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 decrease of NAD(P)H:quinone oxidoreductase (NQO1) activity was reported for a MMC-resistant subline, HCT 116-R30A, 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 pDT20 from HCT 116-R30A, 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 arginine 139 by tryptophan in the enzyme. NQO1 of pDT9 and pDT20 was expressed in Escherichia coli, purified, and shown to have a protein subunit 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 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 cDNA fragment of nucleotide 298-617 revealed two full-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 the same gene and the exon 4 deletion mRNA is a result of alternative splicing. 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 NQO1 activity. Transcriptional 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.
Pan
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SS
Division of Developmental Therapeutics, University of Maryland Cancer Center, Baltimore 21201.
Forrest
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GL
Akman
S A
SA
Hu
L T
LT
D001483:Base Sequence; D015153:Blotting, Western; D003110:Colonic Neoplasms / Q000201:enzymology* / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics*; D004351:Drug Resistance; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D007215:Indophenol / Q000031:analogs & derivatives / Q000378:metabolism; D016685:Mitomycin / Q000378:metabolism*; D008969:Molecular Sequence Data; D011808:Quinone Reductases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D004273:DNA, Neoplasm; D012333:RNA, Messenger; D007215:Indophenol; D016685:Mitomycin; D011808:Quinone Reductases