Potential role of DNA polymerase beta in gene therapy against cancer: A case for colorectal cancer

Medical Hypotheses (1996) 47, 1-9 © Pearson Professional Ltd 1996

Potential Role of DNA Polymerase Beta in Gene Therapy Against Cancer: A Case for Colorectal Cancer F. F. SHADAN and L. P. VILLARREAL Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92717, USA (Correspondence to LPV. Tel: (714) 824-6074; Fax: (714) 824-8551)

Abstract - - Genetic instablility characterized by the accumulation of mutations of tumor suppressor genes and oncogenes appears to be associated with carcinogenesis in colorectal and other cancers. Mutations of DNA polymerase beta (pol beta) and related chromosomal alterations appear to be consistent with the causal role of a 'mutator phenotype' in carcinogenesis. However, homozygous knockout pol beta mutations appear to interfere with embryogenesis. Increased pol beta activity (i.e. relative to pol alpha activity) has been associated with cell cycle arrest. The related aphidicolin-resistant DNA replication has been observed primarily in differentiating cells, including the mammalian blastocyst, adrenal cortex, thyroid, anterior pituitary, and the mechanism of endoreduplication (amitotic overreplication of DNA) can be traced to lower eukaryotes. This increased activity in relation to terminal commitment is inconsistent with a simple 'DNA repair' view of pol beta. It is therefore proposed that pol beta may play a more fundamental role in cellular differentiation through involvement in a putative subgenomic DNA replication-based model of terminal gene expression. Thus genetic instability, loss of differentiation, and carcinogenesis may result from aberration(s) or 'derailment' of such replication-based mechanism of terminal gene expression. It is suggested to examine the relationship of DNA pol beta to genomic instability and carcinogenesis using genetic analyses and antisense technology with possible applications for gene therapy against colorectal cancer.

Introduction Colorectal cancer represents almost 15% of all newly diagnosed cancers. The estimated death rate from colorectal cancer ranks second to lung cancer in the USA (1-3). Colon cancers are predominantly adenocarcinomas that exhibit variable degrees of neoplasia and loss of differentiation. These cells either produce Date received 28 June 1995 Date accepted 23 August 1995

fungating masses or elevated plaque-like invasive structures (4). Morphological changes and anaplasia are correlated with the accumulation of mutations of oncogenes and tumor-suppressor genes. Standard treatment procedures routinely employed against colon cancer (2) and other cancers have not fully benefited from the potential advantages of molecular genetics. Development of novel strategies

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will most likely utilize the molecular biology of colorectal cancer to complement current chemotherapeutic and irradiation regimens which are at present genotypenon-specific. Using colorectal cancer as a paradigm for carcinogenesis in which molecular genetics is most intensively studied, consideration of the genetic background and the particular cellular abnormalities may lead to elucidation of better cellular targets for gene therapy and drug design (5,6).

Colorectal cancers exhibit genetic instability and loss of differentiation The molecular genetics of colorectal cancer is among the best studied, and the physical accessibility of these tumors through novel procedures (7,8) identifies these cancers as prime targets for the development of advanced therapies, including combined gene therapy and immunotherapy. Colorectal cancer, as well as many other types of cancer, generally exhibits genetic instability and loss of differentiation. Genetic instability may explain the role of radiation therapy (9) and chemotherapeutic agents (6,10) that may inflict DNA damage. Mutations of the ras oncogene (a G protein observed in growth-factor-stimulated signal transduction) have been reported in about 37-60% of colorectal cancers (11-16). Abnormal over-expression of the myc oncogene has been reported in adenomatous colonic polyps (14,17,18). These oncogenes are among targets of experimental antisense therapy. Abnormalities of tumor-suppressor genes have also been observed in the development of colorectal cancers. Mutations of adenomatous polyposis coli gene (APC) gives rise to familial adenomatous polyposis (FAP). Twenty percent of sporadic tumors were found to have allelic deletions in the long arm of chromosome 5, and 29% of sporadic adenomas plus 36% of sporadic carcinomas were shown to have deletions in the APC gene (14,19). Other studies have increased this value to more than 50% (14,20). Mutations in colorectal cancer (MCC) locus have been found in about 15-40% of colon cancers (14,19,21,22). Deleted in colorectal cancer (DCC) gene was discovered on the long arm of chromosome 18, through analysis of the loss of heterozygosity in colorectal tumors (23), and is thought to be a tumorsuppressor gene. Mutations of p53, a tumor-suppressor gene on chromosome 17p13.1 have been found to be associated with a large number of cancers, including the Li-Fraumeni syndrome, breast cancer, and colorectal cancer (14,24-27). p53 has been implicated in the role of 'checkpoint control', and in the induction of G1 block following DNA damage, presumably to safeguard genetic stability (28). p53 mutations have been implicated in the development of tetraploidy in

MEDICAL HYPOTHESES

colorectal cancers (24). However, large numbers of colorectal cancers have not been reported in p53-/homozygous knockout mice, and p53 mutation can be viewed as a relatively late mutational step in colorectal carcinogenesis (25,29). p53, APC, and DCC are candidates for replacement-gene therapy, although the underlying mechanisms are not fully understood (25,28,30). The accumulation of mutations noted above clearly implicates genetic instability in colorectal tumors. These results do not fully conform to the elegant twohit model of carcinogenesis with the simple requirement of the mutation of both alleles, since several mutations may be identified and implicated in the progression of colon cancers (14,25,31,32). Colorectal carcinogenesis does not require the conservation of any given mutation, as none of the mutations so far discovered have been found to occur in 100% of cancer cells: the APC mutation is not observed in about 71% of sporadic colorectal adenomas nor in about 64% of sporadic colorectal carcinomas (14,19); K-ras mutations are absent in at least 40% of colorectal cancers (11-13,15,16); DCC mutations (allelic loss of 18q) are lacking in about 30% of colorectal carcinomas, and p53 mutations have not been noted in about 40% of all cancers in the presence or absence of aneuploidy (24,33). The occurrence of these mutations does not follow a fixed temporal order (14,25). Thus, the more common characteristic among colorectal tumors (and perhaps other cancers) appears to be genetic instability and loss of differentiation.

What is the relationship between genetic instability and loss of differentiation ? Several lines of evidence suggest a close association between the loss of differentiation, carcinogenesis, and genetic instability (34,35). A large percentage of human and rodent-cell lines consisting of both immortal and transformed cells such as those documented in the American Type Culture Collection (ATCC) manual (36) have aneuploid karyotypes and generally exhibit variable loss of cellular differentiation. In many cancers, aneuploidy is regarded in association with the degree of anaplasia and is often related to prognosis (34,35,37). There are at least two ways to view the relationship between genetic instability and loss of differentiation. One view is the suggestion that the development of a 'mutator phenotype' (loss of repair activity) is needed early in the progression of tumors (38,39). Examples include xeroderma pigmentosum, in which mutations of an excision repair colorectal cancer (ERCC) gene family have been observed, and hereditary nonpolyposis colon cancer (HNPCC), in which the deficiency

DNA POLYMERASE BETA IN GENE THERAPY AGAINST CANCER

of mismatch repair activity (e.g. hMSH2, hMLH1, hPMS2) results in neoplasia (31,38,40-49). The accumulation of a large collection of mutations (APC, DCC, p53, ras, myc) cannot be accounted for by the rate of spontaneous mutations in any given cell with normal repair mechanisms (25,38). Also noteworthy is the potential connection of p53, replication protein A, GADD45 and p21 pathway to the withdrawal from the cell cycle, DNA repair, or apoptosis (28,50). That p53 -/- homozygous knockout mice develop to adulthood argues against the direct involvement of this gene in terminal differentiation or as a 'guardian of genome' during fetal development (29). In these mice, large numbers of colorectal cancer have not been reported, nor are p53 mutations regarded as primary mutations in colorectal tumor progression (25). Deletions of p53 have been seen in about 75% of carcinomas but observed only rarely in the preceding adenomas (14). DNA polymerase beta has been viewed by many as a repair enzyme (refer to (51) and (52) for additional references), in part based on shortpatch DNA repair activity of chemically induced damage and related activity independent of the cell cycle. The pol beta gene has been located to chromosome 8 and this region is one of the most frequently mutated chromosomes in colorectal cancer (38,53,54). In a preliminary analysis of colorectal cancers, using DNA amplification by polymerase chain reaction, cloning, and sequencing, deletion mutations have been mapped to catalytic domains of DNA pol beta in 83% of colorectal cancer patients (5 out of 6) (38). Therefore, pol beta mutational inactivation may result in 'mutator phenotypes'. Mismatch repair deficiency, however, appears to be compatible with phenotypically normal human development (55). As will be discussed below, a number of results may be interpreted to support a different view, suggesting that the role of DNA polymerase beta may be more fundamental to development and to the onset of cellular differentiation than is attributable to DNA repair activity alone. Results that suggest a more fundamental role for pol beta in specific differentiating tissues

Cells can undergo DNA synthesis without commitment to cell division. DNA synthesis in the absence of mitosis is herein referred to as amitotic DNA replication or endoreduplication. DNA repair enzymes and DNA polymerase beta-like activities have generally been implicated in amitotic endoreduplication of DNA. Evidence for amitotic aphidicolin or Ara-Cresistant subgenomic DNA replication (i.e. endoreduplication) has been described in mammalian and dipteran embryogenesis and during cellular differen-

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tiation in culture. DNA pol beta has been identified based on biochemical assays that measure DNA polymerase activity in the presence of aphidicolin, an inhibitor of DNA polymerase alpha and delta but not beta (56-58). As will be discussed, a number of studies have detected aphidicolin-resistant DNA polymerase beta-like activity in terminal differentiation during embryogenesis of lower and higher eukaryotes. The observed increase in DNA pol beta activity (relative to pol alpha activity) in these terminally committed cells does not appear to conform to the general view that pol beta is primarily a DNA repair enzyme involved in the genomic maintenance of proliferating cells. i. Pol beta activity in terminal differentiation of giant trophectoderm of the mammalian blastocyst In higher organisms, such as in the mouse blastocyst, the differentiating trophectoderm (the first cells committed to differentiation) undergo aphidicolin-resistant pol beta-like over-replication of DNA (endoreduplication) in conjunction with terminal differentiation (59-63). In the mouse blastocyst, the inner cell mass is composed of diploid, mitotic, uncommitted toilpotent cells, whereas the trophectoderm consists of polytene terminally committed cells (59,60,62,63). Mouse embryonic stem cells with constitutive homozygous knockout mutations for DNA polymerase beta fail to complete embryogenesis (64,65). The polytenized rodent trophectoderm is insensitive to aphidicolin (an inhibitor of DNA polymerases alpha and delta, but not pol beta), but is sensitive to 2',3'dideoxythyrnidine ( d d ~ P ) , an inhibitor of pol beta (61,66-68). High concentrations of ddTTP inhibit the endoreduplication of trophectoderm in vivo. The endoreduplication of cellular DNA and DNA pol betalike activity in terminally committing giant trophectoderm cells cannot be explained as repair activity alone. ii. Pol beta activity is not required for the proliferation of embryonic stem cells but appears to be essential for embryogenesis Experiments aimed primarily at understanding the role of DNA pol beta in the development of T cells in transgenic mice also indicated that the complete development of the mouse embryo is inhibited when using embryonic stem cells that are unconditional homozygous knockout for pol beta (64,65). The homozygous null mutation for pol beta has not been reported to interfere with the proliferation of the embryonic stem cells that were used unsuccessfully in the initial attempts to generate homozygous knockout mice for pol beta. The embryonic stem cells with wild-type genotype for pol beta, however, completed embryogenesis (64,65). These observations therefore

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suggest that a genetic defect in pol beta interferes with embryogenesis.

MEDICAL HYPOTHESES

terms of DNA repair, particularly given that ACTH inhibits cellular proliferation. In correlation with cellular differentiation, pol beta activity is induced to high levels by ACTH, and is conversely inhibited by hypophysectomy (57). Therefore, in the adrenal gland, ACTH, a hormone that induces the expression of the differentiated function in the fasciculata cells (but inhibits cellular proliferation), also induces a substantial level of pol beta activity. These results again implicate pol beta primarily in the process of terminal commitment, rather than simple DNA repair.

iii. Pol beta in cell cycle control and terminal commitment A simple prediction of the putative involvement of pol beta in terminal differentiation would be the withdrawal from the cell cycle of cells transfected with transcriptionally active exogenous pol beta coding sequences. Such an experiment has been done: mouse 3T3 cells were transfected with exogenous pol beta sequence under the control of metallothionine promotor, and cells maintaining amplified and stable pol b. Pol beta activity reflects the differentiated function beta sequences were selected by a DHFR/methotrexate of the thyroid cells. To actively synthesize and secrete selection scheme to study the effect of variations in thyroid hormone in response to thyroid stimulating the level of pol beta (69). Cells induced to express pol hormone (TSH) epithelial cells transform from somebeta underwent a block of cellular proliferation in what flattened simple cuboidal cells into tall cuboidal correlation with a threefold increase in pol beta DNA cells, containing many mitochondria, increased rough synthetic activity (69), and assumed predominantly endoplasmic reticulum, and prominent golgi apparaspindle-shaped morphology. tus in the basal cytoplasm (75). In hyperthyroidism, High levels of pol beta activity in the absence of DNA pol beta activity is induced threefold relative (or relative to) pol alpha activity have also been to the normal thyroid gland (73). By contrast, in unfound in differentiating thyroid tissue, nervous tissue, differentiated hyperproliferating thyroid tissue such as erythroleukemia cells, rat liver, chicken brain and lens in thyroid carcinoma, the levels of pol beta are lower respectively (70-73) (for additional information see than normal (73). In rats, hypophysectomy inhibits (63). These represent differentiated tissues, examples the production of TSH, and results in the inhibition of which will be discussed further below. However, in of 3.3 S pol beta activity, but not that of DNA pol poorly differentiated prostate cancer cells and colo- alpha activity (72). Daily injection of TSH to hyporectal tumor cells pol beta was found to bear inhibi- physectomized rats reverses the inhibition of 3.3 S pol tory mutations (38,74), particularly in the active site. beta activity (72). Stimulation of the rat thyroid in In our view, therefore, these observations appear to vivo by excess TSH induces the activity of all forms link DNA pol beta activity to the withdrawal from the of DNA pol beta (72). These results again suggest that cell cycle and terminal differentiation. Increased pol levels of DNA pol beta activity correlate with the beta-like activity (relative to or in the absence of pol degree of differentiated function rather than with alpha activity) in differentiating cells rather than pro- hyperproliferation. liferating cells is incongruent with repair activity alone. Rather, pol beta activity is more closely associ- c. Pol beta activity is related to the differentiated ated with withdrawal from the cell cycle and terminal function in the anterior pituitary. The regulation of the histologically distinct differentiated secretory cells commitment. of the anterior pituitary is dependent on the hormonal iv. Expression of differentiated function in feedback from glands receiving trophic pituitary horresponse to hormones mones. The thyroid gland secretes thyroxine in rea. Pol beta activity is related to terminal differentia- sponse to TSH from the pituitary. Thyroxine levels in tion in the adrenal cortex. Adrenocorticotrophic hor- tum modulate the synthesis of TSH by the thyrotroph mone (ACTH) produced by the anterior pituitary cells of the anterior pituitary. Stimulation of the rat gland has a profound stimulatory activity on the anterior lobe pituitary cells to produce TSH by interdifferentiation of the fasciculata cells of the adrenal feting with feedback inhibition from the thyroid recortex. ACTH results in the inhibition of cellular pro- suits in the induction of pol beta activity (58) and liferation of adrenocortical cells, and in the induction increased production of TSH. Again, the level of pol of cellular differentiation in the fasciculata of the beta activity is reflective of the degree of the differenadrenal gland (57) by transforming undifferentiated tiated function. adrenal cells into histologically distinct and functionally differentiated fasciculata cells. In the normally d. Pol beta activity is related to spermatogenesis. active adrenal gland, 80% of the total DNA poly- Somewhat reminiscent of other epithelia (e.g. the epimerase activity is attributable to pol beta. This level of dermis) spermatozoa are produced from uncommitted activity is clearly too high to be explained simply in basal cells into histologically distinct layers of syn-

DNA POLYMERASE BETA IN GENE THERAPY AGAINST CANCER

chronized cells in the seminiferous tubule. The level of pol beta activity has been observed to increase during the development and maturation of the rat testes in correlation with increased spermatogenesis (76), attributed by many to the maintenance of genetic stability in the germline. We propose that spermatogenesis can be viewed alternatively as a model system for the study of terminal differentiation, and meiotic DNA replication might be regarded as a variant of DNA endoreduplication. In an in vitro model of the seminiferous epithelium, expression of temperature sensitive p53 results in the commitment of spermatogonia to the ultimate development of spermatozoa (77,78). In correlation with such process, significant levels of pol beta activity have been observed during sexual maturation in meiotic and postmeiotic cells (76,79). This increased pol beta activity and the generation of mature spermatozoa were inhibited upon hypophysectomy, which prevented the production of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) needed for spermatogenesis (76). v. Pol beta activity and T-cell development DNA pol beta is thought to be involved in the normal maturation of the T-cell receptor, through repair-like activity implicated in the rearrangement of DNA to assemble the T-cell receptor (TCR) or immunoglobufincoding sequences (65,80). However, the maturation of the TCR can also be interpreted as a major step in the commitment to terminal differentiation. T cells with immature T-cell receptors are not capable of performing their differentiated function, such as normal communication with other cells of the immune system and the distinction of 'self' versus 'non-self'. In transgenic mice bearing conditional homozygous knockout mutations for pol beta, T cells are thought to be 'at a disadvantage during development, although some of them survive' and analysis of mice bearing tissue specific heterozygous mutations for pol beta inferred that 'pol beta inactivation results in cell death if it happens to occur before the completion of TCR rearrangement', providing genetic evidence in support of the involvement of pol beta in T-cell development (64,65). vi. Terminal differentiation in lower eukaryotes is associated with endoreduplication The dipteran larva of Drosophila melanogaster generates imaginal discs as it develops adult tissues. In this process, over-replication of DNA (endoreduplication) corresponds with terminal differentiation and the development of the larva. This process is thought to most likely represent DNA pol beta-like activity, resulting in tissue-specific domains which undergo polytene DNA replication at defined times in embryogenesis (61,63,66-68,81-83).

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Conclusion The repair view of pol beta does not provide a convincing explanation for the induced pol beta activity in the differentiated state as is exemplified by observations such as the differentiating blastocyst trophectoderm, and by the ACTH-responsive nonproliferative differentiating fasciculata cells of the adrenal gland. The homozygous knockout mouse embryonic stem cells are viable and proliferative in the absence of pol beta, as are 3T3 cells in which pol beta activity was inhibited using antisense technology. Yet, these homozygous knockout embryonic stem cells failed to undergo complete embryogenesis which requires differentiation in addition to cellular proliferation and repair. Introduction of amplified pol beta gene resulted in cell cycle block in 3T3 cells. Cell-cycle arrest is consistent with the initial withdrawal step in the cellular differentiation pathway. The level of pol beta activity correlates with the degree of differentiated function of specialized secretory cells.

What is the relationship between DNA polymerase beta activity and terminal differentiation ? A number of studies, including those discussed above, have detected aphidicolin-resistant DNA polymerase beta-like activity in terminal differentiation during embryogenesis of lower and higher eukaryotes (for review see (63)). If we consider Darwin's classical view that 'ontogeny recapitulates phylogeny', then clearly the conservation of aphidicolin-resistant DNA polymerase beta-like activity in eukaryotic embryogenesis is likely to be of fundamental significance. Aphidicolin-resistant subgenomic over-replication of DNA has been linked to terminal differentiation. Although the relationship between DNA pol-beta activity and terminal differentiation remains unknown, detection of high levels of pol-beta activity in terminally differentiated cells does not support the 'DNA repair' view of pol beta in association with cellular proliferation. These observations in aggregate have led to the proposal that subgenomic aphidicolin-resistant DNA replication and the related pol beta activity may represent an important step in the expression of terminal genes (63). To summarize this proposal, a basic problem in committed gene expression is the inaccessibility of DNA to trans-acting factors, and repression of transcription by nucleosome occlusion. DNA replication-based chromatin assembly provides a possible solution to the problem of transacting factor accessibility. Endoreduplication is specific to euchromatin and to highly active genes of terminally differentiating cells (82). By contrast, heterochromatin is not

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over-replicated (82,84). A model in which aphidicolinresistant DNA polymerase-beta-like subgenomic DNA replication serves as a basis of expression of terminal genes has been proposed and discussed in detail (63). According to this model, only a very small proportion of genomic DNA representing the terminal genes (a subset of the total transcriptionally active genome, i.e. less than 5% of the total cellular DNA) is thought to be replicated to low copy numbers for gene expression by a pol beta-like activity. Indeed, it has been demonstrated that DNA polymerase beta is involved in the conversion of single-stranded M13 DNA to double-stranded DNA in the Xenopus oocyte (85). The ability of pol beta to synthesize long regions of DNA (i.e. M13 DNA) is more consistent with an activity required for the over-replication of genomic DNA which is clearly distinct from the short-patch repair activity. Further indications of a putative subgenomic DNA replication mechanism are provided by the analysis of polyomavirus DNA replication patterns observed mainly in vivo (63,86-90). Therefore, biochemical as well as genetic evidence closely link pol beta activity with endoreduplication of DNA, embryogenesis, and the related cellular differentiation. Pol beta-like aphidicolin-resistant subgenomic DNA replication may thus form the basis for the expression of the genes of terminal differentiation (63).

What is the linkage between genetic instability and the loss of cellular differentiation ? Generally, cancerous cells exhibit a high degree of concordance between the loss of cellular differentiation, onset of cellular transformation, and the loss of genetic stability or the impairment of DNA repair mechanisms. The development of colorectal cancer, a current paradigm of carcinogenesis, is a case in point. Colorectal carcinogenesis has been associated with the accumulation of mutations of oncogenes (e.g. ras, myc) and tumor suppressor genes (e.g. APC, DCC) as well as the mutation of genes that are thought to 'guard' the genome against instability (e.g. p53 tumorsuppressor gene and repair pathways including pol beta repair activity) (25,28,31,38,49,74). The ability of cancer cells to amplify DNA is a major distinction from precancerous cells (34,35,37) (for additional references see (24)). Genetic instability has often been associated with polyploidy and aneuploidy, which may be related to the aberrant amplification of DNA such as duplications (with or without point mutations), and deletions (viewed as partially or completely aborted amplifications), or translocations (typically involving DNA replication as an initial step). According to a view described earlier, production of a 'mutator' phenotype would explain genetic instability

MEDICAL HYPOTHESES

and the mutation of oncogenes and tumor-suppressor genes (38,39,42,74). However, as supported by the results presented later, if the subgenomic replication of the genes of terminal differentiation is indeed responsible for the expression of the differentiated state as has been proposed (63), then it is possible that an aberration of a putative subgenomic DNA replication mechanism of terminal gene expression possibly mediated by pol beta (aphidicolin-resistant activity) may cause unwarranted, misguided, or error-prone low level DNA replication that could result in genetic instability in conjunction with the loss of differentiation. This possibility provides a potentially intriguing link between the loss of genetic stability and the loss of terminal differentiation.

How might this proposal be assessed? Application of gene delivery, transgenic knockout and anti-sense technologies to study the role of pol beta in carcinogenesis with possible implications for cancer therapy In colorectal cancers, the accumulation of mutations appears to be required for tumor progression and the loss of terminal differentiation. Recently, DNA polymerase beta mutations have been found at a high rate in colorectal cancers as well as in a subset of prostate cancers (38,74), and the presence of pol beta mutations in other cancers deserves further research. The potential linkage of mutations of pol beta to aberrations of terminal DNA replication, the onset of genetic instability, and cellular transformation deserves further study. An outstanding question is whether aberrations of pol beta are causal to the onset of cellular transformation, and whether the loss of pol beta activity is contributing to tumor progression, genetic instability, and loss of differentiation. The preliminary analysis of human colorectal cancers in which deletion mutations have been mapped to the catalytic domain of pol beta (38) is encouraging in this regard. Genetic techniques can serve as powerful tools to test the causality of such mutational events in carcinogenesis, and in the development of genetic instability such as aneuploidy. Development of conditional homozygous genetic knockout of pol beta (64,65) allows investigation into the causality of pol beta mutations in a variety of murine cell types in vivo as well as analysis in vitro by transformation assays in cell culture. Such mutants can be compared to wildtype counterparts with respect to carcinogenesis, tumor progression, genetic instability, and the development of aneuploidy. It is hypothesized that pol beta knockout mutations would promote genetic instability that ultimately leads to tumor suppressor mutations, aneuploidy, loss of differentiated phenotype, and to an

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DNA POLYMERASEBETAIN GENETHERAPYAGAINSTCANCER

increased rate of tumor progression. One should also consider the possibility that the putative endoreduplication pathway implicated in cellular differentiation is likely to involve multiple gene products. Thus, multiple mutations independent from that of pol beta may also play a causal role in carcinogenesis. This may indeed explain the observation that based on polymerase chain reaction, single-stranded conformational polymorphism and sequence analyses, only a subset of prostate cancers examined thus far bear pol beta mutations (74). The introduction of wild-type pol beta gene to homozygous knockout cells for pol beta and to colorectal cells (especially to those that are deficient in pol beta activity) may restore some level of genetic stability and/or the capacity to differentiate. Such effect may prolong or inhibit tumor development. One might further predict that the impact of pol beta provision would depend largely on the pre-existing mutations and the level of differentiation (or tumor grade) prior to the introduction of wild-type pol beta. Based on the observation that the expression of pol beta has resulted in cell-cycle arrest in 3T3 cells (69), it is expected that the expression of wild-type pol beta would result in either cell-cycle block or in the extension of the doubling time of colorectal cancer cells. Since pol beta has been shown to exhibit DNA repair activity, it is expected that the expression of wild-type pol beta may improve cellular resistance to DNA damage and thereby enhance genetic stability (52,91). Cells with mutated pol beta are genetically unstable (38), and factors that inhibit pol beta activity or decrease genetic stability also enhance radiosensitivity (91). Therefore, the introduction of antisense sequence against endogenous pol beta transcripts is likely to inhibit pol beta activity and decrease genetic stability, thereby enhancing cellular susceptibility to the lethal effects of ionizing radiation. Such an approach may be used to enhance the therapeutic ratio of radiation therapy by the delivery of antisense sequences specifically to the anatomic region of tumor invasion. The implication of pol beta in the commitment to terminal differentiation through a putative DNA replicationbased mechanism of terminal gene expression (63) suggests that the expression of wild-type pol beta may alter the tumor grade and enhance the extent of cellular differentiation. Such experimental strategies would shed light on the role of pol beta in the cell cycle, in the maintenance of genetic stability, and in the onset of terminal differentiation. Positive results in cell culture may justify the development of in vivo correlates of the above experiments in animal models to devise gene therapy strategies (92) for therapeutic delivery of sense or antisense sequences of pol beta. Radiosensitivity, tumor grade, level of differentiation,

invasiveness, metastatic potential, survival, as well as potential adverse effects of the strategy can undergo preliminary assessment in tumor-beating rodent model systems treated either with viral vectors bearing pol beta, or with pol beta antisense sequences given in conjunction with radiation. Encouraging results in vivo may lead to clinical trials aimed at the expression of wild-type pol beta to enhance genetic stability, to promote terminal differentiation, and to prolong tumorigenesis. Alternatively, clinical trials may be aimed at improving the therapeutic ratio of palliative radiation therapy (9) through inhibition of pol beta expression in the target cells using antisense technology.

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