IMPORTANCE OF GLUTATHIONE AND ASSOCIATED ENZYMES IN DRUG RESPONSE

Maintenance of cellular homeostasis is a critical survival trait in tu mors when exposed to anticancer drugs. Because conjugation and elimina tion of drugs and their metabolites is dependent upon sequential and c oordinated pathways, acquired drug resistance through a gradual adapti ve response would rarely be expected to be the consequence of changes in the expression of one gene product. We have used a number of drug-r esistant human cell lines to characterize those genes that are implica ted in maintaining a resistant phenotype. Human HT29 colon cancer cell s chronically exposed to ethacrynic acid (EA) [a glutathione (GSH) and glutathione S-transferase (GST) modulator] have acquired resistance t o the drug. Commensurate with resistance, EA is more effectively conju gated to GSH and effluxed from the resistant cells. Using directed and random (differential display) approaches, a number of detoxification and/or protective gene products have been shown to be expressed at ele vated levels. These include: gamma-glutamyl cysteine synthetase (gamma -GCS, the rate-limiting enzyme in GSH biosynthesis); GST pi (the enzym e catalyzing the conjugation reaction); multidrug resistance associate d protein (MRP) (the membrane pump responsible for effluxing the conju gate from the cell interior). In addition, other gene products not dir ectly linked with EA metabolism were induced, including dihydrodiol de hydrogenase (an alpha-ketoreductase) (30-fold), DT-diaphorase (threefo ld), and a transcriptional regulator SSP 3521 (threefold). HL60 cells resistant to a GSH paralog Ter199 also show increased expression of so me of these gene products. Furthermore, an adriamycin-resistant human HL60 cell line also shows overexpression of GST pi, gamma-GCS, and MRP , but in addition has approximately 20-fold more DNA-dependent protein kinase catalytic subunit (DNA-PKcs). This enzyme is an early stress r esponse gene that can phosphorylate and activate downstream transcript ion factors. Such overexpression could impact on the transcriptional c ontrol of the other detoxification gene products. Both adriamycin and a typical drug-GSH conjugate (APA-SG) are inhibitors of DNA-PK. Becaus e cellular levels of these conjugates would presumably be a good indic ator of stress, it would seem reasonable to speculate that DNA-PK may act as a receiver and transmitter of signals that are crucial to the d rug-resistant phenotype. Additionally, this enzyme may prove to be a p otentially important target for drug design based upon the inhibitory activity of GSH conjugates.