The importance of NAD(P)H:quinone oxidoreductase(NQO1) in activating quinone antitumor drugs, causing quinone drug resistance and defending cells against invasive carcinogens has drawn increasing amount of attention. Various tumor cells maintain higher concentrations of NQO1 than their normal tissue. Quinone drug resistant tumor cells further complicate their NQO1 system. Alterations of NQO1 in tumor cells are stable indicating that mutations either to the gene or the gene expression occurred. Studies concerning cellular regulation of NQO1 in human tumor cells are lacking. The objectives of this application are to ascertain 1) the mechanism of NQO1 regulation in human colon cancer HCT 116 cells and its mitomycin C (MMC)-resistant sublines and 2) ways to induce NQO1 in human tumor cells that augment the cytotoxicity of quinone drugs and circumvent drug resistance. The ultimate goal is to provide information that targets NQO1 for clinical applications of quinone antitumor drugs. Enzymatic active NQO1 was cloned from both MMC-sensitive (MMC/s) and MMC-resistant (MMC/r) HCT 116 cells. The full-length mRNA and protein NQO1 were substantially decreased in MMC/r cells in comparison to MMC/s cells. Both, the NQO1 genes of the two cell lines share same copies of the gene, same DNA sequence in the promotor region, and same rate of mRNA decay. The expression of NQO1 in MMC/r cells is altered transcriptionally, probably involving transcription elements. Differences of transcription elements in the two cell lines will be analyzed by 1) the expression of a receptor gene, luciferase, in the two cell lines with a construct regulated by the NQO1 promotor; 2) band shift and super-shift of specific DNA oligonucleotides by nuclear extracts containing the elements; 3) transfecting MMC-resistant cells with NQO1 constructs to study the reversal of MMC-resistance; induction of NQO1, a phase II defence enzyme that protects cells against carcinogens has been reported mainly in mouse cell lines. Little study is conducted in human cells. The applicant believes that induction of this enzyme in human tumor cells can be studied for two goals: 1) to enhance the antitumor activity of MMC and other antitumor quinone drugs by inducers such as oltipraz and iallyl disulfide; and 2) to circumvent quinone drug resistance.
