THE NAD(P)H-QUINONE OXIDOREDUCTASE LOCUS IN HUMAN COLON-CARCINOMA HCT-116 CELLS RESISTANT TO MITOMYCIN-C

Previously, we reported an association of mitomycin C resistance and a deficiency of NAD(P)H:quinone oxidoreductase (NQO(1)) in HCT 116-R30A cells, a subline derived from mitomycin C-sensitive HCT 116 cells. In HCT 116 cells, we found two mRNAs coding full-length cDNAs of NQO(1) differing at codon 139, one with arginine (wild type), and one with tr yptophan. Only the tryptophan 139 form of mRNA was detected in HCT 116 -R30A cells. In addition, an exon 4 deleted mRNA of NQO(1), a product of alternative splicing, was detected in both cell lines. Analysis by semiquantitative reverse transcription-PCR showed that NQO(1) mRNA cod ing full-length cDNAs in HCT 116-R30A cells was 15% of that present in HCT 116 cells. A M(r) 26,000 protein, representing the exon 4 deleted mRNA, was not detected by polyclonal anti-NQO(1) in HCT 116 sublines. Recombinant plasmids of exon 4 deleted cDNA generated a M(r) 26,000 p rotein without enzymatic activity in Escherichia coli but not in Cos7 cells. The function of exon 4 deleted mRNA is yet unknown. The rates o f decay of all NQO(1) mRNAs in HCT 116 and HCT 116-R30A cells were sim ilar. DNA sequences of the promoter regions of the NQO(1) gene (-837 b p) from both cell lines did not differ from each other or from the sam e region of the human liver NQO(1) gene. Sequences of cis elements in the 837-bp region and mRNA stability could not account for the low exp ression of full-length mRNA in HCT 116-R30A cells. Southern blot analy sis shelved the size and the intensity of the NQO(1) gene in the two c ell lines to he similar. This result was confirmed by semiquantitative PCR analysis of a 450-bp fragment in the NQO(1) gene containing codon 139 and the exon 4 region. Digestion of this PCR-amplified fragment b y restriction enzyme MspI revealed that HCT 116 cells have two heteroz ygous NQO(1) alleles, a wild-type and a tryptophan 139 form. The funct ional wild-type NQO(1) allele was not detected in HCT 116-R30A cells. Sensitive and resistant cell lines each contained one normal and one a bnormal chromosome 16. Loss of the mild-type NQO(1) allele in HCT 116- R30A cells did not result from a loss of chromosome 16 or copies of th e NQO(1) gene. Alteration of factor(s) such as trans-acting factors an d DNA methylation may be involved in the down-regulation of NQO(1) in the mitomycin C-resistant HCT 116-R30A cells.