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C E Canman

Publications and source records attributed to C E Canman.

27 records · Page 2Linked to original sources
PubMed1995

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.

Cell Survival↗
PubMed1994

The p53-dependent G1 cell cycle checkpoint pathway and ataxia-telangiectasia.

The p53 protein is a critical participant in a signal transduction pathway which mediates a G1 cell cycle arrest and apoptotic cell death in mammalian cells after ionizing irradiation. Cells from patients with the cancer-prone, radiation-sensitive disorder, ataxia-telangiectasia (AT), exhibit suboptimal (delayed and/or defective) induction of p53 protein after ionizing radiation with some dependence on dose. Other protein products which participate in this signal transduction pathway, including p21WAF1/CIP1, Gadd45, and Mdm2, are also suboptimally induced in AT cells after ionizing radiation. Induction of p53 is also abnormal in AT cells following treatment with methylmethanesulfonate and bleomycin but appears relatively normal following treatment with UV-C irradiation or the topoisomerase inhibitors, etoposide and camptothecin. These results demonstrate a specific defect in this p53-dependent signal transduction pathway in AT cells. Potential models for this observed specificity of the AT defect as measured by p53 induction include problems with responses to: (a) single-strand, but not double-strand, DNA breaks; or (b) chemically, but not enzymatically, generated DNA ends.

Ataxia Telangiectasia↗
PubMed1994

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.

Base Sequence↗
PubMed1994

WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis.

The tumor growth suppressor WAF1/CIP1 was recently shown to be induced by p53 and to be a potent inhibitor of cyclin-dependent kinases. In the present studies, we sought to determine the relationship between the expression of WAF1/CIP1 and endogenous regulation of p53 function. WAF1/CIP1 protein was first localized to the nucleus of cells containing wild-type p53 and undergoing G1 arrest. WAF1/CIP1 was induced in wild-type p53-containing cells by exposure to DNA damaging agents, but not in mutant p53-containing cells. The induction of WAF1/CIP1 protein occurred in cells undergoing either p53-associated G1 arrest or apoptosis but not in cells induced to arrest in G1 or to undergo apoptosis through p53-independent mechanisms. DNA damage led to increased levels of WAF1/CIP1 in cyclin E-containing complexes and to an associated decrease in cyclin-dependent kinase activity. These results support the idea that WAF1/CIP1 is a critical downstream effector in the p53-specific pathway of growth control in mammalian cells.

Alleles↗
PubMed1993

Resistance to fluorodeoxyuridine-induced DNA damage and cytotoxicity correlates with an elevation of deoxyuridine triphosphatase activity and failure to accumulate deoxyuridine triphosphate.

Deoxyuridine triphosphate (dUTP) misincorporation and uracil misrepair have long been implicated in fluoropyrimidine-induced DNA damage; however, the enzymatic activities responsible for these lesions have not been previously identified as critical determinants of overall sensitivity to the antitumor effects of these agents. The purpose of this study was to determine whether differences in uracil misincorporation/misrepair could account for the difference in sensitivity to fluorodeoxyuridine (FdUrd)-induced cytotoxicity and DNA damage in 2 human colorectal tumor cell lines having identical sensitivities to FdUrd-induced thymidylate synthase inhibition. Compared to HT29 cells, SW620 cells were resistant to both cytotoxicity and induction of DNA double-strand breaks, as assessed by pulse field gel electrophoresis. Alkaline elution experiments demonstrated that this resistance coincided with delayed induction of DNA single-strand breaks on parental DNA and, to a lesser extent, on nascent DNA. Following treatment with FdUrd for 24 h, HT29 cells accumulated 904 +/- 273 pmol deoxyuridine triphosphate (dUTP)/10(7) cells, whereas SW620 cells accumulated 20 +/- 7 pmol dUTP. Consistent with this difference in extent of dUTP accumulation was the observation that deoxyuridine triphosphatase levels in SW620 cellular extracts were 4.4-fold higher than in HT29 extracts. The ability to accumulate dUTP, intracellular deoxyuridine triphosphatase activity, and extent of DNA damage appear to be important determinants for predicting the response to FdUrd treatment in these cell lines.

Cell Survival↗
PubMed1992

Variations in patterns of DNA damage induced in human colorectal tumor cells by 5-fluorodeoxyuridine: implications for mechanisms of resistance and cytotoxicity.

We have previously shown that treatment of the HT29 human colorectal tumor (HCT) cell line with 100 nM 5-fluorodeoxyuridine (FdUrd) induces DNA fragments ranging from 50 kilobases to 5 megabases. The studies reported here were conducted to characterize the kinetics, concentration dependence, and pharmacologic specificity of this process and to determine if such fragmentation varies among HCT cell lines. HT29 and SW620 cells yielded similar fragment size distributions upon treatment with either FdUrd or CB3717 [a folate analog inhibitor of thymidylate synthase (TS)]. With either of these agents the SW620 line required higher drug concentrations or longer incubation times than HT29 cells to achieve a given level of fragmentation or cytotoxicity, even though the two cell lines are equally sensitive to FdUrd-induced TS inhibition. These data indicate that SW620 resistance is not due to a lesion in the events leading up to TS inhibition but it may be due to a difference in the steps following TS inhibition. Aphidicolin, a DNA polymerase inhibitor, did not cause substantial fragmentation or cytotoxicity in these two cell lines, demonstrating that the fragmentation response to the other two drugs is not a general consequence of DNA synthesis inhibition. A third HCT line, HuTu80, gave rise only to a smaller and more discrete population of DNA fragments, ranging from approximately 50 to 200 kilobases, following exposure to FdUrd. Similar patterns were seen in this line upon treatment with CB3717 or aphidicolin, indicating that this fragmentation pattern is not specific to TS inhibition and may be characteristic of a more general response than that seen in the other two cell lines. DNA fragments induced by FdUrd in HuTu80 cells did not degrade into smaller pieces, demonstrating that the process by which they are formed is distinct from apoptosis. We conclude that the responses of HCT cells to FdUrd-induced TS inhibition vary significantly, that these differences may reflect heterogeneity in the mechanism of DNA damage formation, and that, in some cases, FdUrd resistance may be due to alterations in the fragmentation process.

Cell Survival↗
PubMed1989

Dependence of etoposide-induced cytotoxicity and topoisomerase II-mediated DNA strand breakage on the intracellular ionic environment.

We have found that blockade of the Na+,K+-pump by the cardiac glycoside ouabain protects human A549 and hamster V79 cells from the cytotoxic effects of the topoisomerase II poison etoposide. One thousand-fold higher concentrations of ouabain were required to protect V79 cells compared to A549 cells. Since this difference parallels previously measured differences in pump sensitivity, it suggests that protection is mediated directly through pump blockade. Ouabain affected neither the cellular influx nor efflux of etoposide. However, pump blockade did decrease the formation of etoposide-induced DNA-topoisomerase, II-cleavable complexes, assessed as single and double strand DNA breaks using alkaline and neutral elution. To determine if this decrease were a direct effect of change in ionic environment produced by pump blockade, experiments with isolated nuclei and partially purified topoisomerase II were performed. Etoposide-induced cleavable complex formation and topoisomerase-mediated decatenation were assessed in buffers which mimicked either normal intracellular ionic conditions or those produced by ouabain. Compared to the buffer which resembled the normal intracellular ionic conditions, the buffer that mimicked the conditions produced by pump blockade produced fewer etoposide-mediated cleavable complexes in isolated nuclei and less decatenating activity of partially purified topoisomerase II. These findings demonstrate that inhibition of the Na+,K+-pump causes an alteration in the intracellular ionic environment which decreases the activity of topoisomerase II, thus producing a decrease in etoposide-induced cleavable complex formation and cytotoxicity. Since ionic changes occur inside normal cells during progression through the cell cycle as well as in cells that have undergone transformation, these data suggest that the intracellular ionic environment plays a role in determining the sensitivity of normal and malignant cells to this group of chemotherapeutic agents.

Animals↗