NAD(P)H-QUINONE OXIDOREDUCTASE EXPRESSION AND MITOMYCIN-C RESISTANCE DEVELOPED BY HUMAN COLON-CANCER HCT-116 CELLS

An association between the resistance to mitomycin C (MMC) and a decre ase of NAD(P)H:quinone oxidoreductase (NQO(1)) activity was reported f or a MMC-resistant subline, HCT 116-R30A, derived from MMC-sensitive H CT 116 cells. Eight NQO(1) cDNA clones were isolated from these two su blines by reverse transcription-PCR. Two clones, pDT9 from HCT 116 and pDT20 from BCT 116-R30A, are the full length of 274 amino acids. Thes e two clones differ by a T to C substitution at nucleotide 464, which results in a replacement of arginine 139 by tryptophan in the enzyme. NQO(1) of pDT9 and pDT20 was expressed in Escherichia coli, purified, and shown to have a protein submit of M(r) 30,000. The change of amino acid 139 resulted in a shift of isoelectric pH from 9.5 to 8.35 and a 60% decrease of activity in reducing MMC. All of the other six clones differ from pDT9 by a deletion of exon 4. On Northern blot, we detect ed two mRNA species of NQO(1) (1.2 and 2.7 kilobases) due to alternati ve polyadenylation in all sublines. MMC-resistant sublines showed 75-9 0% mRNA expression relative to HCT 116 cells. Reverse transcription-PC R amplification of cDNA fragment of nucleotide 298-617 revealed two fu ll-length mRNAs in HCT 116 cells but only one full-length mRNA in HCT 116-R30A cells. An exon 4 deletion mRNA was detected in both sublines. The two full-length mRNAs may be from either alleles or chimeras of t he same gene and the exon 4 deletion mRNA is a result of alternative s plicing, On Western blot, we detected only one M(r) 30,000 protein in all sublines. A substantial decrease of this protein in MMC-resistant sublines (5% of HCT 116) explained the 95% decrease of their NQO(1) ac tivity. Transcriptional regulation and posttranscriptional modificatio n may be responsible for the disparity of gene expression of NQO(1) an d the low concentration of NQO(1) protein in MMC-resistant sublines. R eversal of MMC resistance and the recovery of NQO(1) in two revertants further supports the hypothesis that cellular control of NQO(1) can m odulate the cytotoxicity of MMC.