Identification and proposed mechanism of action of thymidine kinase inhibition associated with cellular exposure to camptothecin analogs

Donna M. Voeller, Jean L. Grem, Yves Pommier, Ken Paull, Carmen J. Allegra

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Purpose: To investigate the effects of several camptothecin analogs including 9-aminocamptothecin (9-AC), SN38, topotecan, and irinotecan (CPT- 11) on the enzymes involved in the pyrimidine salvage pathway including thymidylate synthase (TS). A COMPARE analysis using the NCI 60 cell line drug-screening panel suggested that there were similarities in the mechanisms of action of camptothecin analogs and TS inhibitors. Methods: TS enzymatic activity was measured by both an in situ tritium release assay using both the H630 colon cancer cell line and the CEM human leukemia cell line, and by a radiolabelled in vitro assay using partially purified human TS as the enzyme source. Thymidine kinase (TK) activity was measure by a radiolabelled in vitro assay using H630 colon cancer cell lysates as the enzyme source. Results: In vitro studies indicated that none of the analogs directly inhibited TS enzymatic activity; however, utilization of a coupled TS/TK in situ assay with radiolabelled deoxyuridine as the precursor revealed marked inhibition by the camptothecin analogs. 9-AC, SN38, and topotecan yielded IC 50 values of 1.3, 1.6, and 1.1 μM respectively. In contrast, there was no inhibition detected when deoxycytidine was used as the radiolabelled nucleoside precursor, suggesting that the drug effect was through inhibition of TK, rather than inhibition of TS. In vitro studies using cell lysates from H630 human colon cancer cells to measure TK activity showed no decrease in TK activity after 9-AC treatment. In addition, no changes were detected in the dATP and dTTP nucleotide pools. Permeabilizing the cell membranes with saponin did not abolish the inhibitory effect of the camptothecins indicating that altered cell transport was not responsible for the decreased activity in the in situ assay in intact cells. Conclusion: These studies suggest that there is inhibition of TK in intact cells associated with topoisomerase I inhibition by camptothecin analogs, and the inhibition of TK is the result of an indirect effect not related to feedback inhibition by changes in dTTP pools.

Original languageEnglish (US)
Pages (from-to)409-416
Number of pages8
JournalCancer Chemotherapy and Pharmacology
Volume45
Issue number5
DOIs
StatePublished - Jan 1 2000

Fingerprint

Camptothecin
Thymidylate Synthase
Thymidine Kinase
9-aminocamptothecin
irinotecan
Assays
Cells
Colonic Neoplasms
Topotecan
Cell Line
Enzymes
Salvaging
Type I DNA Topoisomerase
Deoxyuridine
Deoxycytidine
Preclinical Drug Evaluations
Tritium
Saponins
Prodrugs
Cell membranes

Keywords

  • Camptothecin analogs
  • Cross- inhibition
  • Thymidine kinase
  • Thymidylate synthase inhibitors
  • Topoisomerase I

ASJC Scopus subject areas

  • Oncology
  • Toxicology
  • Pharmacology
  • Cancer Research
  • Pharmacology (medical)

Cite this

Identification and proposed mechanism of action of thymidine kinase inhibition associated with cellular exposure to camptothecin analogs. / Voeller, Donna M.; Grem, Jean L.; Pommier, Yves; Paull, Ken; Allegra, Carmen J.

In: Cancer Chemotherapy and Pharmacology, Vol. 45, No. 5, 01.01.2000, p. 409-416.

Research output: Contribution to journalArticle

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abstract = "Purpose: To investigate the effects of several camptothecin analogs including 9-aminocamptothecin (9-AC), SN38, topotecan, and irinotecan (CPT- 11) on the enzymes involved in the pyrimidine salvage pathway including thymidylate synthase (TS). A COMPARE analysis using the NCI 60 cell line drug-screening panel suggested that there were similarities in the mechanisms of action of camptothecin analogs and TS inhibitors. Methods: TS enzymatic activity was measured by both an in situ tritium release assay using both the H630 colon cancer cell line and the CEM human leukemia cell line, and by a radiolabelled in vitro assay using partially purified human TS as the enzyme source. Thymidine kinase (TK) activity was measure by a radiolabelled in vitro assay using H630 colon cancer cell lysates as the enzyme source. Results: In vitro studies indicated that none of the analogs directly inhibited TS enzymatic activity; however, utilization of a coupled TS/TK in situ assay with radiolabelled deoxyuridine as the precursor revealed marked inhibition by the camptothecin analogs. 9-AC, SN38, and topotecan yielded IC 50 values of 1.3, 1.6, and 1.1 μM respectively. In contrast, there was no inhibition detected when deoxycytidine was used as the radiolabelled nucleoside precursor, suggesting that the drug effect was through inhibition of TK, rather than inhibition of TS. In vitro studies using cell lysates from H630 human colon cancer cells to measure TK activity showed no decrease in TK activity after 9-AC treatment. In addition, no changes were detected in the dATP and dTTP nucleotide pools. Permeabilizing the cell membranes with saponin did not abolish the inhibitory effect of the camptothecins indicating that altered cell transport was not responsible for the decreased activity in the in situ assay in intact cells. Conclusion: These studies suggest that there is inhibition of TK in intact cells associated with topoisomerase I inhibition by camptothecin analogs, and the inhibition of TK is the result of an indirect effect not related to feedback inhibition by changes in dTTP pools.",
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T1 - Identification and proposed mechanism of action of thymidine kinase inhibition associated with cellular exposure to camptothecin analogs

AU - Voeller, Donna M.

AU - Grem, Jean L.

AU - Pommier, Yves

AU - Paull, Ken

AU - Allegra, Carmen J.

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N2 - Purpose: To investigate the effects of several camptothecin analogs including 9-aminocamptothecin (9-AC), SN38, topotecan, and irinotecan (CPT- 11) on the enzymes involved in the pyrimidine salvage pathway including thymidylate synthase (TS). A COMPARE analysis using the NCI 60 cell line drug-screening panel suggested that there were similarities in the mechanisms of action of camptothecin analogs and TS inhibitors. Methods: TS enzymatic activity was measured by both an in situ tritium release assay using both the H630 colon cancer cell line and the CEM human leukemia cell line, and by a radiolabelled in vitro assay using partially purified human TS as the enzyme source. Thymidine kinase (TK) activity was measure by a radiolabelled in vitro assay using H630 colon cancer cell lysates as the enzyme source. Results: In vitro studies indicated that none of the analogs directly inhibited TS enzymatic activity; however, utilization of a coupled TS/TK in situ assay with radiolabelled deoxyuridine as the precursor revealed marked inhibition by the camptothecin analogs. 9-AC, SN38, and topotecan yielded IC 50 values of 1.3, 1.6, and 1.1 μM respectively. In contrast, there was no inhibition detected when deoxycytidine was used as the radiolabelled nucleoside precursor, suggesting that the drug effect was through inhibition of TK, rather than inhibition of TS. In vitro studies using cell lysates from H630 human colon cancer cells to measure TK activity showed no decrease in TK activity after 9-AC treatment. In addition, no changes were detected in the dATP and dTTP nucleotide pools. Permeabilizing the cell membranes with saponin did not abolish the inhibitory effect of the camptothecins indicating that altered cell transport was not responsible for the decreased activity in the in situ assay in intact cells. Conclusion: These studies suggest that there is inhibition of TK in intact cells associated with topoisomerase I inhibition by camptothecin analogs, and the inhibition of TK is the result of an indirect effect not related to feedback inhibition by changes in dTTP pools.

AB - Purpose: To investigate the effects of several camptothecin analogs including 9-aminocamptothecin (9-AC), SN38, topotecan, and irinotecan (CPT- 11) on the enzymes involved in the pyrimidine salvage pathway including thymidylate synthase (TS). A COMPARE analysis using the NCI 60 cell line drug-screening panel suggested that there were similarities in the mechanisms of action of camptothecin analogs and TS inhibitors. Methods: TS enzymatic activity was measured by both an in situ tritium release assay using both the H630 colon cancer cell line and the CEM human leukemia cell line, and by a radiolabelled in vitro assay using partially purified human TS as the enzyme source. Thymidine kinase (TK) activity was measure by a radiolabelled in vitro assay using H630 colon cancer cell lysates as the enzyme source. Results: In vitro studies indicated that none of the analogs directly inhibited TS enzymatic activity; however, utilization of a coupled TS/TK in situ assay with radiolabelled deoxyuridine as the precursor revealed marked inhibition by the camptothecin analogs. 9-AC, SN38, and topotecan yielded IC 50 values of 1.3, 1.6, and 1.1 μM respectively. In contrast, there was no inhibition detected when deoxycytidine was used as the radiolabelled nucleoside precursor, suggesting that the drug effect was through inhibition of TK, rather than inhibition of TS. In vitro studies using cell lysates from H630 human colon cancer cells to measure TK activity showed no decrease in TK activity after 9-AC treatment. In addition, no changes were detected in the dATP and dTTP nucleotide pools. Permeabilizing the cell membranes with saponin did not abolish the inhibitory effect of the camptothecins indicating that altered cell transport was not responsible for the decreased activity in the in situ assay in intact cells. Conclusion: These studies suggest that there is inhibition of TK in intact cells associated with topoisomerase I inhibition by camptothecin analogs, and the inhibition of TK is the result of an indirect effect not related to feedback inhibition by changes in dTTP pools.

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KW - Thymidylate synthase inhibitors

KW - Topoisomerase I

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