
Mrel 1 activity.
The modulator may be an artificially synthesized compound or a naturally
occurnng compound.
The modulator may be a low molecular weight compound, oligonucleotide,
polypeptide or
peptide, or a fragment, analog, homolog, variant or derivative thereof.
[0061] An oligonucleotide modulator may be an oligonucleotide with at least
about 50% to
about 100% nucleotide sequence identity with (TTAGGG)n, wherein n is from
about 1 to about
333. The oligonucleotide may be of a form including, but not limited to,
single-stranded,
double-stranded, or a combination thereof. The oligonucleotide preferably
comprises a single-
stranded 3'-end of from about 2 to about 2000 nucleotides, more preferably
from about 2 to about
200 nucleotides. The oligonucleotide may also be an EST. Also specifically
contemplated is an
analog, derivative, fragment, homolog or variant of the oligonucleotide.
[0062] As shown in the Examples, certain oligonucleotides of the present
invention caused the
inhibition of proliferation and induction of apoptosis in cells, whereas other
oligonucleotides of
the present invention cause the inhibition of growth arrest and inhibition of
apoptosis. The
difference in the activity of the oligonucleotides was dependent on the number
of 3' hydrolyzable
internucleotide linkages. By varying the number of 3' hydrolyzable
internucleotide bonds, the
effect of the oligonucleotides was varied.
[0063] Not being bound by theory, we believe that the oligonucleotides are
recognized by the
MRN complex and serve as a substrate for the 3'-exonuclease Mrel 1. The
corollary is that
substrate oligonucleotides that comprise 3'-nonhydrolyzable internucleotide
bonds act as
antagonists or inhibitors of Mrel 1. Other factors determining the level of
Mrel l activity
include, but are not limited to, the total concentration of 3'-hydrolyzable
internucleotide bonds,
base sequence and G content.
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[0064] An internucleotide bond is considered hydrolyzable for purposes of the
present invention
if (i) it is a phosphodiester linkage or an analog thereof that is
hydrolyzable by Mrel 1 under
physiological conditions, and (ii) all internucleotide bonds 3' thereto are
also hydrolyzable. An
internucleotide bond is considered nonhydrolyzable for purposes of the present
invention if it is
not hydrolyzable by Mrel 1 under physiological conditions, regardless of the
number of
hydrolyzable internucleotide linkages that are 3' thereto. Representative
examples of
nonhydrolyzable internucleotide linkages include, but are not limited to,
phosphorothioate
linkages and peptide nucleic acid linkages (PNA).
[0065] In one embodiment of the invention, the oligonucleotide comprises
hydrolyzable
internucleotide bonds. The oligonucleotide may comprise from about 1 to about
200
hydrolyzable internucleotide bonds. The oligonucleotide may also comprise
nonhydrolyzable
internucleotide bonds. The oligonucleotide may comprise from about 0 to about
199
nonhydrolyzable internucleotide bonds.
[0066] In another embodiment, the oligbnucleotide comprises nonhydrolyzable
bonds. The
oligonucleotide may comprise from about 1 ~to about 200 nonhydrolyzable
internucleotide bonds.
The oligonucleotide may also comprise hydrolyzable internucleotide bonds. The
oligonucleotide
comprise from about 0 to about 5 hydrolyzable internucleotide bonds. Preferred
oligoriucleotides are T-oligos described herein and as described in co-pending
U.S. Patent
Application No. 10/122,630, filed April 12, 2002, which is incorporated herein
by reference.
b. Modulator of Tankyrase
[0067] The present invention also relates to a modulator of tankyrase
activity. The modulator
may induce tankyrase activity. The modulator may also inhibit tankyrase
activity. The
modulator maybe an artificially synthesized compound-or a naturally occurnng
compound. The
modulator may be a low molecular weight compound, polypeptide or peptide, or a
fragment,
analog, homolog, variant or derivative thereof.
c. Modulator of the DNA Damage Pathway
(0068] The present invention also relates to a modulator of the DNA damage
pathway. The
modulator may induce the DNA damage pathway. The modulator may also inhibit
the DNA
damage pathway. The modulator may be an artificially synthesized compound or a
naturally
occurring compound. The modulator may be a low molecular weight compound,
polypeptide or
peptide, or a fragment, analog, homolog, variant or derivative thereof.
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d. Modulator of MRN complex formation
[0069] The present invention also relates to a modulator of M1RIV complex
formation. The
modulator may induce formation of the M1RN complex. The modulator may also
inhibit
formation of the M1RN complex. The modulator may be an artificially
synthesized compound or
a naturally occurring compound. The modulator may be a low molecular weight
compound,
polypeptide or peptide, or a fragment, analog, homolog, variant or derivative
thereof.
3. Composition
[0070] The present invention also relates to a composition comprising a
modulator as described
above. The composition may comprise an activator of Mrel 1. The composition
may also
comprise an activator of tankyrase. The composition may also comprise an
inhibitor of Mrel 1.
The composition may also comprise an inhibitor of tankyrase. The composition
may also
comprise more than one modulator of the present invention. The composition may
also comprise
one or more modulators together with an additional therapeutic.
[0071] In one embodiment of the present invention, the composition comprises
an
oligonucleotide of the present invention. The oligononucleotide may comprise
hydrolyzable
irlternucleotide bonds or nonhydrolyzable internucleotide bonds, or a
combination' thereof. In a
preferred embodiment, the oligonucleotide is an activator of Mrel 1. In
another preferred
embodiment, the oligonucleotide is an inhibitor of Mrel 1. As discussed above,
the activity of the
oligonucleotide may be adjusted to induce or inhibit Mrel 1 based on the total
concentration of
hydrolyzable internucleotide bonds.
a. Formulation
[0072] Compositions of the present invention may be in the form of tablets or
lozenges
formulated in a conventional manner. For example, tablets and capsules for
oral administration
may contain conventional excipients including, but not limited to, binding
agents, fillers,
lubricants, disintegrants and wetting agents. Binding agents include, but are
not limited to,
syrup, accacia, gelatin, sorbitol, tragacanth, mucilage of starch and
polyvinylpyrrolidone. Fillers
include, but are not limited to, lactose, sugar, microcrystalline cellulose,
maizestarch, calcium
phosphate, and sorbitol. Lubricants include, but are not limited to, magnesium
stearate, stearic
acid, talc, polyethylene glycol, and silica. Disintegrants include, but are
not limited to, potato
starch and sodium starch glycollate. Wetting agents include, but are not
limited to, sodium lauryl
sulfate). Tablets may be coated according to methods well known in the art.
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[0073] Compositions of the present invention may also be liquid formulations
including, but not
limited to, aqueous or oily suspensions, solutions, emulsions, syrups, and
elixirs. The
compositions may also be formulated as a dry product for constitution with
water or other
suitable vehicle before use. Such liquid preparations may contain additives
including, but not
limited to, suspending agents, emulsifying agents,. nonaqueous vehicles and
preservatives.
Suspending agent include, but are not limited to; sorbitol syrup, methyl
cellulose, glucose/sugar
syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminum
stearate gel, and
hydrogenated edible fats. Emulsifying agents include, but are not limited to,
lecithin, sorbitan
monooleate, and acacia. Nonaqueous vehicles include, but are not limited to,
edible oils, almond
oil, fractionated coconut oil, oily esters, propylene glycol, and ethyl
alcohol. Preservatives
include, but are not limited to, methyl or propyl p-hydroxybenzoate and sorbic
acid.
[0074] Compositions of the present invention may also be formulated as
suppositories, which
may contain suppository bases including, but not limited to, cocoa butter or
glycerides.
Compositions of the present invention may also be formulated for inhalation,
which may be in a
form including, but not limited to, a solution, suspension; or emulsion that
may be administered
as a dry powder or in the form of an aerosol .using a propellant, such as
dichlorodifluororiiethane
or trichlorofluoromethane. Compositions of the present invention may also be
formulated
transdermal formulations comprising aqueous or nonaqueous vehicles 'including,
but not limited
to, creams, ointments, lotions, pastes, medicated plaster, patch, or membrane.
[0075] Compositions of the present invention may also be formulated for
parenteral
administration including, but not limited to, by injection or continuous
infusion. Formulations
for injection may be in the form of suspensions, solutions, or emulsions in
oily or aqueous
vehicles, and may contain formulation agents including, but not limited to,
suspending,
stabilizing, and dispersing agents. The composition may also be provided in a
powder form for
reconstitution with a suitable vehicle including, but not limited to, sterile,
pyrogen-free water.
[0076] Compositions of the present invention may also be formulated as a depot
preparation,
which may be administered by implantation or by intramuscular injection. The
compositions
may be formulated with suitable polymeric or hydrophobic materials (as an
emulsion in an
acceptable oil, for example), ion exchange resins, or as sparingly soluble
derivatives (as a
sparingly soluble salt, for example).
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[0077] Compositions of the present invention may also be formulated as a
liposome preparation.
The liposome preparation can comprise liposomes which penetrate the cells of
interest or the
stratum corneum, and fuse with the cell membrane, resulting in delivery of the
contents of the
liposome into the cell. For example, liposomes such as those described in U.S.
Patent No.
5,077,211 of Yarosh, U.S. Patent No. 4,621,023 of Redziniak et al. or U.S.
Patent No. 4,508,703
of Redziniak et al. can be used. The compositions of the invention intended to
target skin
conditions can be administered before, during, or after exposure of the skin
of the mammal to
UV or agents causing oxidative damage. Other suitable formulations can employ
niosomes.
Niosomes are lipid vesicles similar to liposomes, with membranes consisting
largely of non-ionic
lipids, some forms of which are effective for transporting compounds across
the stratum
corneum.
4. Methods of Treatment
a. Activator of Mrel l, Tankyrase, the DNA Damage Pathway or MRN Complex
Formation
[0078] The modulators of the present invention that induce or increase the
activity of Mrel 1, .
tankyrase, the DNA damage pathway or MRN complex formation may be used alone
or in
combination with other treatments to treat conditions associated with failure
of growth arrest,
apoptosis or proliferative senescence. Representative examples of such
conditions include, but
are not limited to, hyperproliferative diseases, such as cancer and the benign
growth of cells
beyond a normal range as, for example, keratinocytes in psoriasis or
fibroblast hypertrophic scars
and keloids, or certain subsets of lymphocytes in the case of certain
autoimmune disorders. The
forms of cancer to be treated by these methods are manifested in various forms
and arising in
various cell types and organs of the body, for example, cervical cancer,
lymphoma,
osteosarcoma, melanoma and other cancers arising in the skin, and leukemia.
Also among the
types of cancer cells to which the therapies are directed are breast, lung,
liver, prostate,
pancreatic, ovarian, bladder, uterine, colon, brain, esophagus, stomach, and
thyroid. The
modulators may also be used to inhibit tanning, to promote cellular
differentiation and for
immunosuppresion.
[0079] In one embodiment of the present invention, an oligonucleotide of the
present invention
comprising hydrolyzable internucleotide bonds is used to treat a condition
associated with failure
of growth arrest, apoptosis or proliferative senescence by administering the
oligonucleotide to a
patient in need of such treatment. The oligononucleotide may also comprise
nonhydrolyzable
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internucleotide bonds. As discussed above, the activity of the oligonucleotide
may be adjusted to
induce growth arrest or apoptosis based on the total concentration of
hydrolyzable
internucleotide bonds. The oligonucleotide may be administered in combination
with
modulators of the present invention or other treatments.
[0080] In a preferred embodiment, the oligonucleotide is used to treat a
cancer selected from the
group consisting of cervical, lymphoma, osteosarcoma, melanoma, skin,
leukemia, breast, lung,
liver, prostate, pancreatic, ovarian, bladder, uterine, colon, brain,
esophagus, stomach, and
thyroid.
[0081] T-oligos are capable of blocking induction or elicitation of allergic
contact
hypersensitivity as effectively as UV irradiation in a mouse model, through
upregulation of TNF-
a and IL10, known mediators of immunosuppression. A topical or systemic
activator of Mrel 1
may, therefore, replace steroid therapy, for example, in treatment of
lymphocyte-mediated skin
diseases, such as psoriasis or eczema as well as lymphocyte-mediated systemic
diseases such as
rheumatoid arthritis, multiple sclerosis, lupus erythematosis, and many other
diseases.
b. Inhibitor of Mrel l, Tankyrase, the DNA Damage Pathway 'or M18N Complex
Formation
[0082] The modulators of the present invention that inhibit or decrease the
activity of Mrel l,
tankyrase, the DNA damage pathway or MRN complex formation may be used alone
or in
combination with other treatments to treat conditions associated with growth
arrest, apoptosis or
proliferative senescence. Representative examples of such conditions include,
but are not limited
to, exposure to UV radiation and side effects of cancer treatments on normal
tissues, such as
chemotherapy and radiation therapy, or promoting the tanning response in sun
exposed normal
skin. The modulators may also be used to inhibit cellular differentiation.
[0083] In another embodiment, an oligonucleotide of the present invention
comprising
nonhydrolyzable internucleotide bonds is used to treat a condition associated
with growth arrest
or apoptosis by administering the oligonucleotide to a patient in need of such
treatment. The
oligononucleotide may also comprise hydrolyzable internucleotide bonds. As
discussed above,
the activity of the oligonucleotide may be adjusted to inhibit growth arrest
or inhibit apoptosis
based on the total concentration of hydrolyzable internucleotide bonds. The
oligonucleotide may
be administered in combination with modulators of the present invention or
other treatments.
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[0084] In a preferred embodiment, the oligonucleotide is used to treat a
condition selected from
the group consisting of exposure to UV radiation and side effects of cancer
treatments, such as
chemotherapy and radiation therapy.
c. Administration
[0085] Compositions of the present invention may be administered in any manner
including, but
not limited to, orally, parenterally, sublingually, transdermally, rectally;
transmucosally,
topically, via inhalation, via buccal administration, or combinations thereof.
Parenteral
administration includes, but is not limited to, intravenous, intraarterial,
intraperitoneal,
subcutaneous, intramuscular, intrathecal, and intraarticular.
d. Dosage
[0086] A therapeutically effective amount of the composition required for use
in therapy varies
with the nature of the condition being treated, the length of time that
activity is desired, and the
age and the condition of the patient, and is ultimately determined by the
attendant physician. In
general, however, doses employed for adult human treatment typically are in
the range of 0.001
mg/kg to about 200 mg/kg per day. The dose may be about 1 pglkg to about 100
p.g/kg; per day.
The desired dose may be conveniently administered in a single dose, or as
multiple doses
administered at appropriate intervals, for example as two, three, four or more
subdoses per day.
Multiple doses often are desired, or required.
[0087] The dosage of a modulator may be at any dosage including, but not
limited to, about
1 p,g/kg, 25 p,g/kg, 50 p.g/kg, 75 pg/kg, 100 pg/kg, 125 pg/kg, 150 pg/kg, 175
pg/kg, 200 pg/kg,
225 pg/kg, 250 p,g/kg, 275 pg/kg, 300 p.g/kg, 325 ~g/kg, 350 pg/kg, 375 pg/kg,
400 pglkg,
425 p,g/kg, 450 pg/kg, 475 p,g/kg, 500 pg/kg, 525 pg/kg, 550 pg/kg, 575 pg/kg,
600 p,g/kg,
625 pglkg, 650 p,g/kg, 675 pg/kg, 700 pg/kg, 725 p.g/kg, 750 pg/kg, 775 ~glkg,
800 pg/kg,
825 p.g/kg, 850 pg/kg, 875 pg/kg, 900 p,g/kg, 925 pg/kg, 950 pg/kg, 975 pg/kg
or 1 mg/kg.
5. Screening Methods
[0088] The present invention also relates to screening methods of identifying
modulators of
Mrel l activity. The present invention also relates to screening methods of
identifying
modulators of tankyrase activity. The present invention further relates to
screening methods of
identifying modulators of MltN complex formation. Furthermore, the present
invention relates
to screening methods of identifying modulators of the DNA damage pathway. The
screening
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methods may be performed in a variety of formats including, but not limited
to, in vitro, cell-
based, genetic and in vivo assays.
[0089] Modulators of Mrel 1 or tankyrase may be identified by screening for
substances that
specifically bind to Mrel l or tankyrase, as the case may be. Specific binding
substances may be
identified in vitro by one of ordinary skill in the art using a number of
standard techniques
including, but not limited to, immunoprecipitation and affinity
chromatography. Specific
binding substances may also be identified using genetic screens by one of
ordinary skill in the art
using a number of standard techniques including, but not limited to, yeast two-
hybrid and phage
display. Specific binding substances may also be identified using high
throughput screening
methods including, but not limited to, attaching Mrel l or tankyrase to a
solid substrate such as a
chip (e.g., glass, plastic or silicon).
[0090] Modulators of Mrel l or tankyrase may also be identified by screening
in vitro for
substances that modulate the activity of Mrel l or tankyrase, as the case may
be. Modulators
may be identified by contacting Mrel 1 or tankyrase with a suspected modulator
and determining
whether the suspected modulator alters the activity of Mrel 1 or tankyrase, as
the case maybe.
The activity of Mrel 1 may be determined by measuring the hydrolysis of a
nucleic acid substrate
of Mrel l :- Hydrolysis of a nucleic acid substrate may be measured by methods
including, but not .
limited to, measuring UV absorbance and, preferably, gel analysis of labeled
oligos or recovery
of non-precipitatable nucleotide bases.. The activity of tankyrase may be
determined by
measuring the phosphorylation of a peptide or polypeptide including, but not
limited to, TRF1.
(0091] A modulator of MRN complex formation may be identified in vitro by
combining Mrel l,
Rad50 and Nbsl and determining the effects of candidate modulators on MRN
complex
formation compared to a control. Formation of the MRN complex may be measured
using a
number of standard techniques known to one of ordinary skill in the art
including, but not limited
to, centrifugation, coprecipitation and nondenaturing electrophoresis.
[0092] A modulator of Mrel 1 or tankyrase may be identified by screening for
substances that
modulate the activity of Mrel 1 or tankyrase in cell-based assays. A modulator
of the DNA
damage pathway may similarly be identified. Modulators may be identified by
contacting cells
with a suspected modulator and determining whether the suspected modulator
alters the level of
apoptosis, senescence, or phosphorylation of p53 or p95. The candidate
modulator may be a
substance that specifically binds to Mrel 1 or tankyrase, as discussed above.
Modulation of
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apoptosis may be measured by methods including, but not limited to, measuring
the size of the
sub-Go~G~ peak in FACS analysis, TUNEL assay, DNA ladder assay, annexin assay,
or ELISA
assay. Modulation of senescence may be determined by measuring senescence-
associated (3-
galactosidase activity or failure ~to increase cell yields or to phosphorylate
pRb or to incorporate
3H-thymidine after mitogenic stimulation. Modulation of p53 activity may be
determined by
measuring phosphorylation of p53 at serine 15 or serine 37 by gel shift assay
by p53 promoter
driven CAT or luciferase construct read-out, or by induction of a p53-
regulated gene product
such as p21. Modulation of p95 activity may be determined by measuring
phosphorylation of
p95 at serine 343 by shift in the p95 band in a western blot analysis, or by
FACS analysis to
detect an S phase arrest. A modulator of Mrel l or tankyrase may also be
identified by screening
for substances that modulate in vivo tumorigenecity.
[0093] Any cells may be used with cell-based assays. Preferably, cells for use
with the present
invention include mammalian cells, more preferably human and non-human primate
cells.
Representative examples of suitable cells include, but are not limited to,
primary (normal) human
dermal f broblasts, epidermal keratinocytes, melanocytes, and corresponding
immortalized or
transformed cell lines; and primary, immortalized or transformed marine cells
lines. The amount
of protein phosphorylation may be measured using techniques standard in the
art including, but
not limited to, colorimetery, luminometery, fluorimetery, and western
blotting.
[0094] The conditions under which a suspected modulator is added to a cell,
such as by mixing,
are conditions in which the cell can undergo apoptosis or signaling if
essentially no other
regulatory compounds are present that would interfere with apoptosis or
signaling. Effective
conditions include, but are not limited to, appropriate medium, temperature,
pH and oxygen
conditions that permit cell growth. An appropriate medium is typically a solid
or liquid medium
comprising growth factors and assimilable carbon, nitrogen and phosphate
sources, as well as
appropriate salts, minerals, metals and other nutrients, such as vitamins, and
includes an effective
medium in which the cell can be cultured such that the cell can exhibit
apoptosis or signaling.
For example, for a mammalian cell, the media may comprise Dulbecco's modified
Eagle's
medium containing 10% fetal calf serum.
[0095] Cells may be cultured in a variety of containers including, but not
limited to tissue culture
flasks, test tubes, microtiter dishes, and petri plates. Culturing is carried
out at a temperature, pH
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and carbon dioxide content appropriate for the cell. Such culturing conditions
are also within the
skill in.the art.
[0096] Methods for adding a suspected modulator to the cell include
electroporation,
microinjection, cellular expression (i.e., using an expression system
including naked nucleic acid
molecules, recombinant virus, retrovirus expression vectors and adenovirus
expression), adding
the agent to the medium, use of ion pairing agents.and use of detergents for
cell
permeabilization.
[0097] Candidate modulators may be naturally-occurnng molecules, such as
carbohydrates,
monosaccharides, oligosaccharides, polysaccharides, amino acids, peptides,
oligopeptides,
polypeptides, proteins, nucleosides, nucleotides, oligonucleotides,
polynucleotides, including
DNA and DNA fragments, RNA and RNA fragments and the like, lipids, retinoids,
steroids,
glycopeptides, -glycoproteins, proteoglycans and the like; or analogs or
derivatives of naturally-
occurring molecules, such peptidomimetics and the like; and non-naturally
occurring molecules,
such as "small molecule" organic compounds. The term "small molecule organic
compound"
refers to organic compounds generally having a molecular weight less than
about 1000,
preferably less than about 500.
[0098] Candidate modulators may be present within a library (i.e., a
collection of compounds),
which may be prepared or obtained by any means including, but not limited to,
combinatorial
chemistry techniques, fermentation methods, plant and cellular extraction
procedures and the
like. Methods for making combinatorial libraries are well-known in the art.
See, for example, E.
R. Felder, Chimia 1994, 48, 512-541; Gallop et al., J. Med. Chem. 1994, 37,
1233-1251; R. A.
Houghten, Trends Genet. 1993, 9, 235-239; Houghten et al., Nature 1991, 354,
84-86; Lam et al.,
Nature 1991, 354, 82-84; Carell et al., Chem. Biol. 1995, 3, 171-183; Madden
et al., Perspectives
in Drug Discovery and Design 2, 269-282; Cwirla et al., Biochemistry 1990, 87,
6378-6382;
Brenner et al., Proc. Natl. Acad. Sci. USA 1992, 89, 5381-5383; Gordon et al.,
J. Med. Chem.
1994, 37, 1385-1401; Lebl et al., Biopolymers 1995, 37 177-198; and references
cited therein.
[0099] The present invention has multiple aspects, illustrated by the
following non-limiting
examples.
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EXAMPLES
Example 1
Oligonucleotides can induce apoptosis
[0100] Oligonucleotides homologous to the telomere overhang repeat sequence
(TTAGGG; SEQ
ID NO: 1), sequence (l lmer-1: pGTTAGGGTTAG; SEQ ID NO: 2), complementary to
this
sequence (1 lmer-2: pCTAACCCTAAC; SEQ m NO: 3) and unrelated to the telomere
sequence
(1 lmer-3: pGATCGATCGAT; SEQ m NO: 4) were added to cultures of Jurkat cells,
a line of
human T cells reported to undergo apoptosis in response to telomere
disruption. Within 48
hours, 50% of the cells treated with 40 pM of SEQ ID NO: 5 had accumulated in
the S phase,
compared to 25-30% for control cells (p<0.0003, non-paired t-test; see Figures
lA-1D), and by
72 hours, 13% of these cells were apoptotic as determined by a sub-Go/Gl DNA
content,
compared to 2-3% of controls (p<0.007, non-paired t-test; see Figures lE-1H).
At 96 hours,
20+3% of the 1 lmer-1 treated cells were apoptotic compared with 3-5% of
controls (p<0.0001,
non-paired t-test). To exclude preferential uptake of the 1 lmer-1 as an
explanation of its
singular effects, Jurkat cells were treated with oligonucleotides labeled on
the 3' end with
fluorescein phosphoramidite, then subjected to confocal.microscopy:and FACS
analysis. The
fluorescence intensity of the cells was the same after all treatments at 4
hours and 24 hours.
Western analysis showed an increase in p53 by 24 hours after addition of l
lmer-l, but not
l lmer-2 or l lmer -3, with a concomitant increase in the level of the E2F1
transcription factor,
which is known to cooperate with p53 in induction of apoptosis and to induce a
senescent
phenotype in human fibroblasts in a p53-dependent manner as well as to
regulate an S phase
checkpoint.
Example 2
Phosphorothioate Version of the Telomere Overhang Iiomolog llmer-1 Does Not
Induce
Apoptosis
[0101] Cultures of Jurkat human T cells were treated with,either diluent, l
lmer-1 (SEQ m
NO: 1) or the phosphorothioate l lmer-1 (1 lmer-1-S) for 96 hours, then
collected and processed
for FACS analysis. Two concentrations of the oligonucleotides were tested, 0.4
pM (Figures
2A-2C) and 40 ~M (Figures 2D-2F). At 0.4 ~.M, neither of the oligonucleotides
affected the
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expected exponentially growing cell cycle profile of the Jurkat cells. At 40
pM, the 11-mer-1
induced extensive apoptosis, indicated by a sub-Go/Gi peak, while the l liner-
1-S had no effect.
Example 3
Phosphorothioate Version of llmer-1 Blocks Induction of S-Phase Arrest by the
Phosphate
Backbone llmer-1
[0102] Cultures of a keratinocyte cell line (SSC12F, 100,000 cells/38 em2)
were treated for 48
hours with only the l liner-1 (SEQ m NO: 2) or with the l liner-1 in the
presence of increasing
concentrations of the l liner-1-S. As shown previously in Example 1, the l
liner-1 induced an S-
phase arrest as demonstrated by FACS (Becton-Dickinson FacScan). Forty-three
percent of the
cells were in the S phase, compared to 26% of the control, diluent-treated
cells. However, when
increasing concentrations of the phosphorothioate l liner-1 were also added to
these cultures,
fewer cells became arrested (Figures 3A-3G). Complete inhibition of this
arrest was seen with a
ratio of l liner-1: l liner-1-S of 2:_l. The 1 liner-1-S by itself did not
induce the S-phase arrest.
Example 4
Phosphorothioate Forms of the Telomere Oligonucleotides. Reduce Constitutive
and UV-
Induced Pigmentation and Do Not Stimulate Melanogenesis
[0103] Cultures of S91 mouse melanoma cells (100,000 cells/38 emz) were
treated with 100 pM
pTpT or phosphorothioate pTpT (pTspT) (Figure 4) or 40 pM l liner-1 or the
phosphorothioate
l liner-1 (lliner-1-S) (Figure 5) for 6 days and were then collected, counted
and assayed for
melanin content. While the pTpT and l liner-1 (Figure 4 and Figure 5,
respectively) stimulated
melanogenesis in these cells, pTspT and l liner-1-S did not (Figure 4 and
Figure 5, respectively).
Furthermore, both pTspT (Figure 4) and l liner-1-S (Figure S) reduced the
constitutive
pigmentation in these cells, suggesting that chronic exposure of this sequence
during telomere
repair/replication may provide a constant, low level signal for melanogenesis
and this signal is
blocked by pTspT and l liner-1-S.
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Example 5
Phosphorothioate pTspT Inhibits UV-Induced Melanogenesis
[0104] Duplicate cultures of S91 cells (100,000 cells/39 cm2) were either sham-
irradiated or
irradiated with 5 mJ/cm2 solar-simulated light from a 1 kW xenon arc solar-
simulator (XMN
1000-21, Optical Radiation, Azuza, CA) metered at 285 ~ 5 nm using a research
radiometer
(model IL1700A, International Light, Newburyport, MA). Two sham-irradiated
plates were then
supplemented with 100 ~M pTspT and two irradiated cultures were similarly
treated with pTspT.
After one week, cells were collected, counted and analyzed for melanin content
by dissolving the
cell pellets in 1 N NaOH and measuring the optical density at 475 nm. UV
irradiation resulted in
a doubling of melanin content in these cells. However, this response was
blocked by the addition
of pTspT (Figure 6). In addition, the constitutive pigmentation of these cells
was reduced by the
pTspT in the sham-irradiated cultures, similar to the data presented in
Figures 4 and 5.
Example 6
Hydrolysis of the T-oligo is Necessary for Activity
[0105] Oligonucleotides based on SEQ lD NO: 2 were synthesized.
Oligonucleotide 1 was
synthesized entirely with a phosphorothioate backbone. Oligonucleotide 2 had
two
phosphorothioate linkages on each end, with the other linkages in the middle
being
phosphodiester linkages. Oligonucleotide 3 had two phosphorothioate linkages
on the 5' end (S'
end blocked), with the rest of the linkages being phosphodiester linkages.
Oligonucleotide 4 had
two phosphorothioate linkages on the 3' end (3' end blocked), with the rest of
the linkages being
phosphodiester linkages. See Figure 7.
[0106] These oligonucleotides were added to cultures of normal neonatal
fibroblasts. After 48
hours, cells were collected to be analyzed for p53 serine 15 phosphorylation
and p95/Nbsl
phosphorylation by western blot. Other cultures were left in the presence of
the oligonucleotides
for one week and then the cells were stained for senescence-associated (3-
galactosidase activity
(SA-(3-Gal) [3-galactosidase positive cells were scored and presented as a
percent of total cells
(Figure 8).
[0107] Oligonucleotides with a nuclease-accessible 3' terminus are the most
effective at
stimulating "early" responses such as p53 and p95/Nbsl phosphorylation.
However,
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oligonucleotides with a nuclease-accessible 5' terminus can also induce the
senescent phenotype
after one week, but not the phosphorylation reactions at 48 hours, suggesting
that 3' to 5'
nuclease susceptibility is preferable for activity in inducing senescence.
Example 7
Downregulating Mrell Protein Levels Blocks Response of T-Oligos
[0108] Normal human neonatal fibroblasts were treated with either 10 pmoles
Mrel 1 siRNA or
pmoles control (no homology found in expressed human sequences). Cultures
dishes were
approximately 60% confluent on the day of siRNA transfection. Transfections
were carried out
using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the protocol
supplied by the
manufacturer. The transfection cocktail was applied to the cells for 5 hours
and then replaced
with fresh medium alone. The next day, the transfection protocol was repeated.
The following
day, duplicate cultures were treated with the T-oligo or with diluent alone as
a negative control.
Cells were then collected 48 hours later and the protein analyzed by Western
blot using
antibodies specific for phospho-p95 serine 343 (Cell Signaling Technology,
Beverly, MA),
Mrel l (GeneTex, San Antonio, TX), phosphor-p53 serine 15 (Cell Signaling
Technology) and
total p53 (Oncogene, San Diego, CA) (Figure 9). Hela cell lysate vvas used as
a positive control
for Mrel l . Normal fibroblasts exposed to 10 Gy IR or sham irradiated and
collected after one
hour served as positive controls for p53 and p95/Nbsl phosphorylation. The
autoradiographs
were analyzed by densitometry and the values for the T-oligo samples are
expressed relative to
the values for the diluent-treated samples (Figure 10 and Figure 11). After
correcting for loading,
it is apparent that cells with significantly reduced MRE 11 levels have a
reduced phospho-p53
response to T-oligo and an absent phospho-p95/Nbsl response.
Example 8
Inactivation of Both p53 and pRb Pathways is Necessary to Escape T-Oligo-
Induced
Senescence in R2F Fibroblasts
Oligonucleotides
[0109] Two DNA oligonucleotides were used, one homologous to the telomere
overhang (T-
oligo: pGTTAGGGTTAG; SEQ m NO: 2) and one complementary thereto
(pCTAACCCTAAC; SEQ ID NO: 3), which was used as a negative control. These
oligos were
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synthesized by the Midland Certified Reagent Company (Midland, Texas).
Oligonucleotides
were prepared as previously described (Eller et al. [2003] Induction of a
p95/Nbsl-mediated S
phase checkpoint by telomere 3' overhang specific DNA. Faseb J 17, 152-162).
Cell source and culture
[0110] R2F newborn dermal fibroblasts and derived p53DD, cdk4~4c and
p53DD/cdk4~4c
transductants (a generous gift from Dr. James G. Rheinwald of Harvard Medical
School) lack a
functional p53 pathway, pRb pathway, and both pathways respectively.
Senescence-associated [3-galactosidase staining
[0111] Cells were treated once with diluent alone, 40 p.M T-oligo or 40 pM
complementary
oligo for 1 week without re-feeding. Cells were then fixed for 3-5 minutes in
2%
formaldehyde/0.2% glutaraldehyde and incubated at 37°C (ambient COZ)
overnight with fresh
senescence-associated (3-Gal (SA-(3-Gal) stain solution, as described (Dimri
et al. [1995] A
biomarker that identifies senescent human cells in culture and in aging skin
in vivo. Proc Natl
Acad Sci U S A 92, 9363-9367).
Western blot analysis and antibodies ~ .
[0112] Western blot analysis was performed as previously described (Eller et
al. [ 1996] DNA
damage enhances melanogenesis. Proc Natl Acad Sci U S A 93, 1087-1092). The
following
antibodies were used: DO-1 (Ab-6) anti-p53 (Oncogene Research Products,
Cambridge, MA),
anti-phospho-p53 (ser 15) (Cell Signaling Technology Beverly, MA), anti-
phospho-pRb (ser780,
ser795, ser807/811) (Cell Signaling Technology Beverly, MA), anti-cdk4 (Cell
Signaling
Technology Beverly, MA) and anti-actin (Santa Cruz Biotechnology, CA).
Clonogenic assay
(0113] Human fibrosarcoma cells were treated with diluent alone, 40 p,M T-
oligo or 40 pM
complementary oligo for one week and were then trypsinized and counted. 300
cells were seeded
into 60 mm culture dishes in triplicate and then incubated in complete medium
for 2 weeks with
medium changed twice per week. Subsequently, the cells were fixed for 5 min in
100%
methanol. The methanol was then removed and the culture dishes were rinsed
briefly with water.
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The colonies were stained for 10 min in 4% (w/v) methylene blue solution in
PBS, washed once
again with water, and then counted.
BrdU incorporation assay
[0114] HT-1080 fibrosarcoma cells cultured on Permanox chamber slides were
treated with
diluent, 40 ~.M T-oligo or 40 pM complementary oligo for 4 days and DNA
synthesis was
assayed using 5-bromo-2'-deoxy-uridine (BrdU) Labeling and Detection Kit II
(Roche Molecular
Biochemicals, Indianapolis, IN) following the protocol supplied by the
manufacturer. Briefly,
cells were labeled for 1 hour with BrdU, fixed and incubated with anti-BrdU
monoclonal
antibody. After incubation with anti-mouse-Ig-alkaline phosphatase, the color
reaction was
detected by light microscopy.
Telomere Length
(0115] HT-1080 fibrosarcoma cells were treated with diluent, 40 pM T-oligo or
40 pM
complementary oligo for 4 days and then the genomic DNA was isolated using the
DNeasy
Tissue Kit (Qiagen, Valencia, CA). Telomere length was determined using the
Telo TTAGGG
Telomere Length Assay (Roche Molecular Biochemicals, Indianapolis,1N)
following the
protocol supplied by the manufacturer. Briefly, 1 pg of purified genomic DNA
was digested with
Hinf 1/Rsal, the DNA fragments were separated on a 0.8% agarose gel and then
transferred to a
nylon membrane for Southern blotting, hybridized to a digoxigenin (DIG)-
labeled probe specific
for telomeric repeats and incubated with Anti-DIG-Alkaline Phosphatase.
Terminal restriction
fragments (TRF) were detected by chemiluminescence. The mean TRF length was
calculated by
scanning the exposed X-ray film with a densitometer and calculated as
previously described
(Harley et al. [1990] Telomeres shorten during ageing of human fibroblasts.
Nature 345, 458-
460).
Results
[0116] Senescent fibroblasts characteristically exhibit a large, flat
morphology and an increase in
senescence-associated [i-galactosidase (SA-(3-Gal) activity. Ectopic
expression of TRF2DN
disrupts the telomere loop structure and induces senescence in normal human
fibroblasts by
activating the p53 and pRb pathways. Blocking both the p53 and pRb pathways in
human cells
is required to prevent TRF2°N induced senescence.
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[0117] Cell lines engineered to lack the p53 pathway and/or pRb pathways were
used to analyze
the signaling pathways involved in T-oligo-induced senescence. Inactivation of
the p53 pathway
was achieved through ectopic expression of a dominant negative mutant p53
(p53DD) which
lacks the transcriptional transactivation domain of p53 and binds and
inactivates endogenous
wild-type p53 protein. p21/SDI1 protein, a transcriptional target of p53, is
below the level of
detection in R2F fibroblasts transduced to expressed p53 DD (data not shown).
The disruption
of the pRb pathway was achieved through ectopic expression of a p 16-
insensitive mutant cdk4
(cdk4~4~) unable to bind p 16, thus abolishing its control of the pRb protein.
The suppression of
both pathways was achieved through ectopic expression of both mutants (p53DD/
cdk4~4c).
Expression of p53DD and cdk4~4~ was confirmed by Western blot showing the
overexpression
of p53 and cdk4 proteins respectively (Figure 12a), consistent with a previous
report in which
human keratinocytes were transduced with these mutants.
(0118] Cells were treated with either diluent or 40 pM T-oligo for 1 week and
then assessed for
SA-(3-Gal activity. The normal neonatal foreskin fibroblast parental line
(R2F) was used as a
positive control. As expected, T-oligo-treated R2F fibroblasts exhibited a
large, spread
morphology and an increase in SA-[3-Gal, activity as compared with-diluent-
treated control 'cells
(6517% and 8~1% SA-(3-Gal positive cells, respectively, p<0.01) (Figure
l2b,c). Similarly, in
p53DD R2F fibroblasts, one week exposure to T-oligo induced a large, spread
morphology and
an increase in SA-[i-Gal activity as compared with diluent-treated cells
(4514% and 6~2% SA-(3-
Gal positive cells, respectively, p<0.01) (Figure l2b,c), indicating that
inactivation of the p53
pathway alone is not sufficient to suppress T-oligo-induced senescence. T-
oligo also induced a
senescent phenotype in cdk4~4e R2F fibroblasts as compared with diluent-
treated cells (60f5%
and 7t3% SA-(3-Gal positive cells, respectively, p<0.01) (Figure l2b,c),
indicating that the
compromise of the pRb pathway alone is also not sufficient to suppress T-oligo
induced
senescence. However, when R2F fibroblasts were transduced to express both
p53DD and
cdk4~4o, T-oligo was unable to induce a senescent phenotype as compared with
diluent-treated
cells (7t1% and 5~2% SA-(3-Gal positive cells, respectively, p>0.05) (Figure
l2b,c), indicating
that compromise of both the p53 and the pRb pathways is necessary to fully
suppress T-oligo-
induced senescence in human fibroblasts. Therefore, T-oligo-induced senescence
has the same
requirements as replicative senescence following serial passage or senescence
induced by
TRF2DN.
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Example 9
Inactivation of Both p53 and pRb Pathways is Necessary to Escape T-Oligo-
Induced
Senescence in HT-1080 Cells
[0119] TRF2DN has been reported to induce a senescent phenotype in human
fibrosarcoma HT-
1080 cells. To determine whether exposure to the telomere 3' overhang DNA (T-
oligo) also
induces senescence in these cells, HT-1080 cells (American Type Culture
Collection; Manassas,
VA) were treated with either diluent alone, T-oligo or the complementary oligo
as a control, for
4 days and then assessed for SA-[3-Gal activity. Only T-oligo-treated cells
exhibited spread
morphology and an increase in SA-(3-Gal activity (Figure 13a). T-oligo treated
cultures
contained many more SA-(3-Gal positive cells than cultures treated with
diluent or
complementary control oligo (807%, 3~2% and 6t3%, respectively, p<0.01)
(Figure 13b).
Also, only T-oligo-treated cells and not diluent or control oligo-treated
cells were not
proliferating as shown by pronounced reduction of BrdU incorporation (7~2%,
908% and
8510%, respectively, p<0.01) (Figure l3c,d).
Example 10
Telomere Oligonucleotides Prevent Phosphorylation of pRb
[0120] HT-1080 cells are known to have functional pRb, but the p53 pathway is
deficient as a
result of being pl6 deficient. We next examined whether T-oligo treatment
activates pRb by
preventing its phosphorylation in HT-1080 cells. Western blot analysis
revealed that there was a
striking and selective reduction of pRb phosphorylation on serine 780, serine
795 and serine
807/811 in response to T-oligo (Figure 13e). Interestingly, in tumors
deficient in p16, pRb is
often intact and functional. In these cells, the deregulation of cdk4 results
in pRb
hyperphosphorylation and leads to unrestricted cell growth and tumor
formation. Cdk4, but not
cdk2, activation phosphorylates pRb very efficiently on serine 780 and serine
795. The findings
thus suggest that T-oligo inhibits cdk4 activity in the absence of p16,
presumably through the
induction of other INK4 family members, indicating the non-essential role of
p16 in the complex
network of pRb regulation and also suggesting that pRb can not be simply
viewed as an absolute
downstream effector.
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Example 11
The Effects of Telomere Oligonucleotides are not Reversible
[0121] In order to test whether the removal of T-oligo would reverse the
senescent phenotype of
fibrosarcoma cells, parallel cultures of HT-1080 cells were treated for 4 days
with diluent or
40 pM T-oligo or 40 ~M complementary control oligo. Cells were then given
fresh complete
media without further oligonucleotide treatment. After 1 and 2 days, T-oligo
pretreated cells still
exhibited an enlarged morphology and an increase in SA-(3-Gal activity (Figure
14a) and did not
resume DNA synthesis (Figure 14b). Western analysis also showed that the pRb
proteins were
sustained in an active, inhibitory state in T-oligo pretreated cells (Figure
14c).
[0122] To determine the long-term effect of T-oligo treatment on cell growth,
HT-1080 human
fibrosarcoma cells were treated with either diluent alone, 40 pM T-oligo or 40
pM
complementary control oligo for one week and then an equal number of cells
were replated and
medium was changed twice per week for 2 weeks with no further treatment, and
then stained
with methylene blue (Figure 15a). Compared with complementary oligo-treated
cells
(90.59.4% of diluent treated control), the clonogenic_ capacity of cells
pretreated with T-oligo
was almost completely suppressed (5.71.9% of diluent-treated control, p<0.01)
(Figure 15b).
These data indicate that T-oligo induced senescence in this malignant cell
line is not reversible.
Example 12
The Affect of Telomere Oligonucleotides on Mean Telomere Lengths
[0123] To determine the affect of T-oligos on the mean telomere length (MTL)
in HT-1080
cells, cells were analyzed after treatment with T-oligo for 4 days which
corresponded to the time
that the senescent phenotype was readily observed. T-oligo did not alter MTL
(5.56 kb) as
compared with diluent-treated (5.61 kb) or complementary oligo treated
controls (5.51 kb)
(Figure 16). The less than 100 by difference in calculated MTL is within the
range of
experimental variation and is not significant. This is consistent with the
observation that
treatment of fibroblasts with T-oligo for up to 1 week does not result in
degradation of the
telomere 3' overhang, as is observed following telomere disruption by
TRF2°N (data not shown).
Because disruption of the telomere loop is known to cause rapid shortening of
MTL and
digestion of the 3' overhang, the fact that T-oligo initiates similar or
identical signaling without
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affecting MTL or causing digestion of the 3' overhang indicates that the T-
oligo mimics the
exposure of the 3' overhang sequence in the absence of telomere loop
disruption, i.e., in the
absence of DNA damage.
Example 13
PARP activity is required for T-oligo responses
[0124] To investigate the role of PARPs in responses to T-oligo, fibroblasts
were pretreated with
one of two different PARP inhibitors, 3-aminobenzamide (3AB, 2.5 mM) or 1,5-
dihydroxyquinoline (IQ, 100 p,M) for 2 hours before addition of 40 pM T-oligo
or an equal
amount of diluent as a control. An additional dose of each inhibitor was given
to the cells 4 hours
after addition of the T-oligo or diluent (D). Fibroblasts were treated with
3AB and T-oligo,.then
collected 48 hours later for western blot. T-oligo-induced upregulation of
total p53, p21,
phosphorylation of p53 serine 15 (indicating p53 activation) were all reduced
in the presence of
3AB (Figure 17A).
[0125) Fibroblasts pretreated with IQ similarly showed reduced induction of
total p53 and p53
phosphorylated on serine 1 S at 16, 20 and 24 hours after addition of T-oligo
(data not shown).
The effect of IQ on blocking T-oligo-mediated inductions of total p53, p53
phosphoserine 15,
and p21 persisted through 48 hours after addition of T-oligo. These data
demonstrate that the p53
responses to T-oligo require upstream PARP activity.
Example 14
PARP Inhibitors Prevent P53 Activation And Induction By T1ZF2Drr
(0126] Neonatal fibroblasts were treated with AdTRF2DN or AdGFP as a negative
control. Two
hours before infection, cells were treated with either diluent 3AB (2.5 mM) or
IQ (100 ~M).
After 3 days cells were collected for western analysis for the c-myc-tagged
TltF2DN (to confirm
infection), p53 serine 15 phosphorylation and p21 induction. Comparing lane 2
to lanes 4 and 6
of Figure 17F indicates that both 3AB and IQ reduced p53 phosphorylation and
p21 induction in
response to T1ZF2Drr.
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Example 15
Effects of T-Oligos are not Dependent on Telomerase
[0127] Saos-2 cells are an osteosarcoma cell line that is reportedly
telomerase negative and
maintain telomeres by the ALT pathway. Saos-2 cell lines were treated with
either diluent or 40
pM of the indicated oligonucleotide and cells were collected after 48 hours
for FACS analysis.
Only the homologous nucleotide causes an S phase arrest of the cells (Figure
18a). Furthermore, .
the telomere overhang oligonucleotide, as well as by IR, induced
phosphorylation of p95/Nbsl
(Figure 18b). The results that the effect of the T-oligo in the telomerase
negative cells is
identical to the response in telomerase positive malignant cell lines.
[0128]
Example 16
Downregulating PARP Tankyrase Protein Levels Blocks Response of T-Oligos
[0129] Paired cultures of human fibroblasts were treated once with tankyrase
siRNA, with a non-
specific siRNA (control) or were mock transfected as a second control. Two
days later, when the
tankyrase levels in tankyrase siRNA-treated cells was markedly reduced, the
cultures were
supplemented with 11-mer-1 (pGTTAGGGTTAG; SEQ lD NO: 2) or the complementary
sequence 11-mer-2. After an additional 24 hours, cells were collected and
processed for western
blotting using an antibody specific for p95 phosphorylated at serine 343,
indicating p95
modification by activated ATM kinase. The film was then subjected to
densitometry and the
diluent control for each group of cells was set at 1.0 in arbitrary units
(Figure 19). As expected,
in cells with normal tankyrase levels the T-oligo treated cells had twice the
amount of
phosphorylated p95, while the control ohgo-treated or diluent-treated cells
had only a 30-40%
increase. However, in the tankyrase knockdown group, the 11-mer-1 treated
cells showed no
increase in p95 phosphorylation (a level of 1.1 versus 1.0 and 1.3 for the
controls). These data
indicate that tankyrase, the telomere-associated PARP, is necessary to
transduce the T-oligo
signal that leads to ATM activation and subsequent modification
(phosphorylation) of p95,
thereby causing S-phase arrest of treated cells (Eller et al., FASEB J 2003).
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Example 17
T-oligo Causes Non-ATM-Mediated Phosphorylation of p53
[0130] Normal neonatal fibroblasts were treated with either diluent or 40 pM
(1 lmer-1) for 4, 6,
8, 19, 24 and 48 hours and then collected for Western blot analysis using an
antibody specific for
p53 phosphoserine 37. Sham and IR-irradiated (10 Gy) fibroblasts were used
used as negative
and positive controls, respectively. Increased band intensity in the western
blot, corresponding
to p53 serine 37, is detected as early as 8 hours and is very prominent at 48
hours in T-oligo (T)-
treated vs diluent (D)-treated samples."
[0131] As shown above, T-oligo causes phosphorylation ofp53 on serine 15.
Phosphorylation
of p53 at serine 1 S is mediated by ATM. Figure 20 indicates that T-oligos
also cause
phosphorylation of p53 on serine 37. Phosphorylation of p53 at serine 37 is
mediated by either
the ATM-related (ATR) kinase or the DNA-PK kinase, but is not known to be
mediated by
ATM. Demonstration of p53 serine 37 is thus another marker of pathway
activation and one or
both of these kinases are downstream targets of Mrel 1 activation. Moreover,
many of the
therapeutic effects of activating the Mrel l pathway are UV-mimetic, and UV is
known to.
activate both ATR and DNA-PK but not ATM.
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SEQUENCE LISTING
<110> Trustees of Boston University
Gilchrest, Barbara A.
Eller, Mark
<120> MODULATION OF TELOMERE-INITIATED CELL SIGNALING
<130> 06225.0003.OOPC04
<150> PCT/US03/11393
<151> 2003-04-11
<160> 4
<170> PatentIn version 3.2
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<213> Homo Sapiens
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ttaggg 6
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1
