Drug resistance during the chemotherapeutic treatment of malignant tumors is a major obstacle in the efficacy of anti-cancer drugs. A number of factors are known to be involved in drug resistance, such as drug transport (efflux pump), metabolic biotransformation, conjugate formation and repair of cellular components. The reduction of intracellular drug content by drug efflux through an elevated function of P-glycoprotein (P-gp) is thought to play a major role in the drug resistance mechanism of natural products, antibiotics and alkaloids. Recent studies strongly suggest that the alteration of genes that regulate cell proliferation and apoptosis affect the response to cancer chemotherapy. Taxol is a potent inhibitor of cell replication by stabilization of polymerized microtubles, resulting in blockage of cells in the G2 / M phase. Taxol mediates cell death by activating the gene-directed pathways of programmed cell death (PCD), which is characterized by internucleosomal DNA fragmentation and morphological features of apoptosis. To study a relationship between drug resistance and cell cycle, two different taxol- resistant cell lines were developed: (I) Long term pulse exposure of taxol- sensitive TAOV human ovarian cancer cells to taxol (TR21 cells), and (II) by transfection with aMDR1 multi-drug resistant gene (5E1 cells). Both cell lines showed equally high expression of P-gp, but the taxol resistance of TR21 cells was higher than that of 5E1 cells. Of many single cell clones isolated from these cell lines, several TR21 clones demonstrated exceptionally rapid proliferation. Doubling times were 15 to 17 h for TR21 derived clones, 24-28 h for 5E1, and 28 to 32 h for the parent TAOV cell lines. Expression of cyclin D1 and Rb cell cycle proteins were extremely high in TR21 rapid proliferating clones, and treatment with taxol further enhanced the high expression as observed by Western blotting. These proteins were undetectable or at low levels in TAOV and 5E1 cells. The high expression of these proteins in TR21 cells was blocked by verapamil (Vp), an inhibitor of P-gp, and the cells underwent programmed cell death (PCD) in a Vp dose-dependent manner. About half of the rapid proliferating TR21 clones were resistant to a 24 h treatment with 8-azaguanine, whereas none of the TAOV- and 5E1- derived clones were viable. About 40 % of surviving clones were resistant to azaserine/hypoxanthine and had amplification of the hypoxanthine-guanine phosphoribosyltransferase RNA. HPLC analysis of 3H-taxol-treated TR21 cells and culture media extracts indicated that a substantially large fraction of 3H-taxol was incorporated into the TR21 cells, partially metabolized and effluxed, leaving a residual amount of the drug in the cells. This residual, mostly metabolically converted to other derivatives, was only detectable after blocking the taxol efflux by Vp. These results suggest that these rapidly proliferating taxol- resistant TR21 cell lines have developed characteristic features such as co-expression of the cell cycle regulatory proteins particularly cyclin D1/Rb; enhanced bio-energetically efficient salvage pathway of nucleotide biosynthesis; and drug metabolism in coordination with active drug efflux derived from P-gp. Inhibition of the transport function of P- gp by Vp may interfere with these multiple mechanisms, leading taxol-resistant TR21 cells to undergo PCD pathways. These multiple mechanisms, lacking in the parent TAOV and 5E1 cells, are therefore less resistant to taxol, proliferate slowly, and are more vulnerable to necrotic and apoptotic cell death. - Apoptosis, Cell Cycle, Drug Metabolism, Drug resistance, Nucleotide synthesis, Taxol, - Human Tissues, Fluids, Cells, etc.
