Abstract
Two camptothecin-resistant cell lines, CPT30 and KB100, were established and characterized previously in our laboratory. Because enhanced sensitivity to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and decreased expression of O6-methylguanine-DNA methyltransferase (MGMT) protein were observed in these lines, we hypothesized that MGMT may be a determinant of cytotoxicity associated with camptothecin-derived DNA topoisomerase I inhibitors (CPTs). We used the Tet-On system to induce expression of MGMT in Chinese hamster ovary (CHO) cells and RNA interference to knock down MGMT expression in human nasopharyngeal carcinoma HONE-1 cells in order to identify any correlations between MGMT expression and CPTs cytotoxicity. CHO-derived Tet-On-inducible cells (S12+) showed MGMT overexpression and statistically significant more resistance to BCNU, camptothecin, 7-ethyl-10-hydrocamptothecin (SN38), and topotecan than parental CHO cells (p < 0.05), but there was less resistance to CPTs than to BCNU. Knockdown of MGMT expression with small interfering RNA in HONE-1 cells conferred increased sensitivity to BCNU and CPTs compared with mock control. Furthermore, alteration of MGMT expression coincides with CPT-induced cell death and poly(ADP-ribose) polymerase cleavage. There were no differences in protein levels and catalytic activity of topoisomerase I between MGMT-proficient and MGMT-deficient cells from the Tet-On-inducible and small interfering RNA (siRNA) systems. Resistance to CPTs coincided with decreased amounts of protein-linked DNA breaks generated by CPTs in MGMT-proficient cells and vice versa in MGMT-deficient cells. Our data indicate that MGMT can modulate cytotoxicity of CPT-derived topoisomerase I inhibitors.
Footnotes
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This work was supported in part by grants from National Health Research Institutes (NHRI), Taipei, Taiwan (NHRI intramural Grant 92A1-CAPP06-1), and the National Science Council, Taipei, Taiwan (NSC 94-2752-B-400-001-PAE).
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These data were presented in abstract at the American Association for Cancer Research 94th Annual Meeting, July 11-14, 2003, in Washington, D.C. (Abstract No. 3706).
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doi:10.1124/jpet.105.095919.
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ABBREVIATIONS: Top I, topoisomerase I; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; CHO, Chinese hamster ovary; CPT, camptothecin; CPTs, CPT-derived topoisomerase I inhibitor(s); Dox, doxycycline; MGMT, O6-methylguanine-DNA methyltransferase; PARP, poly(ADP-ribose) polymerase; α-MEM, α-minimal essential medium; PI, propidium iodide; PLDB, protein-linked DNA break(s); S12, a tetracycline-regulatory MGMT expressing clone derived from CHO cells; S12-, S12 cells without doxycycline addition; S12+, S12 cells with 1 μg/ml doxycycline addition; SN38, 7-ethyl-10-hydrocamptothecin; siRNA, small interfering RNA; TPT, topotecan; tTs, transcriptional silencer; Vec, vector.
- Received September 20, 2005.
- Accepted October 27, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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