Reverse transcription-PCR amplification of eDNA fragment of nucleotide 298—617revealed two full-length mRNAs in HCT 116 cells but only one full-length mRNA in HCT 116-R3OAcells. An exon

An association between the resistance to mitomycin C (MMC) and a decrease of NAD(P)H:quinone oxidoreductase (NQO1) activity was re ported for a MMC-resistant subline, HCT 116-R3OA,derived from MMC sensitive HCT 116 cells. Eight NQO1 cDNA clones were IsOlated from these two sublines by reverse transcription-PCR. Two clones, pDT9 from HCT 116 and pDT2Ofrom HCT 116-R3OA,are the full length of 274 amino acids. These two clones differ by a T to C substitution at nucleotide 464, whIch results in a replacement of aa-ginine 139 by tryptophan in the enzyme. NQO1 of pDT9 and pDT2O was expressed in Escherichia coli, purified, and shown to have a protein subunit OfMr 30'000, The change of amino acid 139 resulted In a shift of isoelectric pH from 9.5 to &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 detected two mRNA species of NQO1 (1.2 and 2.7 kilobases) due to alternative polyadenylation in all sublines. MMC resistant sublines showed 75—90% mRNA expression relative to HCT 116 cells. Reverse transcription-PCR amplification of eDNA fragment of nucleotide 298—617revealed two full-length mRNAs in HCT 116 cells but only one full-length mRNA in HCT 116-R3OAcells. An exon 4 deletion mRNA was detected In both sublines. The two full-length mRNAs may be from either alleles or chimeras of the same gene and the exon 4 deletion mRNA is a result of alternative splicing. On Western blot, we detected only one M@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 NQO1 activity. Tran scriptional regulation and posttranscriptional modification may be responsible for the disparity of gene expression of NQO1 and the low concentration of NQO1 protein in MMC-resistant sublines. Reversal of MMC resistance and the recovery of NQO1 in two revertants further supports the hypothesis that cellular control of NQO1 can modulate the cytotoxicity of MMC.