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The NAD(P)H:Quinone Oxidoreductase Locus in Human Colon Carcinoma HCT 116 Cells Resistant to Mitomycin C¹

Division of Developmental Therapeutics, University of Maryland Cancer Center [L-T. H., S-s. P.], Division of Human Genetics, Department of Obstetrics and Gynecology [J. S.], and Department of Pharmacology and Experimental Therapeutics [S-s. P.], University of Maryland School of Medicine, Baltimore, Maryland 21201

Previously, we reported an association of mitomycin C resistance and a deficiency of NAD(P)H:quinone oxidoreductase (NQO₁) 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 NQO₁ differing at codon 139, one with arginine (wild type), and one with tryptophan. Only the tryptophan 139 form of mRNA was detected in HCT 116-R30A cells. In addition, an exon 4 deleted mRNA of NQO₁, a product of alternative splicing, was detected in both cell lines. Analysis by semiquantitative reverse transcription-PCR showed that NQO₁ mRNA coding 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-NQO₁ in HCT 116 sublines. Recombinant plasmids of exon 4 deleted cDNA generated a M_r 26,000 protein without enzymatic activity in Escherichia coli but not in Cos7 cells. The function of exon 4 deleted mRNA is yet unknown. The rates of decay of all NQO₁ mRNAs in HCT 116 and HCT 116-R30A cells were similar. DNA sequences of the promoter regions of the NQO₁ gene (-837 bp) from both cell lines did not differ from each other or from the same region of the human liver NQO₁ gene. Sequences of cis elements in the 837-bp region and mRNA stability could not account for the low expression of full-length mRNA in HCT 116-R30A cells. Southern blot analysis showed the size and the intensity of the NQO₁ gene in the two cell lines to be similar. This result was confirmed by semiquantitative PCR analysis of a 450-bp fragment in the NQO₁ gene containing codon 139 and the exon 4 region. Digestion of this PCR-amplified fragment by restriction enzyme MspI revealed that HCT 116 cells have two heterozygous NQO₁ alleles, a wild-type and a tryptophan 139 form. The functional wild-type NQO₁ allele was not detected in HCT 116-R30A cells. Sensitive and resistant cell lines each contained one normal and one abnormal chromosome 16. Loss of the wild-type NQO₁ allele in HCT 116-R30A cells did not result from a loss of chromosome 16 or copies of the NQO₁ gene. Alteration of factor(s) such as trans-acting factors and DNA methylation may be involved in the down-regulation of NQO₁ in the mitomycin C-resistant HCT 116-R30A cells.

¹ This work was supported by Grant CA61862 from the NIH and Special Research Initiative Support from the University of Maryland School of Medicine.

² To whom requests for reprints should be addressed, at University of Maryland Cancer Center, 655 W. Baltimore St., Baltimore, MD 21201. Phone: (410) 328-3685; Fax: (410) 328-6559.

Received 5/23/96. Accepted 9/18/96.

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