U.S. patent application number 10/842718 was filed with the patent office on 2005-05-05 for down-regulation of dna repair to enhance sensitivity to p53-mediated suppression.
Invention is credited to Gjerset, Ruth A..
Application Number | 20050095226 10/842718 |
Document ID | / |
Family ID | 24712371 |
Filed Date | 2005-05-05 |
United States Patent
Application |
20050095226 |
Kind Code |
A1 |
Gjerset, Ruth A. |
May 5, 2005 |
Down-regulation of DNA repair to enhance sensitivity to
p53-mediated suppression
Abstract
The present invention details methods for the treatment of
cancer. In particular, it concerns the induction of apoptosis in
cancer cells following treatment with inhibitors of DNA repair in
combination with p53 gene therapy. Treatment of glioblastoma and
breast tumor cells with inhibitors of DNA repair induced growth
suppression that was a result of p53-mediated apoptosis. Thus it
appears that inhibitors of DNA repair in combination with p53 gene
therapy is involved in restoration of p53-mediated apoptosis.
Inventors: |
Gjerset, Ruth A.; (San
Diego, CA) |
Correspondence
Address: |
PERKINS COIE LLP
POST OFFICE BOX 1208
SEATTLE
WA
98111-1208
US
|
Family ID: |
24712371 |
Appl. No.: |
10/842718 |
Filed: |
May 10, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10842718 |
May 10, 2004 |
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09556440 |
Apr 24, 2000 |
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09556440 |
Apr 24, 2000 |
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08675887 |
Jul 5, 1996 |
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6054467 |
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Current U.S.
Class: |
424/93.2 ;
435/456; 514/18.9; 514/19.4; 514/19.5; 514/19.8 |
Current CPC
Class: |
A61K 31/7056 20130101;
A61K 31/203 20130101; A61K 31/166 20130101; A61P 35/00 20180101;
A61K 31/7048 20130101 |
Class at
Publication: |
424/093.2 ;
435/456; 514/012 |
International
Class: |
A61K 048/00; A61K
038/17; C12N 015/861 |
Claims
What is claimed is:
1. A method for the induction of p53-mediated apoptosis in a cell
comprising the step of contacting a cell with at least one
inhibitory agent that inhibits DNA repair.
2. The method of claim 1, further comprising contacting said cell
with a first stimulatory agent that increases the level of a tumor
suppressor in said cell.
3. The method of claim 2, wherein said tumor suppressor is selected
from the group consisting of p53, p21 and MSH-2.
4. The method of claim 2, wherein said first stimulatory agent is
an expression construct that comprises a nucleic acid encoding a
tumor suppressor under the control of a promoter active in
eukaryotic cells.
5. The method of claim 4, wherein said tumor suppressor is p53.
6. The method of claim 5, wherein said expression construct is an
adenoviral expression construct.
7. The method of claim 6, wherein said adenoviral expression
construct lacks a portion of at least one gene essential to
adenoviral replication.
8. The method of claim 7, wherein the essential gene is E1.
9. The method of claim 4, wherein said promoter is a CMV
promoter.
10. The method of claim 1, wherein said inhibitory agent inhibits
the function of a protein selected from the group consisting of
c-jun, c-fos, poly-ADP ribose polymerase, DNA polymerase .beta.,
topoisomerase I, d-TMP synthase, hMTII-A, uracil DNA glycosylase,
alkyl-N-purine DNA glycosylase, DNA ligase IV, DNA ligase III,
Hap-1, Ref-1, poly-ADP ribose polymerase and DNA-dependent protein
kinase.
11. The method of claim 10, wherein said inhibitory agent is a
competitor of a gene product selected from the group consisting of
c-jun, c-fos, poly-ADP ribose polymerase, DNA polymerase .beta.,
topoisomerase I, d-TMP synthase, hMTII-A, uracil DNA glycosylase,
alkyl-N-purine DNA glycosylase, DNA ligase IV, DNA ligase III,
Hap-1, Ref-1, poly-ADP ribose polymerase and DNA-dependent protein
kinase.
12. The method of claim 10, wherein said inhibitory agent is an
antisense construct encoding at least a portion of a gene selected
from the group consisting of c-jun, c-fos, poly-ADP ribose
polymerase, DNA polymerase .beta., topoisomerase I, d-TMP synthase,
hMTII-A, uracil DNA glycosylase, alkyl-N-purine DNA glycosylase,
DNA ligase IV, DNA ligase III, Hap-1, Ref-1, poly-ADP ribose
polymerase and DNA-dependent protein kinase.
13. The method of claim 10, wherein said inhibitory agent is a
retinoid.
14. The method of claim 13, wherein said retinoid is the synthetic
retinoid SR11220.
15. The method of claim 10, wherein said inhibitory agent is
3-aminobenzamide.
16. The method of claim 1, further comprising the step of providing
a DNA-damaging agent.
17. The method of claim 16, wherein said DNA-damaging agent is
selected from the group consisting of cisplatin, carboplatin, VP16,
teniposide, daunorubicin, doxorubicin, dactinomycin, mitomycin,
plicamycin, bleomycin, procarbazine, nitrosourea, cyclophosphamide,
bisulfan, melphalan, chlorambucil, ifosfamide, merchlorehtamine,
taxol, taxotere, anthracyclines and ionizing radiation.
18. The method of claim 1, wherein said cell is a tumor cell.
19. The method of claim 18, wherein said tumor cell is selected
from the group consisting of lung tumor cell, a prostate tumor
cell, a breast tumor cell, a colon tumor cell, a liver tumor cell,
a brain tumor cell, a kidney tumor cell, a skin tumor cell and an
ovarian tumor cell.
20. The method claim 18, wherein said tumor cell is selected from
the group consisting of a squamous cell carcinoma, a non-squamous
cell carcinoma, a glioblastoma, a sarcoma, a melanoma, a papilloma,
a neuroblastoma and a leukemia cell.
21. The method of claim 1, wherein said tumor cell is in a
subject.
22. The method of claim 1, wherein said subject is human.
23. The method of claim 1, wherein said inhibitory agent is
delivered by direct intratumoral injection.
24. The method of claim 2, wherein said stimulatory agent is
delivered by direct intratumoral injection.
25. The method of claim 23, wherein said injection comprises
continuous perfusion.
26. The method of claim 24, wherein said injection comprises
continuous perfusion.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates generally to the field of
cancer therapy. More particularly, it concerns a method of inducing
p53-mediated apoptosis in tumor cells by inhibiting DNA repair.
[0003] 2. Description of Related Art
[0004] Normal tissue homeostasis is achieved by an intricate
balance between the rate of cell proliferation and the rate of cell
death. Disruption of this balance is thought to be a major event in
the development of cancer. The inhibition of apoptosis, or
programmed cell death, has been linked to this disruptive event.
The effects of cancer are catastrophic, causing over half a million
deaths per year in the United States alone.
[0005] Though conventional therapies are available, development of
resistance to such treatment is a major obstacle to treatment of
cancer. For example, glioblastoma multiforme is the most common
intracranial brain tumor and is particularly resistant to therapy,
and rapidly becomes more resistant as therapy continues.
Glioblastoma tumors are highly vascularized, infiltrate the brain
extensively and can attain a very large size. Glioblastomas are
unilaterally fatal and patients have a mean survival time of about
one year from the time of diagnosis.
[0006] Traditional treatment modalities for glioblastoma include
surgery, radiotherapy, and chemotherapy. However, glioblastomas
respond poorly to most chemotherapeutic agents, even though the
blood brain barrier is broken down as a consequence of the disease.
Certain chemotherapeutic agents such as cisplatin, carmustine,
procarbazine and 5-fluororacil are somewhat efficacious in the
treatment of glioblastoma but the tumors are never completely
eradicated by these methods. A major reason for the failure of
traditional treatment therapies of glioblastoma is the development
of resistance in subsets of tumor cells. One reason for this
resistance appears to be a result of genetic changes that accompany
disease progression, including loss of wild-type p53 function.
Mutations in p53 occur in over 50% of adult glioblastoma cases and
are associated more with disease progression.
[0007] The p53 gene is well recognized as a tumor suppressor gene
(Montenarh, 1992). There is now considerable evidence linking
mutations of p53 in the oncogenesis of many human cancers. There
are numerous reports demonstrating that the growth of colon,
glioblastoma, breast cancer, osteosarcoma and lung tumor cells can
be suppressed by the expression of wild-type p53. The introduction
of wild-type p53 into a wide variety of p53-mutated cells, using
viral delivery methods, has resulted in the expression of the
wild-type p53 transgene and a suppression of the malignant
phenotype. These observations demonstrate that a high level of
expression of wild-type p53 is a desirable course for the treatment
of oncogenic malignancy.
[0008] More recently, p53 has been shown to be a trigger of
apoptosis (Yonish-Rouach et al., 1991; Shaw et al., 1992; Lowe et
al., 1993; Lotem and Sachs, 1993; Clarke et al., 1993) which
suggests that the disruption of p53 in tumors has significant
consequences for cancer therapy. The desensitization of tumor cells
to the effects of traditional cancer therapies as a result of p53
mutation may aid in the progression of disease. In addition to p53
mutations, cancer therapies such as radiotherapy and chemotherapy
that induce DNA damage to a tumor cell contribute to the
development of resistance of tumors. Several studies suggest that
treatment of tumors with DNA damaging agents results in
up-regulated DNA damage repair mechanisms, which could account for
increased resistance to DNA damaging therapy. In normal cells, DNA
damage results in cell cycle arrest and induction of DNA repair
mechanisms, so as to prevent the transfer of damaged DNA to the
next generation of cells. Cells that sustain high levels of DNA
damage, such as tumor cells that exhibit high levels of karyotypic
instability, or cells that are treated with DNA damaging agents,
are induced to undergo apoptosis. This switch from either arrest
and DNA repair or apoptosis is mediated by p53. These effects,
among others, show that there remains a need for improved methods
of cancer therapy.
SUMMARY OF THE INVENTION
[0009] It is, therefore, a goal of the present invention to provide
improved methods for the treatment of cancer. More particularly, it
is a goal to provide methods for overcoming or limiting the
therapy-inhibiting effects of DNA repair in cancer cells. These
methods facilitate the function of the tumor suppressor, p53, in
the induction of apoptosis in cells sustain DNA damage.
[0010] There is provided, according to the present invention a
method for the induction of p53-mediated apoptosis in a cell
comprising the step of contacting a cell with at least one
inhibitory agent that inhibits DNA repair. This method may further
comprise contacting the cell with a first stimulatory agent that
increases the level of a tumor suppressor in said cell. The tumor
suppressor may be p53, p21 or MSH-2, and preferably is p53. The
stimulatory agent may be an expression construct that comprises a
tumor suppressor gene under the control of a promoter active in
eukaryotic cells. The expression construct may be an adenoviral
expression construct. Preferably, the said adenoviral expression
construct lacks a portion of at least one gene essential to
adenoviral replication, such as the El gene. A preferred promoter
is the CMV promoter.
[0011] Though any inhibitory agent of DNA repair may be used, the
method advantageously employs and said inhibitory agent that
inhibits the function of a protein selected from the group
consisting of c-jun, c-fos, poly-ADP ribose polymerase, DNA
polymerase .beta., topoisomerase I, d-TMP synthase, hMTII-A, uracil
DNA glycosylase, alkyl-N-purine DNA glycosylase, DNA ligase IV, DNA
ligase III, Hap-1, Ref-1, poly-ADP ribose polymerase and
DNA-dependent protein kinase.
[0012] In one embodiment, the inhibitory agent is a non-functional
version of an agent involved with DNA repair. For example, a mutant
jun protein that competitively inhibits c-jun may be employed. In
another embodiment, the inhibitory agent is an antisense construct
encoding at least a portion of a gene such as c-jun, c-fos,
poly-ADP ribose polymerase, DNA polymerase .beta., topoisomerase I,
d-TMP synthase, hMTII-A, uracil DNA glycosylase, alkyl-N-purine DNA
glycosylase, DNA ligase IV, DNA ligase III, Hap-1, Ref-1, poly-ADP
ribose polymerase and DNA-dependent protein kinase. In another
embodiment, the inhibitory agent is a retinoid, for example, the
synthetic retinoid SR11220. In yet another embodiment, the
inhibitory agent is 3-aminobenzamide.
[0013] The method may also comprise the step of providing a
DNA-damaging agent. Suitable DNA-damaging agents included
cisplatin, carboplatin, VP16, teniposide, daunorubicin,
doxorubicin, dactinomycin, mitomycin, plicamycin, bleomycin,
procarbazine, nitrosourea, cyclophosphamide, bisulfan, melphalan,
chlorambucil, ifosfamide, merchlorehtamine, taxol, taxotere,
anthracyclines and ionizing radiation.
[0014] Tumor cells such as a lung tumor cell, a prostate tumor
cell, a breast tumor cell, a colon tumor cell, a liver tumor cell,
a brain tumor cell, a kidney tumor cell, a skin tumor cell and an
ovarian tumor cell all are contemplated targets of the method.
These tumors may be a squamous cell carcinoma, a non-squamous cell
carcinoma, a glioblastoma, a sarcoma, a melanoma, a papilloma, a
neuroblastoma and a leukemia cell. The tumors may be treated ex
vivo or in a subject, such as a human subject.
[0015] Delivery of the inhibitory agent, the stimulatory agent
and/or the DNA damaging agent is advantageously via direct
intratumoral injection. In a more specific embodiment, the
injection comprises continuous perfusion of the tumor.
[0016] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0018] FIG. 1. Cell viability assay. Cell growth was measured after
7 days of untreated parental cells (T98G), parental cells
transfected with empty vector (T98GLHCX), and parental cells
transfected with a vector expressing mutant jun (T98G-mutant jun)
after infection with p53 adenovirus. Percentage viability is
relative to the same subclones infected with .beta.gal-
adenovirus.
[0019] FIG. 2. Cell viability assay. Cell growth was measured after
7 days of .beta.gal adenovirus or p53 adenovirus-infected T47D
breast cancer cells under different condition (left to right): no
treatment; in the presence of 10.sup.-8 M SR11220; following 1 hour
exposure to 10 .mu.M cisplatin; in the presence of 10.sup.-8 M
SR11220 and following a 1 hour exposure to cisplatin. Percentage
viability is relative to the same conditions in untreated cells
infected with .beta.gal- adenovirus.
[0020] FIG. 3A. DNA damage assay of cisplatin-treated T98G parental
cells. DNA damage after zero (closed circles) and six (open circle)
hours after a 1.5 hour cisplatin treatment was measured by PCR.TM.
of a 2.7 Kbp region of the HPRT gene and expressed as the
percentage of signal observed after PCR.TM. of a 150 base pair
region within the HPRT gene. Refer to Example I for methods and
materials.
[0021] FIG. 3B. DNA damage assay of cisplatin-treated T98G parental
cells in the presence (closed circles) or absence (open circles) of
10 mM 3-aminobenzamide. DNA damage was measured 6 hours after a 1.5
hour treatment with cisplatin. DNA damage was measured by PCR.TM.
of a 2.7 Kbp region of the HPRT gene and expressed as the
percentage of signal observed after PCR.TM. of a 150 base pair
internal control region. Refer to Example I for methods and
materials.
[0022] FIG. 3C. DNA damage assay of cisplatin treated T98G-mutant
jun cells. DNA damage after zero (closed circles) and six (open
circle) hours after a 1.5 hour cisplatin treatment was measured by
PCR.TM. of a 2.7 Kbp region of the HPRT gene and expressed as the
percentage of signal observed after PCR.TM. of a 150 base pair
internal control region. Refer to Example I for methods and
materials.
[0023] FIG. 4. Model of AP-1 involvement in DNA repair. Wild-type
fos and jun form a transcription factor complex AP-1, which upon
DNA damage is phosphorylated on jun at position 63 and 73.
Phosphorylated AP-1 is involved in the initiation of transcription
of DNA repair enzymes. Mutant jun has substitutions of alanine for
serine at positions 63 and 73, and thus forms an inactive AP-1
complex upon DNA damage, and cannot induce transcription of DNA
repair enzymes.
[0024] FIG. 5. Cell growth of 9L rat glioblastoma cells expressing
an endogenous mutant p53 was measured after infection with a human
wild-type p53 adenovirus (light bars) or .beta.gal adenovirus (dark
bars) and treated one day later for one hour with (right set of
bars) or without (left set of bars) 50 mM cisplatin. Percentage
viability was measured 7 days later. Refer to Example I for methods
and materials.
[0025] FIG. 6. Cell growth of 9L rat glioblastoma cells expressing
endogenous mutant p53 were stably modified with vector only (pCEP4,
light bars) or human wild-type p53 (pCEPp53 dark bars), and either
(sets of bars left to right): untreated; treated for 6 days with 5
mM 3-aminobenzamide (ABZ); or treated for 6 days with 10 mM
3-aminobenzamide (ABZ). Percentage viability was measure at 6 days.
Refer to Example I for methods and materials.
[0026] FIG. 7A. Cell growth of T47D breast cancer cells after
infection with .beta.-gal adenovirus (closed circles) or p53
adenovirus (open circles) and one hour treatment with various
concentrations of cisplatin two days after infection. Cell
viability was measured 7 days post-infection. Refer to Example I
for methods and materials.
[0027] FIG. 7B. Cell growth of T98G glioblastoma cells after
infection with .beta.-gal adenovirus (closed circles) or p53
adenovirus (open circles) and one hour treatment with various
concentrations of cisplatin two days after infection. Cell
viability was measured 7 days post-infection. Refer to Example I
for methods and materials.
[0028] FIG. 7C. Cell growth of T98G glioblastoma cells stably
modified with vector only (PLRNL, closed circles) or vector
expressing human wild-type p53 (pLp53RNL, open circles) and one
hour treatment with various concentrations of cisplatin. Cell
viability was measured 7 days post-treatment. Refer to Example I
for methods and materials.
[0029] FIG. 7D. Cell growth of 9L rat glioblastoma cells stably
modified with vector only (9LpCEP4, closed circles) or vector
expressing human wild-type p53 (9LpCEPp53, open circles) and one
hour treatment with various concentrations of cisplatin. Cell
viability was measured 7 days post-treatment. Refer to Example I
for methods and materials.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0030] Cancer accounts for the death of over half a million people
each year in the United States alone. The causes for cancer are
multifactorial, but it is known that aberrations in mechanisms
involved with controlled cell death (apoptosis) may result in
uncontrolled cell proliferation and, hence, contribute to many
cancers. Conventional cancer therapies such as radiotherapy and
chemotherapy that result in DNA damage are poorly effective or lose
their effectiveness over time in some cancers. A consequence of the
treatment of tumor cells with such DNA damaging agents is the
induction of DNA repair mechanisms. Up-regulation of DNA repair
mechanisms contributes to the increasing resistance of tumors to
sustained therapy.
[0031] The present invention provides a means of increasing levels
of apoptosis in tumor cells through inhibition of DNA damage repair
mechanisms. The present invention relies, in part, on the
observation that agents that inhibit the expression of DNA repair
enzymes sensitize tumor cells to p53-mediated apoptosis. This
finding can be employed in a number of ways. First, treatment of
tumors which express wild-type p53 with DNA repair inhibitory
agents can sensitize the tumors to p53-mediated apoptosis. Second,
the delivery of combination therapy, where p53 gene therapy is used
in combination with agents that inhibit DNA repair are used to
inhibit the DNA repair mechanisms, thereby inducing apoptosis. And
third, in a three-part combination therapy, one can deliver a p53
gene, DNA repair inhibitors and DNA damaging agents. Thus, for the
purposes of the this application, a "functional" p53 or p53 gene is
one that confers this apoptosis-inducing ability on cells. A
"wild-type" p53 will accomplish this function. The details of these
embodiments of the present invention, as well as others, are
described in more detail in the following sections.
A. Role of p53 in Assessing DNA Damage and Therapy
Sensitization
[0032] In normal cells, DNA damage spontaneously occurs in the form
of nucleotide additions, deletions, or substitutions. Upon damage
to DNA, the cell cycle is arrested and mechanisms are induced that
repair the damaged DNA before completion of the cell cycle and
passage of the DNA message to the next generation of cells. Normal
expression of wild-type p53 may play a role in determining the end
result of DNA damage. WAF1, one of the targets of p53 , encodes a
p21.sup.WAFI/CIP1 protein of 21,000 Dalton molecular weight, which
is an inhibitor of cyclin-dependent kinase 2 required for the
G1-to-S transition (E1-Deiry et al., 1993). Low levels of DNA
damage may result in p53-mediated induction of this cell cycle
inhibitor, which prevents the cell from progressing through the
cell cycle and passing on damaged DNA to the next generation.
However, DNA damage that is extensive and not easily repaired may
preferentially result in the induction of the apoptotic
pathway.
[0033] Karyotypic instability is a hallmark of cancer and is
particularly apparent in glioblastoma (Bigner et al., 1981; Bigner
et al., 1990). This instability manifested by an increased level of
chromosomal breakage and rearrangement, including translocations,
additions, deletions, amplifications, loss of heterozygosity and
aneuploidy. Tumor cells show elevated rates of spontaneous gene
amplification compared to normal cells, where gene amplification is
rarely detected (Tisty, 1990). Spontaneous deletions also occur
with increased frequency in tumor cells (Kaden et al., 1989). Loss
of p53 function, it particular its ability to monitor DNA
conditions, may favor the growth of karyotypically unstable cells
by removing a trigger for apoptosis that could eliminate cells with
unstable genomes (Lane, 1992). Thus, loss of p53 may contribute to
the marked aneuploidy and karyotypic instability observed in
tumors, for example, glioblastoma.
[0034] Genomic instability accompanied by loss of p53-mediated
apoptosis also can lead to cancer therapy resistance. Studies of
p53 null transgenic mice have shown that normal transgenic
hematopoetic cells (Lotem and Sachs, 1993), E1A-expressing
transgenic fibroblasts (Lowe et al., 1993) and transformed
transgenic fibroblasts (Lowe et al., 1994) were all more resistant
to apoptosis following treatment with a variety of anti-cancer
agents.
[0035] The data presented herein show that glioblastoma cells and
other tumor cells lacking functional p53 are significantly more
sensitive to the DNA damaging effect of cisplatin and radiation
following introduction of am exogenous, wild-type p53 gene. This
enhancement of cell death has been shown to be the result of
apoptosis. Expression of exogenous wild-type p53 in cells
containing an intact normal p53 does not affect the growth of these
cells. Since mutation of p53 is widespread in cancer, the use of
wild-type p53 as a therapy sensitizer may have far reaching
consequences in treating p53-negative associated cancers as well as
cancers that contain a functional p53 gene, including but not
limited to breast, lung, prostate, colon, liver, brain, skin,
ovarian, pancreatic, kidney, lymphoid and renal.
[0036] In some cells lacking a functional p53, introduction of
exogenous wild-type p53 may achieve only a slowing of growth or
reversible growth arrest. Transfer of wild-type p53 into T98G
glioblastoma cells slows the growth of the cells but does not
induce apoptosis. Similarly, GM47 glioblastoma cells which express
an inducible wild-type p53 undergo reversible growth arrest at high
levels of p53 induction. However, after de-induction, the cells
begin cell cycling (Mercer et al., 1990).
[0037] However, because wild-type p53 sensitizes tumor cells to DNA
damaging agents, long term expression may not be necessary in
certain combined therapies. Transient expression of p53, followed
by induction of DNA damage by chemotherapeutic agents of ionizing
radiation or other DNA damaging agents, could provide the
appropriate trigger to direct the cell into the irreversible
apoptotic pathway. Support for this concept has been seen in recent
studies involving transient expression of wild-type p53 delivered
by an adenovirus that was shown to increase drug sensitivity in
culture and induce partial sensitivity when transferred into tumor
in vivo (Fugiwara et al., 1994). This phenomenon may further be
exploited by use of inhibitors of DNA repair.
[0038] Tumor suppression and therapy sensitization through p53
appears to be selective for tumors cells (Baker et al., 1990),
possibly because p53 levels in normal cells are kept at low level
by rapid turnover (Rogel et al., 1985). In addition, the intrinsic
genomic instability of tumor cells, which would normally induce the
apoptotic pathway, may also be a significant factor in contributing
to the induction p53-mediated apoptosis in response to artificially
induced DNA damage, such as chemotherapy or radiation therapy. Thus
the apparent specificity of p53 for tumor cells would allow the
selective targeting of p53-specific suppression, which would be
less toxic than tumor cell suppression protocols that aim at a
general cell cycle block. While conventional therapies such as
chemotherapy and radiotherapy are by themselves non-specifically
toxic to normal dividing cells, targeting of tumors that have
unstable genomes with inhibitors of DNA repair mechanisms may
prevent tumor cells from repairing DNA damage caused by DNA
damaging agents, and thus proceed to the p53-mediated apoptotic
pathway.
[0039] It also is contemplated that other tumor suppressors, for
example p21 and MSH-2, will function in an analogous manner to p53
for the purpose of inducing apoptosis. Thus, reference to the use
of p53 implicitly includes reference to these and other tumor
suppressors to the extent that they are involved with apoptotic
mechanisms.
B. DNA Repair Mechanisms and Inhibition of DNA Repair
[0040] Treatment of tumor cells with DNA damaging agents results in
the induction of DNA repair mechanisms. The success or failure of
DNA repair may have a significant role in determining the
consequences of p53 expression in a cell subjected to DNA damage.
Tumor cells that fail to repair DNA damage, arising from either
intrinsic genomic instability or from external DNA damaging agents,
may be more susceptible to p53-mediated apoptosis. Several studies
suggest that up-regulated DNA repair occurs in cells that have
become resistant to DNA damaging agents such as cisplatin. This is
evidenced by the fact that expression of DNA repair enzymes,
including dTMP synthetase, DNA polymerase B, topoisomerase I, and
hMTII-A, is up-regulated. Some of these DNA repair enzymes are
subject to transcriptional transactivational by the transcription
factor, AP-1, which consists of subunits fos and jun.
[0041] Inhibition of DNA repair mechanisms results in the increased
sensitivity of tumor cells to apoptosis induced by DNA damaging
agents. For example, DNA repair in tumor cells is down-regulated by
a dominant-negative inhibitor of c-jun (mutant jun or m-jun), which
fails to be phosphorylated due to amino acid substitutions at two
critical phosphorylation sites that are associated with cellular
transformation. The m-jun competes with wild-type jun for binding
to c-fos so that when m-jun is bound to c-fos, it forms an inactive
AP-1 complex, which cannot carry out transactivation of DNA repair
genes (FIG. 4.). However, normal cellular activity of AP-1 is not
dependent on DNA damage-induced phosphorylation of jun, thus m-jun
does not interfere with normal cellular transcription involving
AP-1. Cells having the:mutant jun gene are more sensitive to
p53-induced apoptosis.
[0042] Other mechanisms of DNA repair inhibition include inhibitors
of other transcription factors, such as Sp1, E2F-1 and jun/ATF2
that may be involved with transcription of DNA damage repair
proteins. The included, but are not limited to, uracil DNA
glycosylase, alkyl-N-purine DNA glycosylase, DNA ligase IV, DNA
ligase III, Hap-1 (Ref-1), poly-ADP-ribose polymerase, dTMP
synthetase, DNA polymerase B, topoisomerase I, hMTII-A and
DNA-dependent protein kinase. Tissue-specific transcription factors
that are involved in DNA repair may also be targeted to provide
specific therapy for a particular type of cancer. In one
embodiment, nucleic acids encoding antisense inhibitory agents
relating to each of these targets, or any of the other
transcription factors and DNA repair enzymes, may be used to
inhibit the induction of DNA repair.
[0043] Organochemical compounds also may be employed to inhibit
repair of DNA repair. Such compounds include retinoids like
SR11220, and 3-aminobenzamide. Retinoids in general induce a block
in the cell cycle. SR11220 is a synthetic retinoid that is specific
for AP-1, and thus has less toxicity than other retinoids such as
retinoic acid, which has broad specificity (Fanjul et al., 1994;
incorporated herein by reference). Other retinoids which may be
used to inhibit DNA repair include, but are not limited to,
trans-retinoic acid, 9-cis retinoic acid, known to inhibit AP-1,
and the synthetic retinoids SR11105, SR11217, SR11238, SR11235,
SR11302, SR11220, SR11327, SR11228, SR11324 (Fanjul et al., 1994).
The use of retinoids with broad specificity, while, if administered
intravenously could be significantly toxic, may exhibit only local
toxicity it administered intratumorally or at the.
[0044] 3-aminobenzamide is an inhibitor of poly-ADP ribose
polymerase that inhibits the repair of both single- and
double-strand breaks induced by DNA damaging agents. It has been
shown that p53 is induced following irradiation of 3-aminobenzamide
treated cells (Lane, 1992). Compounds with similar specificity
could be used according to the present invention. Again, although
such compounds may exhibit marked toxicity if used systematically,
the regional or local delivery may obviate much of the toxicity.
For example, delivery of 3-aminobenzamide as a bolus injection into
the tumor mass or the tumor vasculature may result in induction of
apoptosis in tumor cells with only minor effects on the surrounding
normal tissues.
C. Assays for other Agents Capable of Inhibiting DNA Repair
Activity
[0045] In certain embodiments, the present invention concerns a
method for identifying compounds that will inhibit DNA repair
activity without affecting p53 function. It is contemplated that
this screening technique will prove useful in the general
identification of compounds that will induce an increase in
p53-mediated apoptosis in cancer cells.
[0046] Useful compounds may include fragments or parts of the
enzymes or factors listed above, including antisense
oligonucleotides corresponding to DNA repair-related enzymes.
Common antisense targets are regions involve with transcription
initiation, translation initiation and splicing. Alternatively,
compounds isolated from natural sources, such as animals, bacteria,
fungi, plant sources, including leaves and bark, and marine samples
may be assayed as potentially useful pharmaceutical agents. It will
be understood that the pharmaceutical agents to be screened also
could be derived or synthesized from chemical compositions or
man-made compounds.
[0047] A method for determining the ability of a candidate
substance to inhibit the DNA repair activity of cancer cells and to
concomitantly induce apoptosis in said cells, the method including
generally the steps of:
[0048] (a) providing a cell with wild-type p53 function;
[0049] (b) admixing a candidate substance and a DNA-damaging agent
with the cell;
[0050] (c) determining the condition of the cell; and
[0051] (d) comparing the condition of the cell with a genetically
similar cell treated with the DNA-damaging agent in the absence of
the candidate substance.
[0052] In a preferred embodiment, the cell is a cancer cell that
has heightened DNA repair mechanisms. Because of these mechanisms,
the cancer cell is resistant, to a certain extent, to p53-induced
apoptosis. In an alternative embodiment, the cell may be
p53-negative, but a p53 transgene may be provided in step (b) to
facilitate induction of apoptosis.
[0053] In one embodiment, the candidate screening assay relies on
the formation of lesions in DNA, such as those caused by DNA
damaging agents. These lesions block the progression of the Taq
polymerase used in PCR.TM. and thus decrease the yield of the
PCR.TM. product. It has been shown that the level of DNA damage
induced by cisplatin, for example, correlates closely with the
level of amplified PCR.TM. product obtained. Measurement of the
PCR.TM. signal obtained from amplification of a significantly large
region of DNA, for example 2.7 kB, will decrease in relation to the
signal from a small region of amplified DNA, for example 150 bp,
whose signal will not be affected due to its small size, after
treatment with DNA damaging agents.
[0054] Alternatively, it may be desirable simply to measure
inhibition of growth of cancer cells, for example, by measuring
growth according to the MTT assay. A significant inhibition in
growth is represented by decreases of at least about 30%-40% as
compared to uninhibited, and most preferably, of at least about
50%, with more. significant decreases also being possible. Growth
assays as measured by the MTT assay are well known in the art.
Assays may be conducted as described by Mosmann et al., (1983) and
Rubinstein et al. (1990) (incorporated herein by reference).
Therefore, if a candidate substance exhibited inhibition of growth
of cancer cells in this type of study, it would likely be a
suitable compound for use in the present invention.
[0055] Another method of measuring the effects of candidate
compounds will be the determination of apoptosis by TUNEL assay.
Terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end
labeling (TUNEL) assay measures the integrity of DNA and is
performed as described by Fujiwara et al., 1994. Briefly, the cells
are fixed and cytospun on the slide. Cells are incubated in TdT
buffer (30 mM Tris Hcl, pH 7.2; 140 mM cacodylate, 1 mM cobalt
chloride) and incubated with biotinylated dUTP (Boehringer
Mannheim, Indianapolis, Ind) and 100 U/ml TdT enzyme (Bethesda
Research Laboratory) for 1 h at 37.degree. C. The avidin-biotin
complex was detected using the Vectastain Elite kit (Vector
Laboratory, Burlingame, Calif.), by the
diaminobenzidine-H.sub.2O.sub.2 method.
[0056] Quantitative in vitro testing of the DNA repair inhibiting
agents is not a requirement of the invention as it is generally
envisioned that the agents will often be selected on the basis of
their known properties or by structural and/or functional
comparison to those agents already demonstrated to be effective.
Therefore, the effective amounts will often be those amounts
proposed to be safe for administration to animals in another
context.
D. p53 Mutations in Cancer
[0057] p53 currently is recognized as a tumor suppressor gene
(Montenarh, 1992). The p53 gene is a frequent target of mutational
inactivation in a wide variety of human tumors and is already
documented to be the most frequently-mutated gene in common human
cancers (Mercer, 1992). It is mutated in over 50% of human NSCLC
(Hollestein et al., 1991) and in a wide spectrum of other
tumors.
[0058] The p53 gene encodes a 393-amino-acid phosphoprotein that
can form complexes with host proteins such as large-T antigen and
E1B. The wild-type protein is found in normal tissues and cells,
but at concentrations which are minute by comparison with levels of
mutant protein in transformed cells or tumor tissue. Interestingly,
wild-type p53 appears to be important in regulating cell growth and
division. Overexpression of wild-type p53 has been shown in some
cases to be anti-proliferative in human tumor cell lines. Thus p53
can act as a negative regulator of cell growth (Weinberg, 1991) and
may directly suppress uncontrolled cell growth or indirectly
activate genes that suppress this growth. Thus, absence or
inactivation of wild-type p53 may contribute to transformation.
However, some studies indicate that the presence of mutant p53 may
be necessary for full expression of the transforming potential of
the gene.
[0059] Wild-type p53 is recognized as an important growth regulator
in many cell types. Mis-sense mutations are common for the p53 gene
and are essential for the transforming ability of the oncogene. A
single genetic change prompted by point mutations can create
carcinogenic p53. Unlike other oncogenes, however, p53 point
mutations are known to occur in at least 30 distinct codons, often
creating dominant alleles that produce shifts in cell phenotype
without a reduction to homozygosity. Additionally, some of these
negative alleles appear to be tolerated in the organism and passed
on in the germ line. Various mutant alleles appear to range from
minimally dysfimctional to strongly penetrant (Weinberg, 1991).
[0060] Casey and colleagues have reported that transfection of DNA
encoding wild-type p53 into two human breast cancer cell lines
restores growth suppression control in such cells (Casey et al.,
1991). A similar effect has also been demonstrated on transfection
of wild-type, but not mutant, p53 into human lung cancer cell lines
(Takahasi et al., 1992). p53 appears dominant over the mutant gene
and will select against proliferation when transfected into cells
with the mutant gene. Normal expression of the transfected p53 does
not affect the growth of cells with endogenous p53. Thus, such
constructs might be taken up by normal cells without adverse
effects.
E. Treatment of p53-Positive Cancers Using Agents that Inhibit DNA
Repair
[0061] According to the present invention, a patient with cancer
will be treated with various agents that permit p53 induced
apoptosis to occur. These agents include DNA repair inhibitory
agents and, in certain cases, DNA damaging agents. Because p53
function is necessary, it is desirable that the p53 status of the
tumor cells be determined. This may be accomplished using
conventional methods, examples of which are described below.
Optimally, patients will have adequate bone marrow function
(defined as peripheral absolute granulocyte count
of>2,000/mm.sup.3 and platelet count of 100,000/mm.sup.3),
adequate liver function (bilirubin.ltoreq.1.5 mg/dl) and adequate
renal function (creatinine<1.5 mg/dl).
[0062] The patient will be treated with a pharmaceutically
acceptable form of the DNA repair inhibitory agent. These agents
are described above. This administration could be in the form of,
for example, an intratumoral injection, or indeed any other method
of application that is routinely used and well know to one of skill
in the art. A more detailed discussion of formulations and routes
of administration is provided below.
[0063] 1. Determination of p53 Status of Cells.
[0064] A wide variety of detection methods can be employed in the
present invention to detect the p53 status of a cell. There are
numerous antibodies to the p53 protein, hence any assay that
utilizes antibodies for detection, for example, ELISAs, Western
Blotting, immunoassay techniques, etc. Alternatively, assays that
employ nucleotide probes may be used to identify the
presence/absence of p53, for example, Southern blotting, Northern
blotting or PCR.TM. techniques. All the above techniques are well
known to one of skill in the art and could be utilized in the
present invention without undue experimentation.
[0065] i. ELISAs, Immunoassay and Immunohistological Assay.
[0066] Immunoassays encompassed by the present invention include,
but are not limited to those described in U.S. Pat. No. 4,367,110
(double monoclonal antibody sandwich assay) and U.S. Pat. No.
4,452,901 (western blot). Other assays include immunoprecipitation
of labeled ligands and immunocytochemistry, both in vitro and in
vivo.
[0067] Immunoassays generally are binding assays. Certain preferred
immunoassays are the various types of enzyme linked immunosorbent
assays (ELISAs) and radioimmunoassays (RIA) known in the art.
Immunohistochemical detection using tissue sections is also
particularly useful.
[0068] In one exemplary ELISA, the anti-p53 antibodies are
immobilized on a selected surface, such as a well in a polystyrene
microtiter plate, dipstick or column support. Then, a test
composition suspected of containing the desired antigen, such as a
clinical sample, is added to the wells. After binding and washing
to remove non-specifically bound immune complexes, the bound
antigen may be detected. Detection is generally achieved by the
addition of another antibody, specific for the desired antigen,
that is linked to a detectable label. This type of ELISA is known
as a "sandwich ELISA". Detection also may be achieved by the
addition of a second antibody specific for the desired antigen,
followed by the addition of a third antibody that has binding
affinity for the second antibody, with the third antibody being
linked to a detectable label.
[0069] Variations on ELISA techniques are know to those of skill in
the art. In one such variation, the samples suspected of containing
the desired antigen are immobilized onto the well surface and then
contacted with the antibodies of the invention. After binding and
appropriate washing, the bound immune complexes are detected. Where
the initial antigen specific antibodies are linked to a detectable
label, the immune complexes may be detected directly. Again, the
immune complexes may be detected using a second antibody that has
binding affinity for the first antigen specific antibody, with the
second antibody being linked to a detectable label.
[0070] Competition ELISAs are also possible in which test samples
compete for binding with known amounts of labeled antigens or
antibodies. The amount of reactive species in the unknown sample is
determined by mixing the sample with the known labeled species
before or during incubation with coated wells. The presence of
reactive species in the sample acts to reduce the amount of labeled
species available for binding to the well and thus reduces the
ultimate signal.
[0071] Irrespective of the format employed, ELISAs have certain
features in common, such as coating, incubating or binding, washing
to remove non-specifically bound species, and detecting the bound
immune complexes. These are described as below.
[0072] Antigen or antibodies may also be linked to a solid support,
such as in the form of plate, beads, dipstick, membrane or column
matrix, and the sample to be analyzed applied to the immobilized
antigen or antibody. In coating a plate with either antigen or
antibody, one will generally incubate the wells of the plate with a
solution of the antigen or antibody, either overnight or for a
specified period. The wells of the plate will then be washed to
remove incompletely adsorbed material. Any remaining available
surfaces of the wells are then "coated" with a nonspecific protein
that is antigenically neutral with regard to the test antisera.
These include bovine serum albumin (BSA), casein and solutions of
milk powder. The coating allows for blocking of nonspecific
adsorption sites on the immobilizing surface and thus reduces the
background caused by nonspecific binding of antisera onto the
surface.
[0073] In ELISAs, it is more customary to use a secondary or
tertiary detection means rather than a direct procedure. Thus,
after binding of the antigen or antibody to the well, coating with
a non-reactive material to reduce background, and washing to remove
unbound material, the immobilizing surface is contacted with the
clinical or biological sample to be tested under conditions
effective to allow immune complex (antigen/antibody) formation.
Detection of the immune complex then requires a labeled secondary
binding ligand or antibody, or a secondary binding ligand or
antibody in conjunction with a labeled tertiary antibody or third
binding ligand.
[0074] "Under conditions effective to allow immune complex
(antigen/antibody) formation" means that the conditions preferably
include diluting the antigens and antibodies with solutions such as
BSA, bovine gamma globulin (BGG) and phosphate buffered saline
(PBS)/Tween. These added agents also tend to assist in the
reduction of nonspecific background.
[0075] The suitable conditions also mean that the incubation is at
a temperature and for a period of time sufficient to allow
effective binding. Incubation steps are typically from about 1 to 2
to 4 hours, at temperatures preferably on the order of 25.degree.
to 27.degree. C. or may be overnight at about 4.degree. C. or
so.
[0076] Following all incubation steps in an ELISA, the contacted
surface is washed so as to remove non-complexed material. Washing
often includes washing with a solution of PBS/Tween, or borate
buffer. Following the formation of specific immune complexes
between the test sample and the originally bound material, and
subsequent washing, the occurrence of even minute amounts of immune
complexes may be determined.
[0077] To provide a detecting means, the second or third antibody
will have an associated label to allow detection. Preferably, this
will be an enzyme that will generate color development upon
incubating with an appropriate chromogenic substrate. Thus, for
example, one will desire to contact and incubate the first or
second immune complex with a urease, glucose oxidase, alkaline
phosphatase or hydrogen peroxidase-conjugated antibody for a period
of time and under conditions that favor the development of further
immune complex formation, e.g., incubation for 2 hours at room
temperature in a PBS-containing solution such as PBS-Tween.
[0078] After incubation with the labeled antibody, and subsequent
to washing to remove unbound material, the amount of label is
quantified, e.g., by incubation with a chromogenic substrate such
as urea and bromocresol purple or
2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS] and
H.sub.2O.sub.2, in the case of peroxidase as the enzyme label.
Quantification is then achieved by measuring the degree of color
generation, e.g., using a visible spectra spectrophotometer.
[0079] Alternatively, the label may be a chemiluminescent one. The
use of such labels is described in U.S. Pat. Nos. 5,310,687,
5,238,808 and 5,221,605.
[0080] Assays for the p53 status of the cell may be performed
directly on biopsy samples. Methods for in vitro situ analysis are
well known and involve assessing binding of antigen-specific
antibodies to tissues, cells or cell extracts. These are
conventional techniques well within the grasp of those skilled in
the art. For example, the antibodies to p53 may be used in
conjunction with both fresh-frozen and formalin-fixed,
paraffin-embedded tissue blocks prepared for study by
immunohistochemistry (IHC). Each tissue block may consist of 50 mg
of residual "pulverized" tumor. The method of preparing tissue
blocks from these particulate specimens has been successfully used
in previous IHC studies of various prognostic factors, e.g., in
breast cancer, and is well known to those of skill in the art.
(Abbondanzo et al., 1990; Allred et al., 1990; Brown et al.,
1990)
[0081] Briefly, frozen-sections may be prepared by rehydrating 50
ng of frozen pulverized tumor at room temperature in PBS in small
plastic capsules; pelleting the particles by centrifugation;
resuspending them in a viscous embedding medium (OCT); inverting
the capsule and pelleting again by centrifugation; snap-freezing in
-70.degree. C. isopentane; cutting the plastic capsule and removing
the frozen cylinder of tissue; securing the tissue cylinder on a
cryostat microtome chuck; and cutting 25-50 serial sections
containing an average of about 500 remarkably intact tumor
cells.
[0082] Permanent-sections may be prepared by a similar method
involving rehydration of the 50 mg sample in a plastic microfuge
tube; pelleting; resuspending in 10% formalin for 4 hours fixation;
washing/pelleting; resuspending in warm 2.5% agar; pelleting;
cooling in ice water to harden the agar; removing the tissue/agar
block from the tube; infiltrating and embedding the block in
paraffin; and cutting up to 50 serial permanent sections.
[0083] ii. Southern and Northern Blotting Techniques
[0084] Southern and Northern blotting are commonly used techniques
in molecular biology and well within the grasp of one skilled in
the art.
[0085] The DNA and RNA from test cells is recovered by gentle cell
rupture in the presence of a cation chelator such as EDTA. The
proteins and other cell milieu are removed by admixing with
saturated phenol or phenol/chloroform and centrifugation of the
emulsion. The DNA and RNA is in the upper aqueous phase, it is
deproteinised and mixed with ethanol. This solution allows the DNA
and RNA to precipitate, the DNA and RNA can then be recover using
centrifugation. In the case of RNA extraction, RNAse inhibitors
such as DEPC are needed to prevent RNA degradation.
[0086] Electrophoresis in agarose or polyacrylamide gels is the
most usual way to separate DNA and RNA molecules. Southern blotting
will confirm the identity of the p53 encoding DNA. This is achieved
by transferring the DNA from the intact gel onto nitrocellulose
paper. The nitrocellulose paper is then washed in buffer that has
for example, a radiolabelled cDNA containing a sequence
complementary to wild-type-P53 DNA. The probe binds specifically to
the DNA that encodes a region of p53 and can be detected using
autoradiography by contacting the probed nitrocellulose paper with
photographic film. p53-encoding mRNA can be detected in a similar
manner by a process known as Northern blotting. For a more detailed
description of buffers gel preparation, electrophoresis condition
etc., the skilled artisan is referred to Sambrook, 1989.
[0087] iii. Polymerase Chain Reaction (PCR.TM.)
[0088] PCR.TM. is a powerful tool in modern analytical biology.
Short oligonucleotide sequences usually 15-35 bp in length are
designed, homologous to flanking regions either side of the p53
sequences to be amplified. The primers are added in excess to the
source DNA, in the presence of buffer, enzyme, and free
nucleotides. The source DNA is denatured at 95.degree. C. and then
cooled to 50-60.degree. C. to allow the primers to anneal. The
temperature is adjusted to the optimal temperature for the
polymerase for an extension phase. This cycle is repeated 25-40
times.
[0089] In particular the present invention uses PCR.TM. to detect
the p53 status of cells. Mutations in the p53 gene are first
detected with Single Strand Conformation Polymorphism (SSCP) which
is based on the electrophoretic determination of conformational
changes in single stranded DNA molecules induced by point mutations
or other forms of slight nucleotide changes. To identify where the
mutation is located at within the p53 gene, each exon is separately
amplified by PCR.TM. using primers specific for the particular
exon. After amplification, the PCR.TM. product is denatured and
separated out on a polyacrylamide gel to detect a shift in mobility
due to a conformational change which resulted because of a point
mutation or other small nucleotide change in the gene. Mutations
result in a change in the physical conformation of the DNA as well
as change in the electrical charge of the molecule. Thus during
electrophoresis when an electrical charge is applied to the
molecule, DNA that is slightly different in shape and charge as
compared to wild-type will move at a different rate and thus occupy
a different position in the gel.
[0090] After determination of which DNA fragment contains the
mutation, the specific nucleotide changes are detected by DNA
sequencing of the amplified PCR.TM. product. Sequencing of linear
DNA breaks down the DNA molecule into its individual nucleotides in
the order with which they are assembled in the intact molecule.
Separation of the individual nucleotides by electrophoresis on a
sequencing gel allows detection of individual nucleotide changes
compared to wild-type and is used to determine homo- or
heterozygocity of a mutation, which is easily distinguished by the
appearance of a single or double band in the sequencing gel.
[0091] 2. Pharmaceutical Compositions and Routes of
Administration
[0092] Aqueous compositions of the present invention will have an
effective amount of a p53 expression vector or p53 protein, along
with a compound that inhibits DNA repair. Such compositions will
generally be dissolved or dispersed in a pharmaceutically
acceptable carrier or aqueous medium.
[0093] The phrases "pharmaceutically or pharmacologically
acceptable" refer to molecular entities and compositions that do
not produce an adverse, allergic or other untoward reaction when
administered to an animal, or human, as appropriate. As used
herein, "pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents and the like. The
use of such media and agents for pharmaceutical active substances
is well known in the art. Except insofar as any conventional media
or agent is incompatible with the active ingredients, its use in
the therapeutic compositions is contemplated. Supplementary active
ingredients, such as other anti-cancer agents, can also be
incorporated into the compositions.
[0094] In addition to the compounds formulated for parenteral
administration, such as those for intravenous or intramuscular
injection, other pharmaceutically acceptable forms include, e.g.,
tablets or other solids for oral administration; time release
capsules; and any other form currently used, including cremes,
lotions, mouthwashes, inhalants and the like.
[0095] The active compounds of the present invention will often be
formulated for parenteral administration, e.g., formulated for
injection via the intravenous, intramuscular, sub-cutaneous, or
even intraperitoneal routes. The preparation of an aqueous
composition that contains a compound or compounds that increase the
expression of wild-type p53 will be known to those of skill in the
art in light of the present disclosure. Typically, such
compositions can be prepared as injectables, either as liquid
solutions or suspensions; solid forms suitable for using to prepare
solutions or suspensions upon the addition of a liquid prior to
injection can also be prepared; and the preparations can also be
emulsified.
[0096] Solutions of the active compounds as free base or
pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0097] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions; formulations including
sesame oil, peanut oil or aqueous propylene glycol; and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases the form must be sterile and
must be fluid to the extent that it may be easily injected. It also
should be stable under the conditions of manufacture and storage
and must be preserved against the contaminating action of
microorganisms, such as bacteria and fungi.
[0098] The active compounds may be formulated into a composition in
a neutral or salt form. Pharmaceutically acceptable salts, include
the acid addition salts (formed with the free amino groups of the
protein) and which are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as
acetic, oxalic, tartaric, mandelic, and the like. Salts formed with
the free carboxyl groups can also be derived from inorganic bases
such as, for example, sodium, potassium, ammonium, calcium, or
ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like.
[0099] The carrier also can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and vegetable oils. The proper
fluidity can be maintained, for example, by the use of a coating,
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. The
prevention of the action of microorganisms can be brought about by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminum
monostearate and gelatin.
[0100] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0101] In certain cases, the therapeutic formulations of the
invention also may be prepared in forms suitable for topical
administration, such as in cremes and lotions. These forms may be
used for treating skin-associated diseases, such as various
sarcomas.
[0102] Administration of therapeutic compositions according to the
present invention will be via any common route so long as the
target tissue is available via that route. This includes oral,
nasal, buccal, rectal, vaginal or topical. Topical administration
would be particularly advantageous for treatment of skin cancers,
to prevent chemotherapy-induced alopecia or other dermal
hyperproliferative disorder. Alternatively, administration will be
by orthotopic, intradermal subcutaneous, intramuscular,
intraperitoneal or intravenous injection. Such compositions would
normally be administered as pharmaceutically acceptable
compositions that include physiologically acceptable carriers,
buffers or other excipients. For treatment of conditions of the
lungs, aerosol delivery to the lung is contemplated. Volume of the
aerosol is between about 0.01 ml and 0.5 ml. Similarly, a preferred
method for treatment of colon-associated disease would be via
enema. Volume of the enema is between about 1 ml and 100 ml. Direct
intratumoral injection is the preferred mode, with continuous
intratumoral perfusion a more specific embodiment.
[0103] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal administration. In this connection, sterile aqueous
media which can be employed will be known to those of skill in the
art in light of the present disclosure. For example, one dosage
could be dissolved in 1 mL of isotonic NaCl solution and either
added to 1000 mL of hypodermoclysis fluid or injected at the
proposed site of infusion, (see for example, Remington's
Pharmaceutical Sciences, 1990). Some variation in dosage will
necessarily occur depending on the condition of the subject being
treated. The person responsible for administration will, in any
event, determine the appropriate dose for the individual
subject.
[0104] An effective amount of the therapeutic composition is
determined based on the intended goal. The term "unit dose" or
"dosage" refers to physically discrete units suitable for use in a
subject, each unit containing a predetermined-quantity of the
therapeutic composition calculated to produce the desired
responses, discussed above, in association with its administration,
i e., the appropriate route and treatment regimen. The quantity to
be administered, both according to number of treatments and unit
dose, depends on the protection desired.
[0105] Precise amounts of the therapeutic composition also depend
on the judgment of the practitioner and are peculiar to each
individual. Factors affecting dose include physical and clinical
state of the patient, the route of administration, the intended
goal of treatment (alleviation of symptoms versus cure) and the
potency, stability and toxicity of the particular therapeutic
substance. For the instant application, it is envisioned that the
amount of therapeutic composition comprising a unit dose will range
from about 5-30 mg of polynucleotide.
[0106] 3. Kits
[0107] All the essential materials and reagents required for
determining wild-type p53 in a sample or for inhibiting the DNA
repair mechanisms in tumor cells may be assembled together in a
kit. When the components of the kit are provided in one or more
liquid solutions, the liquid solution preferably is an aqueous
solution, with a sterile aqueous solution being particularly
preferred.
[0108] For the detection of wild-type p53, the kit may contain
materials for PCR.TM. analyses, such primers, buffers and
appropriate solvents. Alternatively, if the detection is via
immunologic means, the kit may contain antibodies directed to the
p53, secondary antibodies that binding primary antibodies, labels
or signal generating compounds (either conjugated or unconjugated)
and various reagents for the generation and detection of
signals.
[0109] For in vivo use, an inhibitor of DNA repair, in combination
with an p53 expression vector may be formulated into a single or
separate pharmaceutically acceptable syringeable composition. In
this case, the container means may itself be an inhalant, syringe,
pipette, eye dropper, or other such like apparatus, from which the
formulation may be applied to an infected area of the body, such as
the lungs, injected into an animal, or even applied to and mixed
with the other components of the kit.
[0110] The components of these kits may also be provided in dried
or lyophilized forms. When reagents or components are provided as a
dried form, reconstitution generally is by the addition of a
suitable solvent. It is envisioned that the solvent also may be
provided in another container means. The kits of the invention may
also include an instruction sheet defining administration of the
inhibitor of DNA repair and/or the p53 status determining agents,
for explaining the assays for determining p53 levels in
samples.
[0111] The kits of the present invention also will typically
include a means for containing the vials in close confinement for
commercial sale such as, e.g., injection or blow-molded plastic
containers into which the desired vials are retained. Irrespective
of the number or type of containers, the kits of the invention also
may comprise, or be packaged with, an instrument for assisting with
the injection/administration or placement of the ultimate complex
composition within the body of an animal. Such an instrument may be
an inhalant, syringe, pipette, forceps, measured spoon, eye dropper
or any such medically approved delivery vehicle. Other
instrumentation includes devices that permit the reading or
monitoring of reactions in vitro.
F. Treatment of p53-Negative Cancers: Combining Agents that Inhibit
DNA Repair and Gene Therapy
[0112] In a separate embodiment of the present invention, it is
envisioned that inhibitors of DNA repair will be used in
combination with gene therapy in the treatment of those cancers
that do not express a functional p53. It is clear that delivery of
wild-type p53 into tumors that express a mutated p53 gene can
overcome the deleterious effects of the p53 mutation. In the
present embodiment of the invention, an inhibitor of DNA repair can
be administered concurrently with the gene therapy, before the gene
therapy or after the gene therapy. The components need for gene
therapy, as well as the therapeutic inhibitors of DNA repair, can
be assembled in a kit form as described above. The inhibitors of
DNA repair also have been described above, so the remaining
discussion relates to the elements relating to gene delivery.
[0113] 1. Expression Vectors
[0114] Throughout this application, the term "expression construct"
is meant to include any type of genetic construct containing a
nucleic acid coding for a p53 product. In order for the construct
to effect expression, the polynucleotide encoding the p53
polynucleotide will be under the transcriptional control of a
promoter. A "promoter" refers to a DNA sequence recognized by the
synthetic machinery of the host cell, or introduced synthetic
machinery, that is required to initiate the specific transcription
of a gene. The phrase "under transcriptional control" means that
the promoter is in the correct location in relation to the
polynucleotide to control RNA polymerase initiation and expression
of the polynucleotide.
[0115] The term promoter will be used herein to refer to a group of
transcriptional control modules that are clustered around the
initiation site for RNA polymerase II. Much of the thinking about
how promoters are organized derives from analyses of several viral
promoters, including those for the HSV thymidine kinase (tk) and
SV40 early transcription units. These studies, augmented by more
recent work, have shown that promoters are composed of discrete
functional modules, each consisting of approximately 7-20 bp of
DNA, and containing one or more recognition sites for
transcriptional activator or repressor proteins.
[0116] At least one module in each promoter functions to position
the start site for RNA synthesis. The best known example of this is
the TATA box, but in some promoters lacking a TATA box, such as the
promoter for the mammalian terminal deoxynucleotidyl transferase
gene and the promoter for the SV40 late genes, a discrete element
overlying the start site itself helps to fix the place of
initiation.
[0117] Additional promoter elements regulate the frequency of
transcriptional initiation. Typically, these are located in the
region 30-110 bp upstream of the start site, although a number of
promoters have recently been shown to contain functional elements
downstream of the start site as well. The spacing between promoter
elements frequently is flexible, so that promoter function is
preserved when elements are inverted or moved relative to one
another. In the tk promoter, the spacing between promoter elements
can be increased to 50 bp apart before activity begins to decline.
Depending on the promoter, it appears that individual elements can
function either cooperatively or independently to activate
transcription.
[0118] The particular promoter that is employed to control the
expression of a p53 polynucleotide is not believed to be critical,
so long as it is capable of expressing the polynucleotide in the
targeted cell. Thus, where a human cell is targeted, it is
preferable to position the polynucleotide coding region adjacent to
and under the control of a promoter that is capable of being
expressed in a human cell. Generally speaking, such a promoter
might include either a human or viral promoter.
[0119] In various embodiments, the human cytomegalovirus (CMV)
immediate early gene promoter, the SV40 early promoter and the Rous
sarcoma virus long terminal repeat can be used to obtain high-level
expression of the p53 polynucleotide. The use of other viral or
mammalian cellular or bacterial phage promoters which are
well-known in the art to achieve expression of polynucleotides is
contemplated as well, provided that the levels of expression are
sufficient to produce a growth inhibitory effect.
[0120] By employing a promoter with well-known properties, the
level and pattern of expression of a polynucleotide following
transfection can be optimized. For example, selection of a promoter
which is active in specific cells, such as tyrosinase (melanoma),
alpha-fetoprotein and albumin (liver tumors), CC10 (lung tumor) and
prostate-specific antigen (prostate tumor) will permit
tissue-specific expression of p53. polynucleotides. Table 1 lists
several promoters which may be employed, in the context of the
present invention, to regulate the expression of p53 constructs.
This list is not intended to be exhaustive of all the possible
elements involved in the promotion of p53 expression but, merely,
to be exemplary thereof.
[0121] Enhancers were originally detected as genetic elements that
increased transcription from a promoter located at a distant
position on the same molecule of DNA. This ability to act over a
large distance had little precedent in classic studies of
prokaryotic transcriptional regulation. Subsequent work showed that
regions of DNA with enhancer activity are organized much like
promoters. That is, they are composed of many individual elements,
each of which binds to one or more transcriptional proteins.
[0122] The basic distinction between enhancers and promoters is
operational. An enhancer region as a whole must be able to
stimulate transcription at a distance; this need not be true of a
promoter region or its component elements. On the other hand, a
promoter must have one or more elements that direct initiation of
RNA synthesis at a particular site and in a particular orientation,
whereas enhancers lack these specificities. Promoters and enhancers
are often overlapping and contiguous, often seeming to have a very
similar modular organization.
[0123] Additionally any promoter/enhancer combination (as per the
Eukaryotic Promoter Data Base EPDB) could also be used to drive
expression of a p53 construct. Use of a T3, T7 or SP6 cytoplasmic
expression system is another possible embodiment. Eukaryotic cells
can support cytoplasmic transcription from certain bacteriophage
promoters if the appropriate bacteriophage polymerase is provided,
either as part of the delivery complex or as an additional genetic
expression vector.
1TABLE 1 PROMOTER Immunoglobulin Heavy Chain Immunoglobulin Light
Chain T-Cell Receptor HLA DQ .alpha. and DQ .beta.
.beta.-Interferon Interleukin-2 Interleukin-2 Receptor MHC Class II
5 MHC Class II HLA-DR.alpha. .beta.-Actin Muscle Creatine Kinase
Prealbumin (Transthyretin) Elastase I Metallothionein Collagenase
Albumin Gene .alpha.-Fetoprotein .tau.-Globin .beta.-Globin c-fos
c-HA-ras Insulin Neural Cell Adhesion Molecule (NCAM)
.alpha..sub.1-Antitrypsin H2B (TH2B) Histone Mouse or Type I
Collagen Glucose-Regulated Proteins (GRP94 and GRP78) Rat Growth
Hormone Human Serum Amyloid A (SAA) Troponin I (TN I)
Platelet-Derived Growth Factor Duchenne Muscular Dystrophy SV40
Polyoma Retroviruses Papilloma Virus Hepatitis B Virus Human
Immunodeficiency Virus Cytomegalovirus Gibbon Ape Leukemia
Virus
[0124] Further, selection of a promoter that is regulated in
response to specific physiologic signals can permit inducible
expression of the p53 construct. For example, with the
polynucleotide under the control of the human PAI-1 promoter,
expression is inducible by tumor necrosis factor. Table 2
illustrates several promoter/inducer combinations:
2 TABLE 2 Element Inducer MT II Phorbol Ester (TPA) Heavy metals
MMTV (mouse Glucocorticoids mammary tumor virus) .beta.-Interferon
poly(rI)X poly(rc) Adenovirus 5 E2 Ela c-jun Phorbol Ester (TPA),
H.sub.2O.sub.2 Collagenase Phorbol Ester (TPA) Stromelysin Phorbol
Ester (TPA), IL-1 SV40 Phorbol Ester (TPA) Murine MX Gene
Interferon, Newcastle Disease Virus GRP78 Gene A23187
.alpha.-2-Macroglobulin IL-6 Vimentin Serum MHC Class I Gene
Interferon H-2kB HSP70 Ela, SV40 Large T Antigen Proliferin Phorbol
Ester (TPA) Tumor Necrosis Factor FMA Thyroid Stimulating Thyroid
Hormone Hormone .alpha. Gene
[0125] In certain embodiments of the invention, the delivery of an
expression vector in a cell may be identified in vitro or in vivo
by including a marker in the expression vector. The marker would
result in an identifiable change to the transfected cell permitting
identification of expression. Usually, the inclusion of a drug
selection marker aids in cloning and in the selection of
transformants. Alternatively, enzymes such as herpes simplex virus
thymidine kinase (tk) (eukaryotic) or chloramphenicol
acetyltransferase (CAT) (prokaryotic) may be employed. Immunologic
markers also can be employed. The selectable marker employed is not
believed to be important, so long as it is capable of being
expressed along with the polynucleotide encoding p53. Further
examples of selectable markers are well known to one of skill in
the art.
[0126] One will typically include a polyadenylation signal to
effect proper polyadenylation of the transcript. The nature of the
polyadenylation signal is not believed to be crucial to the
successful practice of the invention, and any such sequence may be
employed. The inventor has employed the SV40 polyadenylation signal
in that it was convenient and known to function well in the target
cells employed. Also contemplated as an element of the expression
construct is a terminator. These elements can serve to enhance
message levels and to minimize read through from the construct into
other sequences.
[0127] In preferred embodiments of the present invention, the
expression construct comprises a virus or engineered construct
derived from a viral genome. The ability of certain viruses to
enter cells via receptor-mediated endocytosis and, in some cases,
integrate into the host cell chromosomes, have made them attractive
candidates for gene transfer in to mammalian cells. However,
because it has been demonstrated that direct uptake of naked DNA,
as well as receptor-mediated uptake of DNA complexes (discussed
below), expression vectors need not be viral but, instead, may be
any plasmid, cosmid or phage construct that is capable of
supporting expression of encoded genes in mammalian cells, such as
pUC or Bluescript.TM. plasmid series.
[0128] i. Retroviruses
[0129] The retroviruses are a group of single-stranded RNA viruses
characterized by an ability to convert their RNA to double-stranded
DNA in infected cells by a process of reverse-transcription
(Coffin, 1990). The resulting DNA then stably integrates into
cellular chromosomes as a provirus and directs synthesis of viral
proteins. The integration results in the retention of the viral
gene sequences in the recipient cell and its descendants. The
retroviral genome contains three genes--gag, pol, and env--that
code for capsid proteins, polymerase enzyme, and envelope
components, respectively. A sequence found upstream from the gag
gene, termed .PSI., functions as a signal for packaging of the
genome into virions. Two long terminal repeat (LTR) sequences are
present at the 5' and 3' ends of the viral genome. These contain
strong promoter and enhancer sequences and are also required for
integration in the host cell genome (Coffin, 1990).
[0130] In order to construct a retroviral vector, a nucleic acid
encoding a p53 is inserted into the viral genome in the place of
certain viral sequences to produce a virus that is
replication-defective. In order to produce virions, a packaging
cell line containing the gag, pol and env genes but without the LTR
and .PSI. components is constructed (Mann et al., 1983). When a
recombinant plasmid containing a human cDNA, together with the
retroviral LTR and .PSI. sequences is introduced into this cell
line (by calcium phosphate precipitation for example), the .PSI.
sequence allows the RNA transcript of the recombinant plasmid to be
packaged into viral particles, which are then secreted into the
culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et
al., 1983). The media containing the recombinant retroviruses is
then collected, optionally concentrated, and used for gene
transfer. Retroviral vectors are able to infect a broad variety of
cell types. However, integration and stable expression require the
division of host cells (Paskind et al., 1975).
[0131] A novel approach designed to allow specific targeting of
retrovirus vectors was recently developed based on the chemical
modification of a retrovirus by the chemical addition of lactose
residues to the viral envelope. This modification could permit the
specific infection of hepatocytes via sialoglycoprotein
receptors.
[0132] A different approach to targeting of recombinant
retroviruses was designed in which biotinylated antibodies against
a retroviral envelope protein and against a specific cell receptor
were used. The antibodies were coupled via the biotin components by
using streptavidin (Roux et al., 1989). Using antibodies against
major histocompatibility complex class I and class II antigens,
they demonstrated the infection of a variety of human cells that
bore those surface antigens with an ecotropic virus in vitro (Roux
et al., 1989).
[0133] ii. Adenoviruses
[0134] Human adenoviruses are double-stranded DNA tumor viruses
with genome sizes of approximate 36 kb (Tooze, 1981). As a model
system for eukaryotic gene expression, adenoviruses have been
widely studied and well characterized, which makes them an
attractive system for development of adenovirus as a gene transfer
system. This group of viruses is relatively simple to grow and
manipulate, and exhibit a broad host range in vitro and in vivo. In
lytically infected cells, adenoviruses are capable of shutting off
host protein synthesis, directing cellular machineries to
synthesize large quantities of viral proteins, and producing
copious amounts of virus.
[0135] The E1 region of the genome includes E1A and E1B which
encode proteins responsible for transcription regulation of the
viral genome, as well as a few cellular genes. E2 expression,
including E2A and E2B, allows synthesis of viral replicative
functions, e.g. DNA-binding protein, DNA polymerase, and a terminal
protein that primes replication. E3 gene products prevent cytolysis
by cytotoxic T cells and tumor necrosis factor and appear to be
important for viral propagation. Functions associated with the E4
proteins include DNA replication, late gene expression, and host
cell shutoff. The late gene products include most of the virion
capsid proteins, and these are expressed only after most of the
processing of a single primary transcript from the major late
promoter has occurred. The major late promoter (MLP) exhibits high
efficiency during the late phase of the infection
(Stratford-Perricaudet and Perricaudet, 1991).
[0136] As only a small portion of the viral genome appears to be
required in cis (Tooza, 1981), adenovirus-derived vectors offer
excellent potential for the substitution of large DNA fragments
when used in connection with cell lines such as 293 cells.
Ad5-transformed human embryonic kidney cell lines (Graham, et a.,
1977) have been developed to provide the essential viral proteins
in trans. The inventor thus reasoned that the characteristics of
adenoviruses rendered them good candidates for use in targeting
cancer cells in vivo (Grunhaus and Horwitz, 1992).
[0137] Particular advantages of an adenovirus system for delivering
foreign proteins to a cell include (i) the ability to substitute
relatively large pieces of viral DNA by foreign DNA; (ii) the
structural stability of recombinant adenoviruses; (iii) the safety
of adenoviral administration to humans; and (iv) lack of any known
association of adenoviral infection with cancer or malignancies;
(v) the ability to obtain high titers of the recombinant virus; and
(vi) the high infectivity of adenovirus.
[0138] Further advantages of adenovirus vectors over retroviruses
include the higher levels of gene expression. Additionally,
adenovirus replication is independent of host gene replication,
unlike retroviral sequences. Because adenovirus transforming genes
in the El region can be readily deleted and still provide efficient
expression vectors, oncogenic risk from adenovirus vectors is
thought to be negligible (Grunhaus and Horwitz, 1992).
[0139] In general, adenovirus gene transfer systems are based upon
recombinant, engineered adenovirus which is rendered
replication-incompetent by deletion of a portion of its genome,
such as E1, and yet still retains its competency for infection.
Sequences encoding relatively large foreign proteins can be
expressed when additional deletions are made in the adenovirus
genome. For example, adenoviruses deleted in both E1 and E3 regions
are capable of carrying up to 10 Kb of foreign DNA and can be grown
to high titers in 293 cells (Stratford-Perricaudet and Perricaudet,
1991). Surprisingly persistent expression of transgenes following
adenoviral infection has also been reported.
[0140] Adenovirus-mediated gene transfer has recently been
investigated as a means of mediating gene transfer into eukaryotic
cells and into whole animals. For example, in treating mice with
the rare recessive genetic disorder ornithine transcarbamylase
(OTC) deficiency, it was found that adenoviral constructs could be
employed to supply the normal OTC enzyme. Unfortunately, the
expression of normal levels of OTC was only achieved in 4 out of 17
instances (Stratford-Perricaudet et al., 1990). Therefore, the
defect was only partially corrected in most of the mice and led to
no physiological or phenotypic change. These type of results
therefore offer little encouragement for the use of adenoviral
vectors in cancer therapy.
[0141] Attempts to use adenovirus to transfer the gene for cystic
fibrosis transmembrane conductance regulator (CFTR) into the
pulmonary epithelium of cotton rats have also been partially
successful, although it has not been possible to assess the
biological activity of the transferred gene in the epithelium of
the animals (Rosenfeld et al., 1992). Again, these studies
demonstrated gene transfer and expression of the CFTR protein in
lung airway cells but showed no physiologic effect. In the 1991
Science article, Rosenfeld et al. showed lung expression of
al-antitrypsin protein but again showed no physiologic effect. In
fact, they estimated that the levels of expression that they
observed were only about 2% of the level required for protection of
the lung in humans, i.e., far below that necessary for a
physiologic effect.
[0142] The gene for human al -antitrypsin has been introduced into
the liver of normal rats by intraportal injection, where it was
expressed and resulted in the secretion of the introduced human
protein into the plasma of these rats (Jaffe et al., 1992).
However, and disappointingly, the levels that were obtained were
not high enough to be of therapeutic value.
[0143] These type of results do not demonstrate that adenovirus is
able to direct the expression of sufficient protein in recombinant
cells to achieve a physiologically relevant effect, and they do
not, therefore, suggest a usefulness of the adenovirus system for
use in connection with cancer therapy. Furthermore, prior to the
present invention, it was thought that p53 could not be
incorporated into a packaging cell, such as those used to prepare
adenovirus, as it would be toxic. As E1B of adenovirus binds to
p53, this was thought to be a further reason why adenovirus and p53
technology could not be combined.
[0144] iii. Other Viral Vectors as Expression Constructs
[0145] Other viral vectors may be employed as expression constructs
in the present invention. Vectors derived from viruses such as
vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar
et al., 1988) adeno-associated virus (AAV) (Ridgeway, 1988;
Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984) and herpes
viruses may be employed. These viruses offer several attractive
features for various mammalian cells (Friedmann, 1989; Ridgeway,
1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et
al., 1990).
[0146] With the recent recognition of defective hepatitis B
viruses, new insight was gained into the structure-function
relationship of different viral sequences. in vitro studies showed
that the virus could retain the ability for helper-dependent
packaging and reverse transcription despite the deletion of up to
80% of its genome (Horwich et al., 1990). This suggested that large
portions of the genome could be replaced with foreign genetic
material. The hepatotropism and persistence (integration) were
particularly attractive properties for liver-directed gene
transfer. Chang et al. (1991) recently introduced the
chloramphenicol acetyltransferase (CAT) gene into duck hepatitis B
virus genome in the place of the polymerase, surface, and
pre-surface coding sequences. It was cotransfected with wild-type
virus into an avian hepatoma cell line. Culture media containing
high titers of the recombinant virus were used to infect primary
duckling hepatocytes. Stable CAT gene expression was detected for
at least 24 days after transfection (Chang et al., 1991).
[0147] 2. Alternative Methods for Gene Delivery
[0148] In order to effect expression of p53 constructs, the
expression vector must be delivered into a cell. As described
above, the preferred mechanism for delivery is via viral infection
where the expression vector is encapsidated in an infectious
adenovirus particle.
[0149] Several non-viral methods for the transfer of expression
vectors into cultured mammalian cells also are contemplated by the
present invention. These include calcium phosphate precipitation
(Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al.,
1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et
al., 1986; Potter et al., 1984), direct microinjection (Harland and
Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982;
Fraley et al., 1979) and lipofectamine-DNA complexes, cell
sonication (Fechheimer et al., 1987), gene bombardment using high
velocity microprojectiles (Yang et al., 1990), polycations (Boussif
et al., 1995) and receptor-mediated transfection (Wu and Wu, 1987;
Wu and Wu, 1988). Some of these techniques may be successfully
adapted for in vivo or ex vivo use.
[0150] In one embodiment of the invention, the adenoviral
expression vector may simply consist of naked recombinant vector.
Transfer of the construct may be performed by any of the methods
mentioned above which physically or chemically permeabilize the
cell membrane. For example, Dubensky et al. (1984) successfully
injected polyomavirus DNA in the form of CaPO.sub.4 precipitates
into liver and spleen of adult and newborn mice demonstrating
active viral replication and acute infection. Benvenisty and Neshif
(1986) also demonstrated that direct intraperitoneal injection of
CaPO.sub.4 precipitated plasmids results in expression of the
transfected genes. It is envisioned that DNA encoding an p53
construct may also be transferred in a similar manner in vivo.
[0151] Another embodiment of the invention for transferring a naked
DNA expression vector into cells may involve particle bombardment.
This method depends on the ability to accelerate DNA coated
microprojectiles to a high velocity allowing them to pierce cell
membranes and enter cells without killing them (Klein et al.,
1987). Several devices for accelerating small particles have been
developed. One such device relies on a high voltage discharge to
generate an electrical current, which in turn provides the motive
force (Yang et al., 1990). The microprojectiles used have consisted
of biologically inert substances such as tungsten or gold
beads.
[0152] Selected organs including the liver, skin, and muscle tissue
of rats and mice have been bombarded in vivo (Yang et al., 1990;
Zelenin et al., 1991). This may require surgical exposure of the
tissue or cells, to eliminate any intervening tissue between the
gun and the target organ. DNA encoding a p53 construct may be
delivered via this method.
[0153] In a further embodiment of the invention, the expression
vector may be entrapped in a liposome. Liposomes are vesicular
structures characterized by a phospholipid bilayer membrane and an
inner aqueous medium. Multilamellar liposomes have multiple lipid
layers separated by aqueous medium. Liposomes form spontaneously
when phospholipids are suspended in an excess of aqueous solution.
The lipid components undergo self-rearrangement before the
formation of closed structures and entrap water and dissolved
solutes between the lipid bilayers (Ghosh and Bachhawat, 1991).
Also contemplated are lipofectamine-DNA complexes.
[0154] Liposome-mediated polynucleotide delivery and expression of
foreign DNA in vitro has been very successful. Wong et al. (1980)
demonstrated the feasibility of liposome-mediated delivery and
expression of foreign DNA in cultured chick embryo, HeLa and
hepatoma cells. Nicolau et al. (1987) accomplished successful
liposome-mediated gene transfer in rats after intravenous
injection.
[0155] In certain embodiments of the invention, the liposome may be
complexed with a hemagglutinating virus (HVJ). This has been shown
to facilitate fusion with the cell membrane and promote cell entry
of liposome-encapsulated DNA (Kaneda et al., 1989). In other
embodiments, the liposome may be complexed or employed in
conjunction with nuclear non-histone chromosomal proteins (HMG-1)
(Kato et al., 1991). In yet further embodiments, the liposome may
be complexed or employed in conjunction with both HVJ and HMG-1. In
that such expression vectors have been successfully employed in
transfer and expression of a polynucleotide in vitro and in vivo,
then they are applicable for the present invention. Where a
bacteriophage promoter is employed in the DNA construct, it also
will be desirable to include within the liposome an appropriate
bacteriophage polymerase.
[0156] Another mechanism for transferring expression vectors into
cells is receptor-mediated delivery. This approach takes advantage
of the selective uptake of macromolecules by receptor-mediated
endocytosis in almost all eukaryotic cells. Because of the cell
type-specific distribution of various receptors, the delivery can
be highly specific (Wu and Wu, 1993). Receptor-mediated gene
targeting vehicles generally consist of two components: a cell
receptor-specific ligand and a DNA-binding agent. Several ligands
have been used for receptor-mediated gene transfer. The most
extensively characterized ligands are asialoorosomucoid (AS OR) (Wu
and Wu, 1987) and transferrin (Wagner et al., 1993). Recently, a
synthetic neoglycoprotein, which recognizes the same receptor as
ASOR, has been used as a gene delivery vehicle (Ferkol et al.,
1993; Perales et al., 1994) and epidermal growth factor (EGF) has
also been used to deliver genes to squamous carcinoma cells (Myers,
EPO 0273085).
[0157] In other embodiments, the delivery vehicle ma y comprise a
ligand and a liposome. For example, Nicolau et al. (1987) employed
lactosyl-ceramide, a galactose-terminal asialganglioside,
incorporated into liposomes and observed an increase in the uptake
of the insulin gene by hepatocytes. Thus, it is feasible that an
adenoviral expression vector also may be specifically delivered
into a cell type such as lung, epithelial or tumor cells, by any
number of receptor-ligand systems, with or without liposomes. For
example, epidermal growth factor (EGF) may be used as the receptor
for mediated delivery of p53 construct in many tumor cells that
exhibit upregulation of EGF receptor. Mannose can be used to target
the mannose receptor on liver cells. Also, antibodies to CD5 (CLL),
CD22 (lymphoma), CD25 (T-cell leukernia) and MAA (melanoma) can
similarly be used as targeting moieties.
[0158] In certain embodiments, gene transfer may more easily be
performed under ex vivo conditions. Ex vivo gene therapy refers to
the isolation of cells from an animal, the delivery of a
polynucleotide into the cells, in vitro, and then the return of the
modified cells back into an animal. This may involve the surgical
removal of tissue/organs from an animal or the primary culture of
cells and tissues. Anderson et al., U.S. Pat. No. 5,399,346, and
incorporated herein in its entirety, disclose ex vivo therapeutic
methods. During ex vivo culture, the expression vector can express
the p53 construct. Finally, the cells may be reintroduced into the
original animal, or administered into a distinct animal, in a
pharmaceutically acceptable form by any of the means described
below.
G. Combination Therapy
[0159] Tumor cell resistance to DNA damaging agents represents a
major problem in clinical oncology. One goal of current cancer
research is to find ways to improve the efficacy of chemo- and
radiotherapy by combining it with gene therapy. For example, the
herpes simplex-thymidine kinase (HS-tK) gene, when delivered to
brain tumors by a retroviral vector system, successfully induced
susceptibility to the antiviral agent gancyclovir (Culver, et al.,
1992). In the context of the present invention, it is contemplated
that inhibitors of DNA repair mechanisms and p53 gene therapy could
be used similarly in conjunction with chemo- or radiotherapeutic
intervention.
[0160] To induce apoptosis in cells, such as malignant or
metastatic cells, using the methods and compositions of the present
invention, one would generally contact a "target" cell with an
inhibitor of DNA repair (and additionally with a wild-type p53
protein or expression vector containing wild-type p53 if the cell
is p53-negative), and at least one DNA damaging agent. These
compositions would be provided in a combined amount effective to
induce apoptosis; this may include elimination or diminution of the
related tumor burden, or it may simply inhibit proliferation of the
related tumor.
[0161] This process may involve contacting the cells with an
inhibitor of DNA repair (and optionally wild-type p53 expression
vector) and the DNA damaging agent(s) at the same time. This may be
achieved by contacting the cell with a single composition or
pharmacological formulation that includes both agents, or by
contacting the cell with two distinct compositions or formulations,
at the same time, wherein one composition includes the inhibitor of
DNA repair and a wild-type p53 protein or expression vector
containing wild-type p53, and the other includes the DNA damaging
agent.
[0162] Alternatively, the inhibitor of DNA repair and p53 gene
therapy treatment may precede or follow the DNA damaging agent
treatment by intervals ranging from minutes to weeks. In
embodiments where the DNA damaging factor, and the inhibitor of DNA
repair and p53 gene therapy are applied separately to the cell, one
would generally ensure that a significant period of time did not
expire between the time of each delivery, such that the DNA
damaging agent, and inhibitor of DNA repair and p53 gene therapy
would still be able to exert an advantageously combined effect on
the cell. In such instances, it is contemplated that one would
contact the cell with both agents within about 12-24 hours of each
other and, more preferably, within about 6-12 hours of each other,
with a delay time of only about 12 hours being most preferred. In
some situations, it may be desirable to extend the time period for
treatment significantly, however, where several days (2, 3, 4, 5, 6
or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the
respective administrations.
[0163] It also is conceivable that more than one administration of
either the inhibitor of DNA repair and p53 protein or expression
vector containing p53, or the DNA damaging agent will be desired.
Various combinations may be employed, where "A" is the inhibitor of
DNA repair (and optionally a wild-type p53-encoding expression
vector) and the DNA damaging agent is "B":
3 B/A/A A/B/B A/B/A A/A/B B/B/A B/A/B or: B/B/B/A B/B/A/B A/A/B/B
A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A
A/A/B/A A/B/B/B B/A/B/B
[0164] The terms "contacted" and "exposed", when applied to a cell,
are used herein to describe the process by which a protein or
compound, such as an inhibitor of DNA repair, expression vector and
a DNA damaging agent or factor are delivered to a target cell by
placement in direct juxtaposition with the target cell. To induce
apoptosis, both agents are delivered to a cell in a combined amount
effective to induce apoptosis of the cell.
[0165] DNA damaging agents or factors are defined herein as any
chemical compound or treatment method that induces DNA damage when
applied to a cell. Such agents and factors include radiation and
waves that induce DNA damage such as, .gamma.-irradiation, X-rays,
UV-irradiation, microwaves, electronic emissions, and the like. A
variety of chemical compounds, also described as "chemotherapeutic
agents", function to induce DNA damage, all of which are intended
to be of use in the combined treatment methods disclosed herein.
Chemotherapeutic agents contemplated to be of use, include, e.g.,
adriamycin, 5-fluorouracil (5FU), etoposide (VP-16), camptothecin,
actinomycin-D, mitomycin C, cisplatin (CDDP), carboplatin,
bisulphan, melphalan, chlorambucil, ifosfamide, merchlorehtamine,
nitrosourea, tenoposide, daunorubicin, doxorubicin, dactinomycin,
plicamycin, anthracyclines, taxol, taxotere and even hydrogen
peroxide. The invention also encompasses the use of a combination
of one or more DNA damaging agents, whether radiation-based or
actual compounds, such as the use of X-rays with cisplatin or the
use of cisplatin with etoposide. In certain embodiments, the use of
cisplatin in combination with an inhibitor of DNA repair and a p53
protein or gene is particularly preferred as this compound.
[0166] Any method may be used to contact a cell with an inhibitor
of DNA repair, so long as the method results in inhibition of DNA
repair within the cell. This includes both the direct delivery of
an inhibitor of DNA repair protein to the cell and the delivery of
a gene or DNA segment that encodes the inhibitor of DNA repair,
which gene will direct the expression and production of the
inhibitor of DNA repair within the cell. In that protein delivery
is subject to such drawbacks as protein degradation and low
cellular uptake, it is contemplated that the use of a recombinant
vector that expresses a inhibitor of DNA repair protein will
provide particular advantages.
[0167] In treating cancer according to the invention, one would
contact the tumor cells with a DNA damaging agent in addition to
the inhibitor of DNA repair. This may be achieved by irradiating
the localized tumor site with DNA damaging radiation such as
X-rays, UV-light, y-rays or even microwaves. Alternatively, the
tumor cells may be contacted with the DNA damaging agent by
administering to the subject a therapeutically effective amount of
a pharmaceutical composition comprising a DNA damaging compound.
The DNA damaging agent may be prepared and used as a combined
therapeutic composition, or kit, by combining it with an inhibitor
of DNA repair and p53 expression vector, as described above.
[0168] Agents that directly cross-link nucleic acids, specifically
DNA, are envisaged and are shown herein, to eventuate DNA damage
leading to a synergistic anti-neoplastic combination. Agents such
as cisplatin, and other DNA alkylating may be used. Cisplatin has
been widely used to treat cancer, with efficacious doses used in
clinical applications of 20 mg/m.sup.2 for 5 days every three weeks
for a total of three courses. Cisplatin is not absorbed orally and
must therefore be delivered via injection intravenously,
subcutaneously, intratumorally or intraperitoneally.
[0169] Agents that damage DNA also include compounds that interfere
with DNA replication, mitosis, and chromosomal segregation. Such
chemotherapeutic compounds include adriamycin, also known as
doxorubicin, etoposide, verapamil, podophyllotoxin, and the like.
Widely used in clinical setting for the treatment of neoplasms,
these compounds are administered through bolus injections
intravenously at doses ranging from 25-75 mg/m.sup.2 at 21 day
intervals for adriamycin, to 35-50 mg/m.sup.2 for etoposide
intravenously or dquble the intravenous dose orally.
[0170] Agents that disrupt the synthesis and fidelity of nucleic
acid precursors, and subunits also lead to DNA damage. As such a
number of nucleic acid precursors have been developed. Particularly
useful are agents that have undergone extensive testing and are
readily available. As such, agents such as 5-fluorouracil (5-FU),
are preferentially used by neoplastic tissue, making this agent
particularly useful for targeting to neoplastic cells. Although
quite toxic, 5-FU, is applicable in a wide range of carriers,
including topical, however intravenous administration with doses
ranging from 3 to 15 mg/kg/day being commonly used.
[0171] Other factors that cause DNA damage and have been used
extensively include what are commonly known as .gamma.-rays,
X-rays, and/or the directed delivery of radioisotopes to tumor
cells. Other forms of DNA damaging factors are also contemplated
such as microwaves and UV-irradiation. It is most likely that all
of these factors effect a broad range of damage on the precursors
of DNA, the replication and repair of DNA, and the assembly and
maintenance of chromosomes. Dosage ranges for X-rays range from
daily doses of 50 to 200 roentgens for prolonged periods of time (3
to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosage
ranges for radioisotopes vary widely, and depend on the half-life
of the isotope, the strength and type of radiation emitted, and the
uptake by the neoplastic cells.
[0172] The skilled artisan is directed to Remington's
Pharmaceutical Sciences, 15th Ed., 1990. Some variation in dosage
will necessarily occur depending on the condition of the subject
being treated. The person responsible for administration will, in
any event, determine the appropriate dose for the individual
subject. Moreover, for human administration, preparations should
meet sterility, pyrogenicity, general safety and purity standards
as required by FDA Office of Biologics standards.
[0173] The inventor proposes that the local or regional delivery of
an inhibitor of DNA repair, a DNA damaging agent and/or a gene
therapy vector expressing p53 to target cancer cells will be an
efficient method for therapeutic intervention. Alternatively,
systemic delivery of an inhibitor of DNA repair, or the DNA
damaging agent may be appropriate in certain circumstances, for
example, where extensive metastasis of a has occurred.
[0174] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
H. EXAMPLES
[0175] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skilled the art that the techniques disclosed in the examples which
follow represent techniques discovered by the inventor to function
well in the practice of the invention, and thus can be considered
to constitute preferred modes for its practice. However, those of
skill in the art should, in light of the present disclosure,
appreciate that many changes can be made in the specific
embodiments which are disclosed and still obtain a like or similar
result without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
Example I
Materials and Methods
[0176] Cell Lines and Tissue Culture
[0177] T98G glioblastoma cells were obtained from ATCC and cultured
at 37.degree. C. 10% CO.sub.2 in Dulbecco's Modified Eagles Medium
supplemented with 10% newborn calf serum. 9L rat glioblastoma cells
were obtained from Carol Cruse (University of Colorado) and
cultured at 37.degree. C. in 10% CO.sub.2 in Dulbecco's Modified
Eagles Medium supplemented with 10% fetal Calf serum. The
T98G-LHCmjun clone (termed T98G mutant jun or T98G-mjun), as well
as control empty vector modified clone (termed T98GLHCX) were
obtained from Dr. Dan Mercola (Sidney Kimmel Cancer Center) and
were cultured in the same way as were T98G cells except that 100
.mu.g/ml hygromycin was added to the culture medium. The 98G-mjun
is stably modified to express a dominant negative mutant of c-jun
(the mutant was obtained by site-directed mutagenesis by M. Karin
and colleagues and is described by Smeal et al. (1991). Mutant jun
has ser.fwdarw.ala substitutions at positions 63 and 73, two sites
of DNA damage-induced phosphorylation, and can therefore not be
phosphorylated at these sites. 9LpCEPp53 (or 9LpCEP4, control) were
cultured in the same way at 9L cells except that 50 .mu.g/ml
hygromycin was added to the medium.
[0178] Chemicals
[0179] Cisplatin (Platinol.TM., Bristol Myers/Squibb) was obtained
as a 1 mg/ml aqueous solution through local pharmacies. This
solution was diluted to 0.3 mg/ml (1 mM aliquoted, and stored in
the dark at 20.degree. C. 3-Aminobenzamide was purchased from Sigma
Chemical Company, St. Louis, Mo.). The synthetic retinoid SR11220
was supplied by Dr. Magnus Pfahl, Sidney Kimmel Cancer Center. This
retinoid has been demonstrated to have anti-AP-1 activity (Fanjul
et al., 1994).
[0180] Plasmids
[0181] pCEP4 was purchased from Invitrogen (San Diego, Calif.) When
transduced into mammalian cells, this plasmid replicates as an
independent cytoplasmic episome as a result of its EBV origin of
replication. pCEP4 encodes the EBNA-1 protein needed for
replication, and a hygromycin resistance marker. Transgene
expression is driven by the CMV promoter. The reporter plasmid PG13
(PG.sub.13-CAT) was obtained from Bert Vogelstein (Johns Hopkins
Oncology Center). In this plasmid, the chloramphenicol acetyl
transferase (CAT) gene is under the control of a wild -type
p53-specific binding site. Cells to be assayed for wild-type p53
expression are transduced (using Lipofectamine (GIBCO/BRL,
Gaithersberg,MD) and following the manufacture's instructions) wit
PG13 and incubated for two days. Two days following transfection,
cell lysates are prepared and analyzed for their ability to
acetylate .sup.14C-chloramphenicol by thin-layer chromatography.
(See Ausubel et al., 1992 and Kern et al., 1992). Chromatographs
were analyzed by autoradiography or quantitated with and
Ambis4000Radioanalytic Imaging system (Ambis, Inc., San Diego,
Calif.).
[0182] Selection of Rat 9L Cells Expressing Human Wild-Type p53
[0183] Rat 9L glioblastoma cells express p53 mutated at codon 277
(J. Neuroonal. 19:259-268, 1994.) 9LpCEPp53 (or 9LpCEP.sub.4) were
obtained by transducing 5.times.10.sup.5 9L cells with 15 .mu.G of
pCEPp53 (or pCEP4) using Lipofectamine (GIBCO/BRL, Gaithersberg,
Md.) following the manufacturer's instructions. Clones were
selected in 50 .mu.g/ml hygromycin and characterized for wild-type
p53 expression by immunoprecipitation (immunoprecipitation
procedure described in: Gjerset et al., Molec. Carcinog. 5:190-198,
1992) using the 1801 anti-p53 antibody specific for human p53
(p-Ab-2, Oncogene Science, Uniondale, N.Y.), and by the PG13
functional assay described above. pCEPp53 was derived by inserting
the human wild-type p53 cDNA into the multicloning site of
pCEP4.
[0184] Viruses and Infections
[0185] p53 adenovirus or .beta.-galactosidase (.beta.-gal)
adenovirus were obtained from Canji, Inc. (San Diego, Calif.).
These are replication-defective viruses in which the early region
genes E1A and E1B, which are required for viral replication, are
deleted and replaced with the human wild-type p53 and .beta.-gal
sequences, respectively, each form viral CMV promoter (Wills et
al., 1994).
[0186] Cells were infected when they were at 80% confluence with
either 100 pfu per cell overnight in DMEM containing 2%
heat-inactivated FBS (for T98G and clones derived from T98G) or at
50 pfu per cell for 2 hours (9L cells). The efficiency of infection
was determined by staining a sample of the .beta.-gal
virus-infected cells at 48 hrs post-infection as described
(Dannenerg and Saga, 1981). Under the infection conditions used,
.beta.-gal expression was observed in 95-100% of the cell
population.
[0187] Cell Viability Assays
[0188] Viability assays were performed in 96 wells plates 6-7 days
after plating. Cells were plated at a starting cell number per well
of 1000. Triplicate or quadruplicate wells were set up for each
assay point. For cisplatin treatment, triplicate or quadruplicate
wells were set up for each assay point. For cisplatin treatment,
triplicate or quadruplicate wells were exposed for 1 hour to
various concentrations of cisplatin (Platinol.TM.), and then
incubated in the absence of cisplatin for 7 days, during which time
control (untreated) wells were in exponential phase growth. Viable
cell number was based on the bioconversion of the tetrazolium
compound, MTS, into formazan (Promega, Celltiter96.TM.Aq.sub.eous),
as determined by absorbance at 590 nrm using an ELISA reader. For
3-aminobenzamide (ABZ) treatment, triplicate or quadruplicate wells
were treated with either 5 mM ABZ or 10 mM ABX. Fresh ABZ was added
every two days and viability was scored as described above at 6
days. Assays involving the synthetic retinoid SR11220 were
performed in the presence of charcoal-treated fetal bovine serum.
Medium was replaced every two days with medium containing fresh
retinoid.
[0189] PCR.TM. Stop Assay for DNA Damage
[0190] This assay has been described (Oshita and Saijo, 1994;
Jannerwein and Eastman, 1991). The assay is based on the principle
that every DNA lesion, including adducts produced by cisplatin, can
potentially block the progression of the Taq polymerase and
decrease the yield of a given PCR.TM. product. It has been well
demonstrated that the degree of inhibition of PCR.TM. correlates
with the level of platination, indicating that the polymerase is
inhibited by every lesions (Jannerwein and Eastman, 1991).
Furthermore, when whole cells are incubated with varying levels of
cisplatin, the degree of inhibition of amplification of a specific
PCR.TM. product from DNA purified from these cells, correlates
closely with the amount of DNA damage (level of DNA platination) as
measured by atomic adsorption (Jannerwein and Eastman, 1991). The
following primers (used by Oshita and Saijo, 1994) which amplify a
2.7 Kb fragment of the human hypoxanthine phosphoribosyl
transferase (HPRTase) gene were used:
4 5' primer. 5'-TGGGATTACACGTGTGAACCAACC-3' 3' primer.
5'-GATCCACAGTCTGCCTGAGTCACT-3'
[0191] As an internal control for the efficiency of the PCR.TM.
reaction, a nested 5' primer which amplifies a 150 bp fragment of
the same gene was used:
5 nested 5' primer: 5'-CCTAGAAAGCACATGGAGAGCTAG-3'
[0192] At the cisplatin levels used to treat the cells, damage to
the smaller fragment is undetectable. DNA from cells was prepared
immediately after a 1 hour 15 minute treatment with cisplatin (100
.mu.M or 200 .mu.M) and 6 hours later. In some experiments,
3-aminobenzamide (10 mM), an inhibitor of ADP ribosylation and DNA
repair (see Din et al., 1992) was added during the 6 hour recovery
period. Lysates from 5.times.10.sup.6 cells were prepared by
proteinase K digestion of cells, followed by digestion with BamHl
(which reduces viscosity but does not cut within the HPRTase gene)
and Rnase, followed by phenol-chloroform-isoamyl alcohol
extraction, and alcohol precipitation. DNA was washed with 70% EtOH
to remove salt, followed by 100% EtOH dried briefly, and
resuspended at 1 mg/ml in sterile H.sub.2O. Quantitative PCR.TM.
was performed in 50 .mu.l aliquots using 0.5 .mu.g DNA, 50 pmol of
forward primer for 2.7 Kb fragment, 50 pmol reverse primer, and 5
pmol of forward primer for the 150 base fragment, 50 mM KCL, 10 nmM
Tris pH 8.3, 1.5 mM MgCl.sub.2, 250 mM dNTPs, 0.5 .mu.l Tac
polymerasse (Perkin Elmer), and 1 pmol radioactively end-labeled
reverse primer (labeled with y-.sup.32P-dATP). Amplification
conditions were as follows: 1 cycle: 94.degree. C., 1'30; 25
cycles: 94.degree. (1 min)-57.degree. (1 min)-70.degree. (2'30"); 1
cycle:94.degree. (1 min)-57.degree. (1 min)-70.degree. (7').
Control reactions with known amounts of DNA in two-fold dilutions
were performed to insure that the extent of reaction was directly
proportional to the amount of template Following amplification, 10
.mu.l aliquots were electrophoresed on a 1% agarose gel. The gel
was vacuum-dried for two hours onto filter paper and the
PCR.TM.-amplified 2.7 Kb and 150 bp bands were quantitated using an
Ambis4000 Radioanalytic Imaging system (Ambis, Inc., San Diego,
Calif.).
Example II
The Effects of DNA Repair Inhibition on the Growth of Cancer
Cells
[0193] The effects of DNA repair inhibition on the growth of two
different types of cancer cells with varying status in wild-type
p53 expression were tested.
[0194] In the first instance, the following three cell lines were
tested: T98G, parental cell line; T98GLHXC, empty vector modified
control; T98G-mutant jun, containing jun mutated at positions 63
and 73 from serine to alanine. The cells were infected with
wild-type p53-expressing virus and the growth of the cells was
measured after seven days (FIG. 1). The T98G-mutant jun cells had a
significantly reduced viability compared to the T98G parental cells
and the T98GLHXN empty vector control cells. Viability of the p53
adenovirus infected clones was measured relative to the viability
of .beta.gal adenovirus-infected control cells for each
subclone.
[0195] The transcriptional activation of some DNA repair enzymes
are controlled by the transcription factor AP-1, which is a complex
of the proteins fos and jun. .sup.63Ser and .sup.73Ser are
important phosphorylation sites on jun during the induction of DNA
repair in response to DNA damage. In the present instance, mutation
of these amino acids to alanine results in the decreased viability
of T98G glioblastoma cells. The proposed mechanism of action of
these mutations is through inactivation of AP-1, which then would
not be able to transactivate the transcription of DNA repair
enzymes (FIG. 4).
[0196] In the second instance, 9L rat glioblastoma cells were
stably modified with either a control vector, pCEP4, or a vector
encoding human wild-type p53, pCEPp53. The cells were then treated
with 3-aminobenzamide, an inhibitor of the DNA repair enzyme
poly-ADP ribose polymerase (FIG. 6). 9L rat glioblastoma cells that
expressed human wild-type p53 exhibited reduced viability in a dose
dependent manner, with low viability at 5 mM 3-aminobenzamide and
even lower viability at 10 mM 3-aminobenzamide. In contrast, 9L
cells stably expressing only the control vector, were resistant to
growth inhibition at both 5 mM and 10 mM 3-aminobenzamide. The
viability of neither pCEP4 nor pCEPp53 transfected 9L glioblastoma
cells was affected in the absence of 3-aminobenzamide treatment.
Thus cells that are inhibited for DNA repair are sensitive to
p53-mediated growth suppression.
Example III
The Combined Effects of DNA Damaging
Agents and DNA Repair Inhibitors on Tumor Cells
[0197] The effects of cisplatin alone and in combination with a
synthetic retinoid SR11220 on tumor cells expressing wild-type p53
were tested.
[0198] In the first instance, viability of 9L rat glioblastoma
cells infected with either .beta.gal adenovirus or p53 adenovirus,
and treated one day later in the absence or presence of 50 .mu.M
cisplatin was measured seven days after administration of cisplatin
(FIG. 5). 9L rat glioblastoma cells infected with p53 adenovirus
exhibited a significant decrease in viability compared to 9L cells
infected with .beta.gal adenovirus when exposed to cisplatin. The
viability of 9L rat glioblastoma cells infected with either
.beta.gal adenovirus or p53 adenovirus in the absence of cisplatin
did not significantly decrease. Thus tumor cells that have
sustained DNA damage are more susceptible to growth suppression by
p53.
[0199] In the second instance, T47D breast cancer cells were
infected with .beta.gal-adenovirus or p53 adenovirus and treated
with SR11220 or cisplatin or both. SR11220 is a synthetic retinoid
that specifically down-regulates AP-1. Cisplatin is a common
chemotherapeutic agent that creates DNA adducts in cells. T47D
breast cancer cells infected with p53 adenovirus were slightly
sensitive to the effects of p53, in comparison to T47D breast
cancer cells infected with .beta.gal-adenovirus (FIG. 2). Upon
treatment of p53 adenovirus infected T47D cells with 10.sup.-8 M
SR11220, a further reduction in viability was shown. Treatment of
p53 adenovirus infected T47D cells with 10 .mu.M cisplatin had an
even greater effect on the reduction in viability. Treatment of p53
adenovirus infected T47D cells with a combination if 10.sup.-8 M
SR11220 and 10 .mu.M cisplatin further reduced the viability of the
tumor cells. In contrast the viability of T47D cells infected with
the .beta.gal adenovirus control was not reduced significantly
after treatment with SR11220, cisplatin or a combination thereof.
Thus the combined effects of a DNA damaging agent and an inhibitor
of DNA repair significantly sensitize tumor cells to growth
suppression by p53.
Example IV
Cisplatin Treatment of Tumor Cells Induces Apoptosis
[0200] The effects of cisplatin on inducing apoptosis in T98G
glioblastoma cells were tested. DNA damage was measured by PCR.TM.
of a 2.7 Kbp region of the HPRT gene and compared with an internal
control region of 150 bp, that is too small to show significant
effects of DNA damage. The data is represented as the percentage of
the control PCR.TM. signal. DNA damage was measured in T98G
parental cells at zero and six hours after treatment for 1.5 hours
with varying concentrations of cisplatin (FIG. 3A.). The results
show considerable DNA damage immediately after cisplatin treatment
but after time for repair (6 hours), the level of DNA damage is
partially reduced.
[0201] Treatment of T98G parental glioblastoma cells with 10 mM
3-aminobenzamide, an inhibitor of the DNA repair enzyme poly-ADP
ribose polymerase and subsequent treatment with varying
concentrations of cisplatin resulted in significant DNA damage
after six hours post cisplatin treatment (FIG. 3B). In the absence
of 3-aminobenzamide no significant DNA damage remained after six
hours post-cisplatin treatment. Thus chemical inhibition of a DNA
repair mechanism sensitizes T98G tumor cells to apoptosis induced
by DNA damaging agents.
[0202] T98G-mutant jun cells were exposed to varying concentrations
of cisplatin for 1.5 hours and DNA damage was measured at zero and
6 hours post-cisplatin treatment (FIG. 3C). Expression of the
mutant jun, which contains two non-phosphorylatable alanine
substitutions at critical sites for induction of DNA repair,
induced high levels of DNA damage immediately after cisplatin
treatment (zero hour) which was not able to be repaired even after
six hours. Thus the inhibition of DNA repair at the transcriptional
level through blockage of DNA damage-induced AP-1 activity
significantly sensitizes tumor cells for progression to
apoptosis.
Example V
p53-Mediated Tumor Suppression is Enhanced by Increasing Levels of
Cisplatin Treatment
[0203] The effects of increasing levels of cisplatin on the growth
characteristics of the following tumor cells were tested: T47D
breast carcinoma, T98G glioblastoma, and 9L rat glioblastoma Cell
viability was measured in T47D breast carcinoma cells after
infection with wild-type p53 adenovirus and subsequent treatment
with cisplatin two days post-infection (FIG. 7A). Cell viability
decreased with increasing doses of cisplatin whereas control T47D
breast carcinoma cells infected with .beta.gal-adenovirus were
significantly more refractory to the DNA damaging effects of
cisplatin. Similarly, cisplatin treatment of T98G glioblastoma
cells infected with wild-type p53 adenovirus sensitized the tumor
cells to growth suppression in a dose dependent manner (FIG. 7B).
T98G glioblastoma cells infected with .beta.gal adenovirus
exhibited a slightly reduce growth rate in response to increasing
levels of cisplatin.
[0204] Tumor cells stably modified with constructs expressing
wild-type p53 were also sensitized to the effects of cisplatin.
Cell viability of T98G glioblastoma cells stably modified with a
vector containing wild-type p53, pLp53RNL was reduced in a dose
dependent manner after treatment with increasing doses of cisplatin
(FIG. 7C.) Control T98G glioblastoma cells stably modified with
vector only, pLNRL, were more resistant to the effects of cisplatin
treatment. Similarly, cisplatin treatment of 9L rat glioblastoma
cells stably modified by a vector containing wild-type p53,
pCEPp53, significantly reduced the viability of the tumor cells in
a dose dependent manner as compared to 9L glioblastoma cells stably
modified with the control vector pCEP4 (FIG. 7D.). In general then,
as cisplatin-induced DNA damage increases, tumor cell growth
suppression mediated by p53 concomitantly increases.
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forth herein, are specifically incorporated herein by
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Sequence CWU 1
1
3 1 24 DNA Artificial primer 1 tgggattaca cgtgtgaacc aacc 24 2 24
DNA Artificial primer 2 gatccacagt ctgcctgagt cact 24 3 24 DNA
Artificial primer 3 cctagaaagc acatggagag ctag 24
* * * * *