DOUBLE KNOCKOUT OF THE MRP GENE LEADS TO INCREASED DRUG-SENSITIVITY IN-VITRO

Overexpression of the multidrug resistance-associated protein (MRP) ge ne has been implicated in the resistance of tumor cell lines to a wide array of chemotherapeutic agents, but its normal physiological functi on(s) remains unknown. We have compared the sensitivity to chemotherap eutic drugs and toxins of wild-type W9.5 embryonic stem cells (ES) and of single and double MRP gene knockout cells derived therefrom. MRP e xpression was totally abrogated in the double knockout cell line and p artially abrogated in the single knockout cell line. Reverse transcrip tion-PCR analyses demonstrated that the MDR1, MDR2, and MDR3 genes wer e not expressed in either wild-type or MRP knock-out cells. The cytoto xic activities of etoposide, teniposide, vincristine, doxorubicin, dau norubicin, and sodium arsenite were significantly greater in double kn ockout cells than in parental wild-type ES cells; single knockout ES c ells displayed an intermediate level of sensitivity. Tn contrast, no d ifference in sensitivity to colchicine and 1-beta-D-arabinofuranosylcy tosine existed between the cell lines. Etoposide accumulation in doubl e knockout ES cells was 2-fold higher than in wild-type ES cells. Thes e findings indicate that baseline MRP expression has the capacity to e xert a protective role against the toxicity of multiple chemotherapeut ic agents and natural toxins.