The role of DNA repair in mutagenesis was stud- ied in normal, repair-proficient Chinese hamster ovary cells and in two mutant strains that are deficient in excision repair. By using the mutagen 7-bromomethylbenz(a)anthracene (7-BrMeBA) and the technique of alkaline elution of DNA, the mutants were found to be defective at or before the incision step of excision repair. Dose-responses were determined for cell killing, mutation induc- tion at three loci, and sister chromatid exchanges over a survival range of 1.0-0.1 after 7-BrMeBA treatment. The mutants were 5-fold more sensitive to killing than were the normal cells, but the degree of hypersensitivity to mutation induction varied depending on the mutant strain, the genetic marker, and the dose of mutagen. In each instance, the dose-response curve for mutations was es- sentially linear in the repair-deficient cells. In the normal cells, however, the curves for induced resistance to thioguanine and azaadenine were complex and were curvilinear with increasing slope at low doses. This behavior may be attributable to saturation of the excision repair system. No difference was seen in the ef- ficiency of inducing ouabain-resistant mutations in the repair-de- ficient cells compared to the normal cells, indicating a qualitatively different behavior of this marker. These results are consistent with excision repair of 7-BrMeBA damage being error-free in Chinese hamster ovary cells. Sister chromatid exchange, another manifestation of DNA damage, also.was induced with greater ef- ficiency in the repair-deficient cells.

PURPOSE:Patients with malignant gliomas have a poor prognosis. To explore a novel and more effective approach for the treatment of patients with malignant gliomas, we designed a strategy that combines caspase-8 (CSP8) gene therapy and radiation treatment (RT). In addition, the specificity of the combined therapy was investigated to decrease the unpleasant effects experienced by the surrounding normal tissue. METHODS AND MATERIALS:We constructed the plasmid pEGR-green fluorescence protein that included the radiation-inducible early growth response gene-1 (Egr-1) promoter and evaluated its characteristics. The pEGR-CSP8 was constructed and included the Egr-1 promoter and CSP8 complementary DNA. Assays that evaluated the apoptosis inducibility and cytotoxicity caused by CSP8 gene therapy combined with RT were performed using U251 and U87 glioma cells. The pEGR-CSP8 was transfected into the subcutaneous U251 glioma cells of nude mice by means of in vivo electroporation. The in vivo effects of CSP8 gene therapy combined with RT were evaluated. RESULTS:The Egr-1 promoter yielded a better response with fractionated RT than with single-dose RT. In the assay of apoptosis inducibility and cytotoxicity, pEGR-CSP8 showed response for RT. The pEGR-CSP8 combined with RT is capable of inducing cell death effectively. In mice treated with pEGR-CSP8 and RT, apoptotic cells were detected in pathologic sections, and a significant difference was observed in tumor volumes. CONCLUSIONS:Our results indicate that radiation-inducible gene therapy may have great potential because this can be spatially or temporally controlled by exogenous RT and is safe and specific.

Purpose: To evaluate the preclinical pharmacokinetics and antitumor efficacy of a novel orally bioavailable poly(ADP-ribose) polymerase (PARP) inhibitor, ABT-888. Experimental Design: In vitro potency was determined in a PARP-1 and PARP-2 enzyme assay. In vivo efficacy was evaluated in syngeneic and xenograft models in combination with temozolomide, platinums, cyclophosphamide, and ionizing radiation. Results: ABT-888 is a potent inhibitor of both PARP-1 and PARP-2 with Kis of 5.2 and 2.9 nmol/L, respectively. The compound has good oral bioavailability and crosses the blood-brain barrier. ABT-888 strongly potentiated temozolomide in the B16F10 s.c. murine melanoma model. PARP inhibition dramatically increased the efficacy of temozolomide at ABT-888 doses as low as 3.1 mg/kg/d and a maximal efficacy achieved at 25 mg/kg/d. In the 9L orthotopic rat glioma model, temozolomide alone exhibited minimal efficacy, whereas ABT-888, when combined with temozolomide, significantly slowed tumor progression. In the MX-1 breast xenograft model (BRCA1 deletion and BRCA2 mutation), ABT-888 potentiated cisplatin, carboplatin, and cyclophosphamide, causing regression of established tumors, whereas with comparable doses of cytotoxic agents alone, only modest tumor inhibition was exhibited. Finally, ABT-888 potentiated radiation (2 Gy/d × 10) in an HCT-116 colon carcinoma model. In each model, ABT-888 did not display single-agent activity. Conclusions: ABT-888 is a potent inhibitor of PARP, has good oral bioavailability, can cross the blood-brain barrier, and potentiates temozolomide, platinums, cyclophosphamide, and radiation in syngeneic and xenograft tumor models. This broad spectrum of chemopotentiation and radiopotentiation makes this compound an attractive candidate for clinical evaluation.

Caspase-3 plays a critical role as an executioner of apoptosis. The aim of this study is to evaluate the potential of the combination of caspase-3 gene therapy and radiation treatment. We prepared a plasmid (pCI-CSP3) that contained the human caspase-3 gene and the cytomegalovirus promoter. We introduced this plasmid into U251 and U87 human glioma cells and subjected the cells to radiation treatment. The degree of cell death and apoptosis were evaluated. None of the cell lines underwent apoptosis by the overexpression of caspase-3 alone, but the degree of cell death and apoptosis were markedly enhanced by the addition of radiation treatment. Next, we prepared another plasmid (EGR-CSP3) that contained the caspase-3 gene and a radiation-sensitive promoter. Each treatment system using either pCI-CSP3 or EGR-CSP3 showed radio response. The treatment system using pCI-CSP3 more effectively induced apoptosis than that using EGR-CSP3. Caspase-3 gene therapy in combination with radiation treatment has the potential to serve as a radio-responsive gene therapy without any radiation-sensitive promoter.

Objective To detect the role of implantable biodegradable polymers for the local delivery of 125I radiolabeled idoxuridine (IUdR).Methods Para-carboxyphenoxypropane: sebacic acid (PCPP:SA) polymers (10 mg, 25 μCi of the 125I-IUdR) and polyphosphoester (PPE) polymers (15 μg:25 μCi) were synthesized and the release of the radioactivity was counted by a calibrated scintillation counter both in vitro and in vivo. Results PCPP:SA resulted in controlled, protracted release of 125I-IUdR vs. time in vitro, and the similar outcomes were obtained in vivo. The counting of the blood after implantation of the flank PCPP:SA polymers showed the release was much faster than for the intracranial (IC) polymer. For the counting of flank tumors after ipsilateralral (IP) vs. contralateral (CL) polyphosphoester (PPE) 125I-IUdR injection, the release speed of both sides were similar, but the IP polymer had a much higher local concentration than for the CL polymer. The release of the PPE 125I-IUdR polymer into the blood was similar in both side implanted polymers. Autoradiographic quantification of the activity section showed decreasing activity vs. distance from the polymeric implant and decreasing activity for CL vs. IP 125I-IUdR polymeric implantation. Organs counts showed that the highest content was for the intestine, followed by the spleen measured 2, 4, and 8 days after the polymers implantation.Conclusion The implantable and biodegradable polymers make radiolabled IUdR more concentrate in the implanted site and protracte its release.

Purpose/ObjectiveDNA damage that is not detected and repaired can lead to an increased frequency of chromosomal aberrations, a major contributing factor in the onset of tumorogenesis. Such unrepaired DNA damage can also lead to an increase in mitotic cell death as the cell struggles to successfully segregate its chromosomes. We were interested in investigating whether radiation-induced DNA damage could be introduced into both normal and cancerous human cells at a level and frequency that could evade detection by cellular repair mechanisms, ultimately resulting in increased cell death. We were also interested in studying the molecular mechanisms involved in this processes.Materials/MethodsMultiple human cancer cell lines (HCT116, RKO, DU145 and PC-3) were exposed to either high (acute; ∼4500 cGy/hr) or low dose rate (LDR; 2 or 9.4 cGy/hr) ionizing radiation. Clonogenic survival assays, SDS/PAGE and immunoblot analyses for activated ATM and FACS analyses for activated H2AX were performed following radiation exposure at both acute and LDR radiation exposures.ResultsWe observed an increase in cell death among four different human cancer cell lines following LDR exposures compared to acute exposures of equivalent dose. We demonstrated that this increased cell killing was a consequence of ineffective activation of the critical damage sensor ATM and its downstream target H2AX (a cellular marker of sites of DNA damage). This phenomenon was also observed in normal primary human fibroblast cultures. The apparent reduction in clonogenic survival was not due to accumulation of senescent cells during the protracted radiation exposures prior to plating. The failure of LDR treated cells to sufficiently activate ATM was, in fact, not due to the presence of dysfunctional ATM protein following LDR exposure as cells pre-treated with LDR radiation were shown to elicit a normal ATM response following subsequent acute exposures.ConclusionsThe data presented here are the first to demonstrate that low levels of DNA damage introduced by LDR radiation do not activate the DNA damage sensor ATM, a critical cellular response mechanism. This occurs despite the appearance of activated H2AX, a marker of DNA double-strand breaks. This lack of ATM activation and lower activated H2AX ultimately results in greater amounts of cell killing compared to equivalent doses of acute radiation exposures. These findings may aid the further understanding of the early cellular DNA damage response mechanisms and have broad-range implications for clinical treatment of solid tumors Purpose/ObjectiveDNA damage that is not detected and repaired can lead to an increased frequency of chromosomal aberrations, a major contributing factor in the onset of tumorogenesis. Such unrepaired DNA damage can also lead to an increase in mitotic cell death as the cell struggles to successfully segregate its chromosomes. We were interested in investigating whether radiation-induced DNA damage could be introduced into both normal and cancerous human cells at a level and frequency that could evade detection by cellular repair mechanisms, ultimately resulting in increased cell death. We were also interested in studying the molecular mechanisms involved in this processes. DNA damage that is not detected and repaired can lead to an increased frequency of chromosomal aberrations, a major contributing factor in the onset of tumorogenesis. Such unrepaired DNA damage can also lead to an increase in mitotic cell death as the cell struggles to successfully segregate its chromosomes. We were interested in investigating whether radiation-induced DNA damage could be introduced into both normal and cancerous human cells at a level and frequency that could evade detection by cellular repair mechanisms, ultimately resulting in increased cell death. We were also interested in studying the molecular mechanisms involved in this processes. Materials/MethodsMultiple human cancer cell lines (HCT116, RKO, DU145 and PC-3) were exposed to either high (acute; ∼4500 cGy/hr) or low dose rate (LDR; 2 or 9.4 cGy/hr) ionizing radiation. Clonogenic survival assays, SDS/PAGE and immunoblot analyses for activated ATM and FACS analyses for activated H2AX were performed following radiation exposure at both acute and LDR radiation exposures. Multiple human cancer cell lines (HCT116, RKO, DU145 and PC-3) were exposed to either high (acute; ∼4500 cGy/hr) or low dose rate (LDR; 2 or 9.4 cGy/hr) ionizing radiation. Clonogenic survival assays, SDS/PAGE and immunoblot analyses for activated ATM and FACS analyses for activated H2AX were performed following radiation exposure at both acute and LDR radiation exposures. ResultsWe observed an increase in cell death among four different human cancer cell lines following LDR exposures compared to acute exposures of equivalent dose. We demonstrated that this increased cell killing was a consequence of ineffective activation of the critical damage sensor ATM and its downstream target H2AX (a cellular marker of sites of DNA damage). This phenomenon was also observed in normal primary human fibroblast cultures. The apparent reduction in clonogenic survival was not due to accumulation of senescent cells during the protracted radiation exposures prior to plating. The failure of LDR treated cells to sufficiently activate ATM was, in fact, not due to the presence of dysfunctional ATM protein following LDR exposure as cells pre-treated with LDR radiation were shown to elicit a normal ATM response following subsequent acute exposures. We observed an increase in cell death among four different human cancer cell lines following LDR exposures compared to acute exposures of equivalent dose. We demonstrated that this increased cell killing was a consequence of ineffective activation of the critical damage sensor ATM and its downstream target H2AX (a cellular marker of sites of DNA damage). This phenomenon was also observed in normal primary human fibroblast cultures. The apparent reduction in clonogenic survival was not due to accumulation of senescent cells during the protracted radiation exposures prior to plating. The failure of LDR treated cells to sufficiently activate ATM was, in fact, not due to the presence of dysfunctional ATM protein following LDR exposure as cells pre-treated with LDR radiation were shown to elicit a normal ATM response following subsequent acute exposures. ConclusionsThe data presented here are the first to demonstrate that low levels of DNA damage introduced by LDR radiation do not activate the DNA damage sensor ATM, a critical cellular response mechanism. This occurs despite the appearance of activated H2AX, a marker of DNA double-strand breaks. This lack of ATM activation and lower activated H2AX ultimately results in greater amounts of cell killing compared to equivalent doses of acute radiation exposures. These findings may aid the further understanding of the early cellular DNA damage response mechanisms and have broad-range implications for clinical treatment of solid tumors The data presented here are the first to demonstrate that low levels of DNA damage introduced by LDR radiation do not activate the DNA damage sensor ATM, a critical cellular response mechanism. This occurs despite the appearance of activated H2AX, a marker of DNA double-strand breaks. This lack of ATM activation and lower activated H2AX ultimately results in greater amounts of cell killing compared to equivalent doses of acute radiation exposures. These findings may aid the further understanding of the early cellular DNA damage response mechanisms and have broad-range implications for clinical treatment of solid tumors

Purpose: The potential of halogenated pyrimidines for the radiosensitization of human malignant gliomas remains unrealized. To assess the role of local delivery for radiosensitization, we tested a synthetic, implantablebiodegradable polymer for the controlled release of 5-iodo-2‘-deoxyuridine (IUdR) both in vitro and in vivo and the resultant radiosensitizationof human malignant glioma xenografts in vivo.Materials and methods: In vitro: To measure release, increasing (10%, 30%, 50%) proportions (weight/weight) of IUdR in the polyanhydride [(poly(bis(p-carboxyphenoxy)-propane) (PCPP) :sebacic acid (SA) (PCPP : SA ratio 20 : 80)] polymer discs were incubated (1 ml phosphate-buffered saline, 37° C). The supernatant fractions were serially assayed using high performance liquid chromatography. To measure modulation of release,polymer discs were co-loaded with 20 μCi 5-125-iodo-2‘-deoxyuridine (125-IUdR) and increasing (10%, 30%, or 50%) proportions of D-glucose. To test radiosensitization, cells (U251 human malignant glioma) were sequentially exposed to increasing (0 or 10 μM) concentrations of IUdR and increasing (0, 2.5, 5.0, or 10 Gy) doses of acute radiation. In vivo: To measure release, PCPP : SA polymerdiscs having 200 μCi 125-IUdR were surgically placed in U251 xenografts (0.1—0.2 cc) growing in the flanksof nude mice. The flanks were reproducibly positioned over a collimated scintillation detector and counted. To measure radiosensitization, PCPP : SApolymer discs having 0% (empty) or 50% IUdR wereplaced in the tumor or contralateral flank. After five days, the tumors were acutely irradiated (500 cGy × 2 daily fractions).Results: In vitro: Intact IUdR was released from the PCPP : SA polymer discs in proportion to the percentage loading. After 4 days the cumulative percentages of loaded IUdR that were released were 43.7 $plusmn; 0.1, 70.0 ± 0.2, and 90.2 ± 0.2 (p < 0.001 ANOVA) for the 10, 30, and 50% loadings. With 0, 10, 30,or 50% D-glucose co-loading, the cumulative release of 125-IUdR from PCPP : SA polymers was 21, 70, 92, or 97%(p < 0.001), respectively, measured 26 days after incubation.IUdR radiosensitized U251 cells in vitro. Cell survival (log10) was – 2.02 ± 0.02 and – 3.68± 0.11 (p < 0.001) after the 10 Gy treatment and no (control) or 10 μM IUdR exposures, respectively. In vivo: 125-IUdR Release: The average counts (log10 cpm ± SEM) (hours after implant) were 5.2 ± 0.05 (0.5), 4.3 ± 0.07 (17), 3.9 ± 0.08 (64), and 2.8 ± 0.06 (284). Radiosensitization: Afterintratumoral implantation of empty polymer or intratumoral 50%IUdR polymer, or implantation of 50% IUdR polymers contralateral to tumors, the average growth delays of tumors to4 times the initial volumes were 15.4 ± 1.8, 20.1 + 0.1,and 20.3 + 3.6 (mean + SEM) days, respectively (p = 0.488one-way ANOVA). After empty polymer and radiation treatments,no tumors regressed and the growth delay was 31.1 + 2.1 (p = 0.046 vs. empty polymer alone) days. After implantation of50% IUdR polymers either contralateral to the tumors orinside the tumors, followed by radiation, tumors regressed; growth delays to return to the initial average volumes of 14.0+ 3.6 or 24.2 + 0.2 (p < 0.01) days, respectively.Conclusions: Synthetic, implantable biodegradable polymers hold promise for the controlled release and local delivery ofIUdR for radiosensitization of gliomas.

In response to anticancer therapeutics, human colon cancer cells growing in vitro either enter into a stable arrest or die, depending on the integrity of their cell-cycle checkpoints1. To test whether altered checkpoints can modulate sensitivity to treatment in vivo, xenografts were established from isogenic lines differing only in their p21 checkpoint status. Although all tumors with intact checkpoint function underwent regrowth after treatment with γ-radiation, a significant fraction of checkpoint-deficient tumors were completely cured. This difference in sensitivity was not detected by the clonogenic survival assay, because both arrest and death preclude outgrowth of colonies. These results demonstrate that checkpoint status affects sensitivity to anticancer treatments in vivo, and these findings have important implications for identifying and testing new therapeutic compounds.

Purpose:Manipulation of the cell cycle is a therapeutic goal in many cancers that feature irradiation as a major therapeutic modality.The raf-I protein is an important molecule in transduction of signals from the cell surface to the nucleus.Raf-1 phosphorylates downstream MAPK and its signaling cascade results in transcription factor activity that influences cell transformation and differentiation.c-raf-1 is located at chromosome 3~25, a chromosomal locus deleted in 80% of SCLCs and is a transforming oncogene with constitutive serindthreonine kinase activity when activated.However, large clinical series demonstrate that despite being frequently deleted in lung cancers, activation of the remaining c-raf-1 allele is extremely rare.These data suggested to us that c-raf-1 activation when it occurs, is negatively selected for and might function as a tumor suppressor in SCLC cells.To test this hypothesis, we transduced SCLC cells with an inducible rafconstruct (ARaf-I :ER) and determined the effects of an activated raf molecule on cell cycle, and expression of cyclins, cyclin dependent kinase inhibitors and cyclin dependent kinase activities in the presence and absence of low dose rate irradiation.Materials rind Methods:Rb deficient SCLC cells were transduced with an inducible &molecule, ARaf-1 :ER.This molecule is a fusion of the estrogen receptor and the catalytic domain of c-raf-1.The molecule is made constitutively and is sequestered by heat shock proteins (hsp) Estradiol activates the activity of the molecule by releasing it from its hsp chaperone.(Samuels,et.al.Mol Cell Bio.13:6241,1993)After demonstration of stable expression of ARaf-l:ER in the transduced cells, we determined whether activated ARaf-1:ER could modify cell cycle responses to low dose rate (0.25 Gyihr) irradiation and identified mediators of cell cycle control modified by ARaf-1:ER signaling. Results:Raf-1 :ER causes phosphorylation and activation of p4Up44 MAPK which can be reversed by exposure to small molecule MAPK kinase (MEK) inhibitorsCell cycle distribution and BrdU assays demonstrated that SCLC cells exposed to 0.25 Gy/ hr accumulate in Gl when ARaf-1:ER is activated whereas cells accumulate in G2 in the absence of ARaf-l:ER activation.The Gl cyclin E, and CDK inhibitors p27 and p57 are induced and CDK2 activity is reduced Furthermore, cdc2 p34 and cyclin B levels are reduced resulting in diminished cdc2 activity in cells when ARaf-1:ER is activated prior to exposure to low dose rate irradiation.These phenomena are inhibited by exposure of estradiol activated ARaf-1:ER cells to small molecule MEK inhibitors.Conclusions:Our data show that activation of MAPK by ARaf-1:ER can restore a normal response to DNA damage by low dose rate irradiation in Rb deficient cells and suggest that c-raf-I can function as a tumor suppressor gene when activated in SCLC cells.(Supported by CA5879, CA5884,

Purpose/Objective: Poly(ADP-ribosyl)ation is a postranslational modification of nuclear proteins catalyzed by poly(ADP-ribose)polymerase (PARP), with NAD + serving as substrate.PARP is strongly activated upon recogmtion of DNA stand breaks by its DNA-binding domain.Experiments with low-molecular-weight inhibitors of PARP (e. g. 3-aminobenzamide) have led to the view that PARP activity plays a role in DNA repair and possibly also in DNA replication, cell proliferation, and differentiation.Accumulating evidence for non-specific inhibitor effects prompted us to develope a molecular genetic system to inhibit PARP in living cells, i. e, to overexpress selectively the DNA-binding domaine of PARP as a dominant negative mutant.The radiation response modifying effect of the inhibition of poly(ADP-ribosyl)ation was examined Materials & Methods:The DNA-binding domain (DBD) of PARP under the control of a Dexamethasone ('Dex) inducible promoter was transfected into cells of the SV40transformed Chinese hamster cell line CO60 Stable transfectans with and without treatment with Dex were exposed to ionizing radiation (~°Co, 2.5 Gy/min) and the radiosensidvity determined using the colony forming assay.Survival curves were fitted according to the linear-quadratic model]. Results:Transfected cells exhibited a virtually complete inhibition of poly(ADP-ribosyl)ation after treatment with Dex as shown by immunofluoreseenee.Dex treated stable transfectants showed a marked radiosensitization in comparison to stable transfectants without Dex treatment (dose modification factor at 10% survival = 1.44). Conclusion:This molecular genetic approach of selective overexpression of the DBD of PARP in a trans-dominant fashion led to a specific inhibition of poly(ADP-ribosyl)ation.Cells overexpressing the DBD of PARP exhibited a marked sensitization to ionizing radiation.This effect could represent a mechanism for a gentherapeutie approach in radiation oncology

Purpose: To measure, quantify, and evaluate the planar dose-rate distribution for human tumor xenografts implanted into mice that are treated with Y-90-labeled monoclonal antibodies or bispecific antibodies and Y-90-labeled haptens.Methods and Materials: Twenty-five LS174T human colon carcinoma tumors grown subcutaneously in nude mice were treated with Y-90 by either directly labeled ZCE025 or bispecific ECA001-DBX antibody systems. A simple, quick technique using GAF(TM) radiochromic medium determined the dose-rate distribution in a plane passing through the tumor center. The dose-rate distribution is generated from exposure to activity situated in one-half of the tumor (0.045 to 0.83 g).Results: Planar dose-rate distributions were obtained from the tumor xenografts. Planar dose-rate histograms were computed along with the coefficients of variance and skewness of the distributions. The observed dose-rate distributions were quantitatively compared to those calculated for a uniformly distributed activity in a half-ellipsoid of the same volume and approximate shape as the tumor half. The observed dose-rate distributions were usually broader with a more positive coefficient of skewness than the dose-rate distributions calculated from the uniformly active half-ellipsoids. For Y-90, tumor shape plays an important role in determining the minimum tumor dose. For these tumors, the tumor minimum dose-rate is always observed along the edge, usually where the edge curvature is most convex. Larger tumors tended to have broader dose-rate distributions and more positive coefficients of skewness. Exceptions to this trend were associated with dose-rate maxima displaced from the central regions due to activity heterogeneity or tumor size greatly exceeding the range of emission. Calculations for dose rate from the conventional Medical Internal Radiation Dose (MIRD) formulation exceeded the average and minimum dose rate derived from radiochromic media. The coefficient of skewness became more positive for increasing time between injection and tumor excision, consistent with the activity evolving into a more uniform activity distribution.Conclusion: Using radiochromic media to measure the spatial dose-rate distribution is a valuable method for comparing the dose-rate heterogeneity among experimental tumor xenografts in animals treated with radiolabeled antibodies. Tumor size (relative to the particle range) and changes in activity distribution affect the dose-rate distribution that are reflected by changes in the coefficients of skewness and variation of the dose-rate area histogram. The increase in coefficients of variation and skewness with tumor size and time results from the size of the Y-90 beta particle penetration range that either exceeds or is comparable to the tumor dimensions. The minimum dose rate is more dependent, relative to the average and the maximum dose rates, on the curvature of the tumor surface.

We have outlined a model in which aging may be associated with changes in chromatin structure that produce alterations in the extent of DNA supercoiling. Our model would suggest that the major difference in a short-lived rodent and a long-lived human being would be reflected as the rate at which such changes occur. In support of this model we have presented data that rodent cells as a class are more resistant to PUVA than are human cells. Further, we have outlined corroborating data that would suggest that such resistance may reflect a difference in the extent of psoralen intercalation that in turn is dependent on DNA supercoiling. Since it is known that changes in DNA supercoiling can alter both the expression of genes and the repair of DNA, it is feasible that changes in supercoiling could lead to a deterioration both in gene regulation and in DNA fidelity. Our model relates to multistage carcinogenesis in a straightforward manner, predicting that cancer initiators produce a heritable change in chromatin structure, while cancer promoters induce transient changes in chromatin structure. We propose that this model is consistent with the developing molecular model of cancer as caused by the inappropriate expression of dominant transforming oncogene(s). Indeed, our model would predict that aging and carcinogen exposure would share a common capacity to alter chromatin structure within regions of the genome, with carcinogens perhaps more random than aging in their induction of such alterations.(ABSTRACT TRUNCATED AT 250 WORDS)

The majority of the high (12-fold elevated) baseline sister-chromatid exchanges (SCEs) that occur in the CHO mutant line EM9 appear to be a consequence of incorporated BrdUrd, and they arise during replication of DNA containing BrdUrd in a template strand. In normal CHO cells the alkaline elution patterns of DNA newly replicated on a BrdUrd-containing template are significantly altered compared with those seen during the replication on an unsubstituted template. The nascent DNA synthesized on such an altered template is delayed in reaching mature size, possibly because replication forks are temporarily blocked at sites occurring randomly along the template. Transient blockage of replication forks may be a prerequisite for SCE. The delay in replication on BrdUrd-substituted templates was greater in EM9 cells than in parental AA8 cells and was also greater in AA8 cells treated with benzamide, an inhibitor of poly(ADPR) polymerase, than in untreated AA8 cells. Under these conditions, treatment with benzamide also produced a 7-fold increase in SCEs in AA8. An EM9-derived revertant line that has a low baseline SCE frequency showed less delay in replication on BrdUrd-substituted templates than did EM9. However, under conditions where the template strand contained CldUrd, which was shown to produce 4-fold more SCEs than BrdUrd in AA8 cells, the replication delay in AA8 was not any greater in the CldUrd-substituted cells. Thus, other factors besides the delay appear to be involved in the production of SCEs by the template lesions resulting from incorporation of the halogen-substituted pyrimidine molecules.