To study a possible linkage of drug resistance and cell cycle alteration, we developed taxol resistant cell lines (TR21) after repeated treatment of human ovarian cancer cells (TAOV) with taxol. Other taxol resistant cell lines were developed by transfection of the multi-drug resistant (mdr1) gene in TAOV cells without subsequent taxol treatment (5E1, 2E1). Both cell lines showed equally high expression of P-glycoprotein (P-gp), but taxol resistance of TR cells was higher (IC50=22.4 M) than 5E1 cells (IC50=5.8 - 8 M). Among many single cell clones isolated from all cell lines, several TR clones demonstrated exceptionally fast cell growth, with a doubling time (DT) of 15 to 17 h, compared to 28 to 32 h for the parent TAOV cells. Cell cycle acceleration was modest in 5E1 cell lines (DT=24-28 h). HPLC analysis of 3H-taxol treated TR21 and 5E1 cell extracts indicated that the majority of 3H-taxol and its derivatives were effluxed leaving a residual amount of drug in the cells, which were only detectable after blocking the taxol efflux by Vp. To explore a role of cell cycle in the mechanisms of taxol resistance, we examined the expression of cell cycle regulatory proteins by Western blotting. There was a marked difference between TAOV and 5E1 cell lines in the expression of bcl-2, cdc2/cdk1, cdk5 and cyclin D1despite the fact that the only difference between these cell lines is that the 5E1 cells had been transfected with the mdr1 gene. Bcl-2 and cdk5 expression was high in all taxol resistant cells, but not in TAOV cells. The most remarkable difference was observed in cyclin D1 and Rb, both which were extremely high in TR cells, particularly in the rapidly proliferating TR21-17 clone, as opposed to undetectable level in TAOV and 5E1 cells. Treatment with taxol further enhanced the high expression of cyclin D1. Cyclin E expression was also highly enhanced after treatment with taxol in TR21- 17 cells. Adenovirus 2E1B expression was also higher in TR cells than in TAOV and 5E1 cells. These findings suggest TR cell lines bear a clearly different profile of cell cycle regulatory protein expression, and respond with increased expression of these proteins after treatment with taxol. The high expression of these proteins were blocked by verapamil (Vp), a blocking agent of P-gp, and TR cells underwent a programed cell death (PCD) in a dose dependent manner, suggesting P-gp is participating in the PCD pathways probably through modulating the drug efflux. About half of the first proliferating TR clones were resistant to transient treatment with 8- azaguanine (8-AG, 20 g/ml, 24 h, designated as TRAG clones), whereas all TAOV and 5E1 derived cells were not viable under exposure to drugs. About 40 % of the 8-AG resistant TR clones were resistant to a treatment with azaserine (5.8 M) / hypoxanthine (0.1 mM) (designated as TRAG-R) suggesting that the fast growth of TRAG cells is sustained primarily by a bio-energetically efficient salvage pathway for nucleotide biosynthesis. Over-expression of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene, as detected by Northern blot analysis, was observed in TRAG-R cells, but not in 5E1 cells. These findings suggest that clonal development of rapidly proliferating cell lines have; (1) active drug transport function (efflux) derived from P-gp; (2) different profiles of cell cycle regulatory programs with development of over-expression of the mdr1 gene during prolonged treatment with taxol; and (3) Efficient salvage biosynthetic pathway of nucleotide synthesis. These three functions are in concert to support drug resistance and fast cell growth. Consequently, Vp blocking of the transport function of P-gp may disrupt the well balanced regulatory network, leading TR cells to undergo PCD pathways.
