Increasing evidence has been reported showing the importance of NAD(P)H:oxidoreductase (QR) in activating quinone antitumor drugs leading to cytotoxicity, drug-resistance or detoxification. Various tumor cells have developed quinone drug resistance by regulating their QR concentration. Alterations of QR concentration in tumor cells frequently are stable indicating that mutations either to the gene, to transcription, or to post-proposal is to ascertain the mechanism for cellula regulation of QR concentration in human colon carcinoma HCT 116 cells in the development of mitomycin C (MMC)-resistance and resistance to other quinone drug. Several MMC-resistant (MMC/r) sublines were developed from MMC-sensitive (MMC/s) HCT 116 cells. The MMC/r sublines have unique characteristic in modulating their QR level. QR from both MMC/s and MMC/r sublines have been cloned in this laboratory. MMC/r sublines express the same concentration of QR mRNA but maintain less QR protein than MMC/s cells by 20 fold. This disparity indicates that the alteration of QR in MMC/r cells is probably post-transnational. This problem will be approached as follows: 1) QRs from sublines are purified and peptide analysis, looking for differences on post-transnational modification, will be conducted. 2) The stability of QR in each subline will be studied by pulse-chase labeling with S-methionine. 3) MMC/r cells will be transfected with QR-cloned of MMC/s cells and vice versa. Stably transfected MMC/r cells will be studied for the expression and the stability of MMC/s-clone of QR, and to see if recovery of QR will associated with the return of sensitivity of MMC. 4) QR can be induced with MMC/r cells to evaluate if induction can occur in the MMC/r cells. These information will be useful for strategic design to target QR for clinical applications of quinone antitumor agents. N7-(2-(4-nitrophenyldithio)-ethyl)mitomycin C (NPSSE-MC), a MMC analogue, has more favorable pharmacologic activities than MMC and circumvents MMC- resistance. A P-postlabeling method is used to study DNA adduct formation in cells by this compound. NPSSE-MC will be compared to MMC and quinone antitumor agents such as SR-4233 and EO-9 to look for the mechanism by which this compound circumvents MMC-resistance.
