The role of Bcl-X(S) in radiation sensitivity.

Bcl-X(S) is a pro-apoptosis member of the Bcl2 family that has been shown to induce cell death and enhance chemosensitivity. We have investigated the effect of Bcl-X(S) overexpression on radiation sensitivity. Using a tetracycline-repressible system, we found that removal of tetracycline for 16 h induced Bcl-X(S) and reduced the surviving fraction of NIH 3T3 cells to 25%. However, radiation sensitivity was not significantly affected by Bcl-X(S) expression; the mean inactivation doses for Bcl-X(S) repressed and Bcl-X(S) induced cells were 2.7 +/- 0.3 and 2.3 +/- 0.1 Gy, respectively. We conclude that Bcl-X(S) induces cell death without affecting radiation sensitivity. These results suggest that mitochondrial pathways to apoptosis may not have a significant role in survival after irradiation.

Cytochrome c depletion upon expression of Bcl-XS.

We have shown previously that Bcl-XS causes acute cell death in 3T3 cells without activating caspases (Fridman, J. S., Benedict, M. A., and Maybaum, J. (1999) Cancer Res. 59, 5999-6004). In this study, we determined that the explanation for lack of caspase activation is the cellular depletion of cytochrome c. Electron microscopy revealed gross structural changes in the mitochondria of Bcl-XS-expressing cells; however, cytochrome c was not detected in cytosolic fractions from these cells. Surprisingly, it was determined that cellular cytochrome c levels decreased as Bcl-XS expression levels increased. Experiments performed to eliminate other possible explanations for the lack of caspase activation showed that these 3T3 cells have a functional cytoplasmic apoptosome, a complex of proteins that form a functional trigger capable of activating the proximal caspase in an apoptotic pathway Chinnaiyan, A. M. (1999) Neoplasia 1, 5-15, as cytosolic extracts from these cells were capable of cleaving pro-caspase-9. These cells were also able to release cytochrome c from their mitochondria after appropriate stimulation, other than Bcl-XS expression (i.e. withdrawal from serum for 24 h), and initiate a cell death that is inhibited by a dominant negative caspase-9. We conclude that lack of caspase activation is due to a Bcl-XS-induced depletion of active cytochrome c, a phenomenon that represents an alternative cell death effector pathway and/or a novel mechanism for regulating caspase activation.

Escherichia coli thymidylate synthase expression protects human cells from the cytotoxic effects of 5-fluorodeoxyuridine more effectively than human thymidylate synthase overexpression.

In this study, we compared the relative abilities of human thymidylate synthase (hTS) and Escherichia coli thymidylate synthase (eTS) expression to confer resistance to the cytotoxic effects of treatment with the TS inhibitor 5-fluorodeoxyuridine (FdUrd). G418-selected clones expressing either form of the protein were significantly more resistant than the lacZ-expressing clone, VALZ2, to FdUrd-induced cytotoxicity. Although eTS-expressing clones expressed 2- to 3-fold more TS protein than hTS-overexpressing clones, the representative eTS-expressing clone, VAEG8, and hTS-overexpressing clone, VAHGC, were equally sensitive to an FdUrd-induced loss of clonogenicity; in addition, a large fraction of either form of exogenously expressed TS appeared to be inactive in the intact cell. The clones differed, however, in their responses to leucovorin (LV). Although LV significantly enhanced FdUrd-induced TS inhibition, growth inhibition, and cytotoxicity in VAHGC cells, it had no effect on these parameters in VAEG8 cells. These results suggest that eTS may more efficiently confer resistance to FdUrd plus LV when expressed for the purposes of a "host protection" strategy in vivo.

Expression of endogenously activated secreted or cell surface carboxypeptidase A sensitizes tumor cells to methotrexate-alpha-peptide prodrugs.

Methotrexate (MTX) is one of the most commonly used agents in the treatment of solid malignancies; however, the toxicities of MTX to bone marrow and gastrointestinal tract complicate this therapy. We, therefore, propose a gene-dependent enzyme prodrug therapy to limit these toxicities by localizing the production of MTX to the site of the tumor. The combination of MTX-alpha-peptide prodrugs, which cannot be internalized by the cellular reduced folate carrier, with carboxypeptidase A (CPA), which can remove the blocking peptide, has been demonstrated previously in vitro using antibody-dependent enzyme prodrug therapy. CPA is normally synthesized as a zymogen that is inactive without proteolytic removal of its propeptide by trypsin. Therefore, to adapt this system to gene-dependent enzyme prodrug therapy, a mutant form of CPA was engineered, CPA(ST3), that does not require trypsin-dependent zymogen cleavage but is instead activated by ubiquitously expressed intracellular propeptidases. Purification, peptide sequencing, and kinetic analysis indicated that mature CPA(ST3) is structurally and functionally similar to the trypsin-activated, wild-type enzyme. In addition, CPA(ST3)-expressing tumors cells were sensitized to MTX prodrugs in a dose- and time-dependent manner. To limit diffusion of CPA, a cell surface localized form was generated by constructing a fusion protein between CPA(ST3) and the phosphatidylinositol linkage domain from decay accelerating factor. SDS-PAGE and flow cytometric analysis of infected tumor cells indicated that CPA(DAF) was cell surface localized. Finally, after retroviral transduction, this enzyme/prodrug strategy exhibited a potent bystander effect, even when <10% of the cells were transduced, because extracellular production of MTX sensitized both transduced and nontransduced cells.

bcl-X(S)-induced cell death in 3T3 cells does not require or induce caspase activation.

Using a tetracycline-regulated expression system, we have shown that expression of bcl-X(s) is sufficient to induce acute cell death in 3T3 cells, and that the manner in which these cells die is both morphologically and biochemically different from Fas/CD95-induced apoptosis. bcl-X(s) expression causes loss of the inner mitochondrial membrane potential (deltapsim) but does not induce caspase activation. Loss of viability, as determined by mitochondrial function and ethidium bromide exclusion, was not inhibited by the broad-spectrum caspase inhibitor zVAD-fmk or by expression of a dominant negative caspase 9 (9DN). However, zVAD-fmk was efficacious in inhibiting cell death triggered by an activating anti-Fas/CD95 antibody. In addition, bcl-X(s) does not possess the 5th and 6th alpha-helices (thought to be the membrane-spanning domains in bcl-2, bcl-X(L), and bax) and, therefore, should not be able to form membrane channels, thus eliminating this possible mechanism of action. The finding that bcl-X(s) kills 3T3 cells without caspase activation, along with the absence of membrane spanning domains in bcl-X(s), may, therefore, represent a novel cell death pathway for the pro-death bcl-2 family members.

N-Nitrosodimethylamine-mediated cytotoxicity in a cell line expressing P450 2E1: evidence for apoptotic cell death.

N-Nitrosodimethylamine (NDMA) is an acute hepatotoxin and potent carcinogen. The metabolic activation of NDMA to reactive metabolites is a critical step for the expression of its toxic and carcinogenic potential. We have previously demonstrated a strong correlation between methylation of cellular macromolecules and NDMA-mediated cytotoxicity, and we have demonstrated that reactive oxygen species may partially contribute to the toxic effects in P450 2E1-expressing cells. The mode of cell death in NDMA-treated monolayer cultures exhibited the following characteristics: (i) condensation of nuclear chromatin as demonstrated by using Hoechst 33258 staining, (ii) DNA fragmentation as detected by combining pulsed field and conventional agarose gel electrophoresis, and (iii) DNA double strand breaks determined by using the in situ terminal deoxynucleotidyl transferase assay and flow cytometric analysis. These results indicate that reactive metabolites of NDMA trigger activation of the signal pathway for apoptotic cell death in these P450-expressing cells. The NDMA-mediated cell death was partially prevented by the endonuclease inhibitor, aurintricarboxylic acid, as well as the caspase inhibitors, acetyl-Asp-Glu-Val-Asp-CHO and acetyl-Tyr-Val-Ala-Asp-CHO. The cell cycle distribution was altered in NDMA-treated cells resulting in an increase in the G2/M phase and a decrease in the G1 phase. Our results suggest that DNA degradation, the inability to complete DNA repair, the biochemical events associated with G2/M arrest, and the process of apoptotic death all result from P450 2E1-catalyzed metabolism of NDMA.

A caspase-resistant form of Bcl-X(L), but not wild type Bcl-X(L), promotes clonogenic survival after ionizing radiation.

Bcl-2 and Bcl-X(L) belong to a family of proteins overexpressed in a variety of human cancers which inhibit apoptosis in response to a number of stimuli including chemotherapeutic agents and ionizing radiation. To better understand the role of these polypeptides in modulating the response of cancer cells to ionizing radiation we used cell lines that were engineered to overexpress the two polypeptides. Although Bcl-2 and Bcl-X(L) overexpression resulted in inhibition of radiation-induced apoptosis, it did not result in enhanced clonogenic survival. Consistent with this was the observation that Bcl-2 and Bcl-X(L) protected cells from DNA fragmentation, loss of mitochondrial membrane potential, and caspase activation for up to 72 hours after irradiation. Beyond 72 hours, there was a rapid loss in the ability of Bcl-2 and Bcl-X(L) to inhibit these markers of apoptosis. When Bcl-X(L) was analyzed at 72 hours after irradiation and beyond, a rapid accumulation of a 16-kDa form of Bcl-X(L) was observed. To test the hypothesis that cleavage of the 29-kDa form of Bcl-X(L) by caspases to a 16-kDa polypeptide results in its inability to inhibit apoptosis beyond 72 hours, we constructed a cell line that overexpressed a caspase-resistant form of Bcl-X(L) (Bcl-X(L)-deltaloop). Cells overexpressing Bcl-X(L)-deltaloop were resistant to apoptosis beyond 72 hours after irradiation and did not contain the 16-kDa form at these time points. In addition, Bcl-X(L)-deltaloop overexpression resulted in enhanced clonogenic survival compared with control or Bcl-X(L) overexpressing cells. These results provide a molecular basis for the observation that expression of Bcl-2 or Bcl-X(L) is not a prognostic marker of tumor response to cancer therapy.

Recognition and resolution of potential workplace violence.

OSHA realizes the significance of providing a safe and healthy environment. Its objective is to eliminate or diminish employee exposure to workplace violence by establishing a preventive management program comprised of effective security mechanisms, administrative work practices and other safety control measures. In addition to creating a safer work environment, these systems strengthen employee confidence and productivity and reduce employer workers' compensation fees.

Mechanism and pharmacological specificity of dUTPase-mediated protection from DNA damage and cytotoxicity in human tumor cells.

PURPOSE: We have reported previously that the expression of E. coli dUTPase (dutE) can protect HT29 cells from 5-fluorodeoxyuridine (FdUrd)-induced DNA fragmentation and cytotoxicity. In the study reported here, we further characterized the ability of dutE expression in one HT29 clone, dutE7, to alter the effects of treatment with FdUrd and other thymidylate synthase (TS) inhibitors. In addition, we developed two HuTu80 dutE-expressing clones using a pLNCX-dutE retroviral construct and tested their sensitivity to FdUrd-induced DNA fragmentation and cytotoxicity. METHODS: Both a dutE retroviral expression system and a dutE antibody were developed to facilitate the generation and screening of dutE-expressing clones. HT29 and HuTu80 clones expressing dutE were tested for drug-induced DNA damage with either alkaline elution or pulsed field gel electrophoresis and drug-induced loss of clonogenicity. RESULTS: Following a 24-h treatment with 100 microM CB3717 or 500 nM methotrexate (MTX), dutE7 cells were significantly less sensitive to drug-induced loss of clonogenicity than con3 cells. DutE7 cells were also resistant to CB3717-induced DNA fragmentation at 24 h. However, following a 48-h treatment with CB3717 or MTX there was no difference in survival between con3 and dutE7 cells, even though DNA damage was still greatly attenuated in the dutE7 cell line. In addition, expression of dutE in two HuTu80 clones, 80 C and 80 K, did not protect these cells from FdUrd-induced DNA damage or cytotoxicity. CONCLUSIONS: We conclude that the role of uracil misincorporation and subsequent DNA damage in cytotoxicity induced by TS inhibitors, in HT29 cells, is time dependent, and that cytotoxicity caused by long-term exposure to these drugs is largely independent of resultant DNA damage, in this cell line. The inability of dutE to protect HuTu80 cells from FdUrd further suggests that the significance of uracil misincorporation resulting from TS inhibition varies among cell lines.

Expression of Bcl-XS alters cytokinetics and decreases clonogenic survival in K12 rat colon carcinoma cells.

bcl-XS, a member of the bcl-2 family, has been shown to induce and/or sensitize some cells to undergo programmed cell death, and to negate the anti-apoptotic activity of bcl-XL and bcl-2 by mechanisms which are still uncertain. To help understand these mechanisms we have established stable derivatives of the K12 rat colon carcinoma cell line that express bcl-XS in a tetracycline-regulated manner, using an autoregulatory retroviral cassette. When bcl-XS expression is induced, we observe two phenotypic responses. A small fraction of cells appear to undergo spontaneous apoptosis while the majority of cells undergo a form of cytostasis. In the latter case, the cells stop dividing (or divide a limited number of times at a retarded rate) and swell to many times their original size. These cells can take on a ghostlike appearance and subsequently detach from the culture plates and die or they may remain intact in a hindered state of proliferation. Doubling times were calculated to be 31.4 h in the presence of tetracycline and 50.4 h without tetracycline, bcl-XS expression also causes dramatic alterations in the cell cycle distribution of K12 cells manifesting as a substantial decrease (approximately 50%) in the fraction of S phase cells with a concomitant increase in the G1 population. Continuous expression of bcl-XS, at levels approximately equal to that of bcl-XL, decreased the viability of K12 cells as demonstrated by a log decline in clonogenic survival. This decrease occurred without considerable apoptosis or a compensatory increase in the level of bcl-XL. None of these phenotypes were present in control cells expressing beta-galactosidase in a similar retroviral cassette. These observations demonstrate that bcl-XS can have substantial cytokinetic effects under circumstances that produce relatively little apoptosis.

Prevention of fluorodeoxyuridine-induced cytotoxicity and DNA damage in HT29 colon carcinoma cells by conditional expression of wild-type p53 phenotype.

We have examined the effects of conditionally expressing wild-type p53 activity in HT29 cells on DNA damage and cytotoxicity caused by exposure to fluorodeoxyuridine (FdUrd). Expression of wild-type p53 phenotype for 24 hr before FdUrd treatment provided HT29 cells with virtually complete protection from cytotoxicity caused by this drug. In addition, wild-type p53 expression also prevented FdUrd-induced DNA double-strand breaks and, unexpectedly, single-strand breaks in parental (mature) DNA. Temporary expression of wild-type p53 activity in the absence of drug treatment caused some loss of clonogenicity, although the magnitude of this cytotoxic effect was small compared with the level of cell kill obtained by treatment with cytotoxic drugs for similar periods of time, indicating that HT29 cells are not highly sensitive to induction of programmed cell death by wild-type p53. Because these observations conflict with previously suggested models for FdUrd-induced damage to parental DNA, we propose an alternative model to explain how incorporation of uracil into nascent DNA might result in single-strand breaks in the opposite (parental) strand and how these breaks might be converted to the double-strand breaks that produce cell death.

Expression of wild-type p53 stimulates an increase in both Bax and Bcl-xL protein content in HT29 cells.

It has been shown previously that wild-type p53 activity can simultaneously up-regulate Bax, a protein which predisposes cells to programmed cell death (PCD), and down-regulate Bcl-2, a protein which antagonizes PCD. These findings have been interpreted to suggest that correction of the mutant p53 status of some tumor cells may be a means of increasing their sensitivity to chemotherapeutic agents, by increasing their likelihood of undergoing PCD. We show here that when wild-type p53 activity is expressed in HT29 human colon cancer cells by use of a temperature sensitive p53 mutant, Bax levels rise, but so do levels of Bcl-xL protein. These observations indicate that Bcl-2 and Bcl-xL are regulated differently in response to wild-type p53 activity and that, while correction of mutant p53 phenotype may effectively kill cells having Bcl-2 as their major defense against PCD, this is not necessarily the case in cells using Bcl-xL as their primary defense.

Fluorodeoxyuridine-mediated cytotoxicity and radiosensitization require S phase progression.

The role of cell cycle redistribution in fluoropyrimidine-mediated radiosensitization remains unresolved. To determine if radiosensitization resulted from the redistribution of cells into a sensitive phase of the cell cycle, we assessed fluorodeoxyuridine (FdUrd)-mediated radiosensitization in flow-sorted mid-S phase HT29 human colon cancer cells. We hypothesized that if FdUrd-mediated radiosensitization were strictly the result of cell cycle redistribution, FdUrd-treated mid-S phase cells would remain as radioresistant as mid-S phase cells cultured in the absence of drug. However, we found that the mid-S phase cells from FdUrd-treated populations were markedly radiosensitized. To assess the role of S phase progression in radiosensitization, we exposed FdUrd-treated cells to aphidicolin, an inhibitor of DNA polymerase alpha, prior to irradiation. We found that aphidicolin blocked the radiosensitizing (and cytotoxic) effects of FdUrd. These results, combined with our previous observations that FdUrd-treated cells at the G1/S boundary are minimally sensitized, appear to disprove the hypothesis that sensitization results strictly from cell cycle distribution. Furthermore, they suggest that a key aspect of both FdUrd-mediated radiosensitization and cytotoxicity is S phase progression on a damaged DNA template.

Dependence of fluorodeoxyuridine-induced cytotoxicity and megabase DNA fragment formation on S phase progression in HT29 cells.

The relationship between cell cycle progression and induction of DNA double-strand breaks and cytotoxicity by exposure to fluorodeoxyuridine (FdUrd) was studied in HT29 human colon cancer cells. Fractionation of drug-treated populations by centrifugal elutriation yielded subpopulations having widely divergent abilities to progress through S phase in the presence of the drug. One of these subpopulations, which appeared to undergo coordinated growth arrest, was resistant to FdUrd cytotoxicity and DNA damage. In contrast, the subpopulation which was able to progress furthest through S phase in the presence of FdUrd underwent unbalanced growth arrest (i.e., increase in size and mass out of proportion to DNA synthesis), and displayed both DNA double-strand break formation (assayed by pulsed field gel electrophoresis) and loss of clonogenicity. When cells were elutriated prior to drug treatment, producing fractions enriched in cells at various cell cycle stages, no significant differences in sensitivity to FdUrd-induced cytotoxicity were detected among elutriation fractions. These findings support the model that, in HT29 cells, progression into and through S phase during drug treatment is an important determinant of FdUrd-induced DNA damage and cytotoxicity, but that the cell cycle position at the start of drug exposure is not a critical factor for these effects.

Lack of dependence of 5-fluorodeoxyuridine-mediated radiosensitization on cytotoxicity.

It has been proposed that fluoropyrimidine-mediated cytotoxicity and radiosensitization are closely correlated. We have shown that HT29 human colon cancer cells transfected with the E. coli dUTPase gene are resistant to 5-fluorodeoxyuridine (FdUrd)-mediated cytotoxicity, presumably through more effective elimination of dUTP. We used these cells to assess the association between radiosensitization and cytotoxicity produced by FdUrd. The radiation sensitivities of the clones expressing elevated dUTPase activity (dutE clones) were similar to those of untransfected HT29 cells or HT29 cells which had been transfected with only the expression vector for the E. coli gene (con clones). We found that FdUrd produced similar increases in radiation sensitivity regardless of dUTPase activity. Levels of dUTPase in the dutE clones remained elevated during the entire period of FdUrd exposure, demonstrating that the lack of difference between dutE and Con clones was not a reflection of down-regulation of dUTPase activity by FdUrd. Flow cytometry showed that all clones progressed past the G1/S-phase boundary and into early S phase during FdUrd treatment. These data suggest that the mechanisms of FdUrd-mediated cytotoxicity and radiosensitization are not closely linked. These findings, combined with our previous investigations, are consistent with the hypothesis that radiosensitization occurs in cells which progress past the G1/S-phase boundary in the presence of FdUrd.

Dependence of fluorodeoxyuridine-mediated radiosensitization on S phase progression.

Recent evidence casts doubt on the hypotheses that fluoropyrimidine-mediated radiosensitization is related to cytotoxicity or to cell cycle redistribution into the G1/S boundary. We hypothesized that cells that are capable of progressing into S phase in the presence of fluorodeoxyuridine may also be more susceptible to radiation-induced damage. To test this hypothesis, fluorodeoxyuridine (FdUrd)-treated HT29 human colon cancer cells were separated by centrifugal elutriation into four fractions (1-4) containing a range of cells from those at the G1/S boundary (fraction 1) to those which had progressed approximately 11% into S phase (fraction 4). We found that fraction 4 cells showed significantly greater radiosensitization than fraction 1 cells. We also compared the effects of fluorodeoxyuridine on HT29 and SW620 human colon cancer cells. We found that, in contrast with HT29 cells, SW620 cells arrested at the G1/S boundary and were minimally radiosensitized. Finally, we found that an increase in sensitivity was correlated with a decrease in the rate of repair of DNA double-strand and single-strand breaks (assessed by asymmetric field inversion gel electrophoresis and alkaline elution respectively). These findings are consistent with the hypothesis that fluorodeoxyuridine-mediated radiosensitization depends on S phase progression and a decreased ability to repair radiation-induced DNA damage.

Dependence of 5-fluorouracil-mediated radiosensitization on DNA-directed effects.

PURPOSE: Although 5-fluorouracil (FUra) has been demonstrated to be a radiation sensitizer both in the laboratory and the clinic, it is not known whether radiosensitization results primarily from FUra's DNA or RNA-directed effects. METHODS AND MATERIALS: We studied the radiosensitizing effects of FUra +/- thymidine (dThd)) on HT29 human colon cancer cells, which are relatively sensitive to the DNA-directed action of FUra, in comparison to SW620 and HuTu80 human colon cancer cells, which are relatively resistant to FUra's DNA-directed effects. We hypothesized that if FUra were acting chiefly through DNA dependent mechanisms, HT29 cells would (a) show greater radiosensitization than SW620 and HuTu80 cells under the same conditions of exposure; and (b) demonstrate selective reversal of radiation sensitivity (compared to cytotoxicity) in the presence of FUra + dThd, compared to FUra alone. RESULTS: We found that the enhancement ratio produced by a 24 h exposure to 10 microM FUra was significantly greater in HT29 cells compared to SW620 and HuTu80 cells (enhancement ratios of 2.1 +/- 0.1; 1.1 +/- 0.1, and 1.3 +/- 0.1, respectively). Furthermore, in HT29 cells, dThd blocked FUra-mediated radiosensitization to a greater extent than FUra-mediated cytotoxicity. Thus, our hypotheses were confirmed. CONCLUSION: These findings support the concept that the manipulation of FUra's DNA-dependent actions, for example, through modulators of thymidylate synthase (TS) activity, may increase radiosensitization in clinical trials in the treatment of gastrointestinal cancers. However, since resistance to the DNA-directed effects of fluoropyrimidines can result from mechanisms unrelated to TS inhibition, additional strategies will be required to potentiate fluoropyrimidine-mediated radiosensitization.

Bcl-2 inhibits chemotherapy-induced apoptosis in neuroblastoma.

bcl-2 is the first member of a new class of protooncogenes the products of which inhibit programmed cell death (PCD) or apoptosis. We have previously determined that Bcl-2 is expressed in a significant percentage of untreated primary neuroblastoma (NBL) tumors. In these specimens Bcl-2 expression correlated with other markers of poor prognosis suggesting a role for Bcl-2 in the malignant behavior of NBL tumor cells. To investigate this possibility, a Bcl-2-negative human NBL cell line (Shep-1) was transfected with a bcl-2 expression vector (pSFFVneo-bcl-2). Multiple unique clones were isolated which showed variable levels of Bcl-2 protein by quantitative immunoprecipitation. Vector-transfected controls were generated simultaneously. Clones expressing high levels of Bcl-2 were resistant to cisplatin- and etoposide-induced cytotoxicity in a dose-dependent manner. Analysis of propidium iodide-stained nuclei by flow cytometry after cisplatin or etoposide treatment revealed marked DNA degradation in vector-transfected controls whereas bcl-2 transfectants showed a dose-dependent inhibition of DNA degradation. Analysis by pulsed-field gel electrophoresis revealed relatively large fragment DNA degradation (approximately 50 kilobases) in the absence of internucleosomal degradation in vector-transfected control cells treated with either cisplatin or etoposide. In contrast, Bcl-2-expressing cells showed significantly less DNA degradation at all time points. These single gene transfection experiments have revealed that expression of Bcl-2 renders specific NBL cells resistant to chemotherapy-induced PCD and support the hypothesis that Bcl-2 enhances the malignant phenotype of NBL by promoting tumor resistance to chemotherapy agents.

Kinetics of bromodeoxyuridine elimination from human colon cancer cells in vitro and in vivo.

We have previously shown that the thymidine analogue radiation sensitizer bromodeoxyuridine (BrdUrd) is incorporated into human tumors to a greater extent than into the livers of athymic mice bearing these tumors as xenografts. However, incorporation into the intestine and bone marrow exceeds that of the tumor (T. S. Lawrence, M. A. Davis, J. Maybaum, S. K. Mukhopadhyay, P. L. Stetson, D. P. Normolle, P. E. McKeever, and W. D. Ensminger, Cancer Res., 52: 3698-3704, 1992). We hypothesized that the ratio of tumor incorporation to intestinal or bone marrow incorporation might increase during a period of drug elimination following the termination of an infusion. To test this hypothesis, we infused athymic mice bearing HT29 human colon cancer xenografts with BrdUrd and measured incorporation in the tumor and normal tissues up to 7 days after the infusion was discontinued. In addition, we assessed the effect of exposure to BrdUrd on subsequent incorporation in vitro and in vivo through the use of a stable isotope of BrdUrd ("isotopic BrdUrd"), which could be differentiated from normotopic BrdUrd using the gas chromatographic-mass spectrometric assay. We found a significant increase in the ratio of BrdUrd in the tumor compared to bone marrow and intestine during the drug elimination period. We also found that BrdUrd incorporation slowed the kinetics of subsequent BrdUrd incorporation and elimination. These findings suggest that when the radiation dose-limiting organ is rapidly proliferative, such as the intestine or bone marrow, delivering radiation during a drug elimination period may improve the therapeutic index.

Induction of resistance to fluorodeoxyuridine cytotoxicity and DNA damage in human tumor cells by expression of Escherichia coli deoxyuridinetriphosphatase.

Recent studies from our laboratory suggested that, in some human colorectal tumor cell lines, sensitivity to fluorodeoxyuridine may depend upon the extent of dUTP accumulation that occurs following drug treatment and that elevation of dUTPase activity might be the basis for some instances of resistance to fluoropyrimidines. To test this model, we expressed Escherichia coli dUTPase in an established human tumor cell line (HT29) and measured the effect of this manipulation on response to fluorodeoxyuridine. As predicted, HT29 derivatives containing dUTPase activity 4-5-fold higher than controls were protected from fluorodeoxyuridine-induced loss of clonogenicity and from formation of DNA double strand breaks. These data provide the first direct evidence that alteration in a component of the uracil misincorporation/misrepair pathway can confer resistance to fluoropyrimidines in human tumor cells.