The development of multidrug resistance (MDR) in tumour cells to a wide range of anticancer drugs has become a major obstacle in the chemotherapeutic treatment of cancer. Molecular characterization of MDR tumour cells has led to the identification of several cell-based genetic alterations including the overexpression of a membrane protein, P-glycoprotein (P-gp). P-gp is a ATP dependent drug efflux pump and P-gp ATPase activity has been demonstrated to be essential in drug transport. In an effort to understand how P-gp ATPase activity is coupled to drug binding and transport, we examined the effects of N-ethylmaleimide (NEM), a potent inhibitor of P-gp ATPase, on P-gp drug binding and transport. Our results show that short term treatment of MDR cells with NEM led to a concentration-dependent increase in P-gp drug binding and phosphorylation. In addition, NEM increases [3H]-vinblastine accumulation in drug resistant cells but not in sensitive cells. Our study suggests that inhibition of P-gp ATPase activity, and not increased phosphorylation of P-gp by NEM, is responsible for the observed increase in P-gp-drug binding. / Selection of tumour cell lines in vitro has led to multiple cellular changes that may mediate drug resistance to anticancer drugs. The role of other mechanisms, in addition to P-gp and multidrug resistance protein (MRP) in drug resistance, is supported by evidence from studies with tumour cell lines and clinical tumours. In an effort to identify other cellular changes that may be important in tumour drug resistance to anticancer drugs, we have used a differential immunodot blot method to isolate monoclonal antibodies that bind to proteins in drug resistant but not in drug sensitive cells. By using the immunodot blot method, we have isolated a monoclonal antibody (IPM96) which recognized a 40 kDa protein (P-40) in several MDR cell lines. The expression of P-40 is concurrent with the level of drug resistance. Biochemical characterization showed P-40 to be associated with the cell membrane and in the soluble fraction. Molecular cloning of P40 cDNA revealed that P-40 is identical to annexin I, a substrate for the epidermal growth factor receptor tyrosine kinase. The observed increase in P-40 (or annexin I) protein levels in drug resistant cells is due to the elevation of P-40 transcripts. The pharmacological characterization of P-40 cDNA transfectants (P-40-MCF-7) has demonstrated that overexpression of P-40 in drug sensitive cells is capable of conferring drug resistance to adriamycin, actinomycin D, Taxol and cisplatin. Taken together, our study provides convincing evidence that annexin I is important in the development of drug resistance in cancer cells. In addition, it suggests a novel mechanism of drug resistance that is different from the ATP-dependent drug efflux pumps that mediate P-gp- and MRP-associated MDR
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.35507 |
| Date | January 1998 |
| Creators | Wang, Ying, 1958- |
| Contributors | Georges, Elias (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Institute of Parasitology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001764660, proquestno: NQ55424, Theses scanned by UMI/ProQuest. |
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