Purpose: To identify the molecular mechanism of resistance against topotecan and other topoisomerase (topo) I inhibitors, characterized by reduced cellular accumulation.
Background: Topo I inhibitors are a novel group of potent anticancer agents with a unique mechanism of action and broad spectrum of anti-cancer activity. However, resistance to topo I inhibitors is a major clinical problem. At the cellular level, several mechanisms of resistance to these drugs have been described, e.g. reduced topo I catalytic activity, decreased formation of cleavable complexes, decreased expression of topo I, a point mutation or rearrangement of the topo I gene and overexpression of MDR1 P-glycoprotein (P-gp). We have established a novel topotecan resistant cell line T8, derived from the human ovarian cancer cell line IGROV1, which is highly cross-resistant to SN38 (the active metabolite of CPT11) and 9-aminocamptothecin (9AC) and at a lower level cross-resistant to camptothecin (CPT) and mitoxantrone (MX). The resistance is associated with reduced cellular accumulation of topotecan. Our data are consistent with an energy-dependent efflux system for topotecan in the resistent cell line, which is absent in the parental line. Furthermore, we have established a novel MX resistant cell line MX3, which was also derived from IGROVI. MX3 is cross-resistant to topotecan, SN38 and 9AC. This cell line shows a reduced accumulation of MX as well as of topotecan. No overexpression of P-gp or MRP was observed in T8 and MX3 and no cross-resistance was found to a panel of other anti-cancer agents, including doxorubicin.
Plan of investigation: We aim to identify:
1. the mechanism of reduced accumulation of topotecan at the molecular level; 2. whether reduced accumulation of MX and cross-resistance to MX result from the same molecular mechanism which affects topotecan accumulation and resistance; 3. whether accumulation of other topo I inhibitors is affected by the same pathway. The primary method we will use to compare gene expression in the IGROVI and T8 lines is the cDNA subtraction method, which is based on the representational difference analysis (RDA) principle, combined with a suppression PCR method. This method may reveal, among other genes, a gene which determines cellular accumulation of topotecan, and possibly other topo I inhibitors. The identified cDNAs will be used in transfection experiments to show induction of resistance in the parental IGROVI and other cell lines. The transfected clones will also be analyzed for cross-resistance to other topo I inhibitors and to MX. At an early stage we will aIso compare gene expression in the IGROVI and T8 lines by cDNA hybridization to gene array filters containing large numbers of known genes and expressed sequence tags (ESTs). DNA microarrays ("DNA chips") screening will be used if this technique becomes available in time. For evaluation of the clinical role of the identified resistance gene, we will generate monoclonal antibodies to the resistance protein. They will be used to study subcellular localization, routing and processing of the resistance protein, and to screen normal tissues and patient tumors to elucidate any physiological role or contribution to clinical resistance.
Possible results: Identification of differentially expressed genes, which determine cellular accumulation of topotecan, will give insight into a novel and potentially important mechanism of resistance. lt would provide tools to investigate this mechanism in clinical tumor samples. Assessment of the pattern of cross-resistance should further establish the importance of this pathway for other drugs. |
