Reduced Expression of DNA Topoisomerase I in SF295 Human Glioblastoma Cells Selected for Resistance to Homocamptothecin and Diflomotecan

Abstract

Homocamptothecins (hCPTs) are a novel class of topoisomerase I (Top1) inhibitors with enhanced chemical stability compared with the currently used camptothecin (CPT) analogs irinotecan and topotecan. The hCPT derivative diflomotecan (BN80915) is currently in clinical trials. We established two resistant human glioblastoma cell lines, SF295/hCPT50 and SF295/BN50, by stepwise exposure of the parental SF295 line to increasing concentrations of hCPT and BN80915, respectively. The two resistant cell lines were 15-to 22-fold resistant to hCPT and BN80915 as well as 7-to 27-fold crossresistant to other Top1 inhibitors, including CPT, topotecan, and the indenoisoquinolines MJ-III-65 (NSC 706744) and NSC 724998, but sensitive to the topoisomerase II inhibitors mitoxantrone and etoposide. Neither of the resistant cell lines displayed any detectable expression of the three major drug transporters P-glycoprotien, multidrug resistance-associated protein 1, or ATP-binding cassette, subfamily G (WHITE), member 2, as assessed by immunoblot or flow cytometry. Reduced expression of Top1 protein occurred at the transcriptional level in both of the resistant cell lines, consistent with the reduction of Top1 enzyme level as the major contribution to the resistance phenotype in SF295/ hCPT50 and SF295/BN50 cells. Treatment of the resistant cell lines with the histone deacetylase inhibitor depsipeptide or the DNA methyltransferase inhibitor 5-aza-2Ј-deoxycytidine alone or concomitantly did not result in re-expression of Top1. Our studies suggest that selection for resistance to hCPT or BN80915 is primarily related to reduced Top1 expression at the transcriptional level, resulting in reduced enzyme levels.Homocamptothecins (hCPTs) are a new class of camptothecin (CPT) derivatives. They are different from the conventional CPT in that they bear a seven-membered ␤-hydroxylactone ring instead of the naturally occurring six-membered ␣-hydroxylactone observed in CPT (see Fig. 1). hCPTs are therefore more stable than CPT derivatives (Lavergne et al., 2000a,b) that are readily hydrolyzed to the inactive carboxylate forms (for review, see Pommier, 2006). hCPT, like other CPT analogs, inhibits Top1 by stabilizing the covalent complex between Top1 and DNA, leading to enzyme-linked DNA strand breaks, also referred to as cleavage complexes (Pommier, 2006). Because of their increased chemical stability, hCPTs were expected to exert greater Top1 inhibition and antitumor efficacy than the current FDA-approved CPT analogs irinotecan and topotecan [for review, see Lavergne et al. (2000b);Pommier (2006)]. hCPTs showed very potent antitumor efficacy in many tumors, including brain glioblastoma mouse models. The fluorinated hCPT diflomotecan (BN80915) is one of the most active compounds in terms of antiproliferative activity in the hCPT series (Bailly et al., 1999;Lavergne et al., 2000b). BN80915 has been selected for further development as a highly active hCPT-based Top1

Sensitivity and Acquired Resistance of BRCA1;p53-Deficient Mouse Mammary Tumors to the Topoisomerase I Inhibitor Topotecan

“…Abcg2 −/− ;Brca1 −/− ;p53 −/− tumors can still develop full topotecan resistance, however, and a substantial decrease in the level of Top1 can explain resistance in several of these tumors. Remarkably, this profound decrease in Top1 protein was not accompanied by a corresponding decrease in Top1 mRNA in most tumors, in sharp contrast with the observations on camptothecin-resistant tumor cell lines (12,32,38). If downregulation of Top1 would also be posttranscriptional in human tumors, its detection by gene expression profiling would be impossible.…”

“…However, using Top1 cDNA derived from five individual topotecan-resistant Abcg2 −/− , Brca1 −/− ,p53 −/− tumors (TB1, TB2, TB4, TB5, and TB8), we did not find a single-point mutation analyzing eight independent clones per tumor (not shown). Another alteration reported to cause topotecan resistance is a reduction in the level of the drug target (12,31,32). Yet, in both Abcg2-proficient and -deficient tumors, we did not observe a significant decline of Top1 transcripts ( Fig.…”

Topoisomerase I levels in the NCI-60 cancer cell line panel determined by validated ELISA and microarray analysis and correlation with indenoisoquinoline sensitivity

“…Expression of Top1 is associated with tumor growth, tumor differentiation, and poor prognosis for survival in colorectal cancers (2, 3). High levels of Top1 also correlate with response to the Top1 inhibitor irinotecan (CPT-11) (3, 4), whereas reduced expression of Top1 is linked to resistance to Top1 inhibitors (5–7). However, to date, a highly quantitative assay for measuring Top1 levels in a (semi) high throughput manner has been lacking.…”

Microscopic modes and free energies for topoisomerase I-DNA covalent complex binding with non-camptothecin inhibitors by molecular docking and dynamics simulations

“…The binding of CPT to the cleavage complex is considered to be reversible, but the compound becomes lethal due to the collision of the stalled enzyme-DNA complex with the replication fork. 29–31 The CPT and its derivatives, such as topotecan, bind to the binary complex by intercalating between the bases of the DNA substrate at the cleavage site.…”