This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pan, S.-s. Articles by Hu, L.-T. Search for Related Content PubMed PubMed Citation Articles by Pan, S.-s. Articles by Hu, L.-T.

NAD(P)H:Quinone Oxidoreductase Expression and Mitomycin C Resistance Developed by Human Colon Cancer HCT 116 Cells¹

Division of Developmental Therapeutics, University of Maryland Cancer Center, Baltimore, Maryland 21201 [S-S. P., L-T. H.]; Division of Biology, Beckman Research Institute Research Institute [G. L. F.], and Department of Medical Oncology and Therapeutics Research, City of Hope National Medical Center [S. A. A.], Duarte, California 91010

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.

¹ This work was supported by Grant CH-412 from the American Cancer Society (to S. P.)

² To whom requests for reprints should be addressed, at University of Maryland Cancer Center, 655 West Baltimore Street, Baltimore, MD 21201.

Received 6/23/94. Accepted 11/ 9/94.

This article has been cited by other articles:

				A. Begleiter, D. Hewitt, A. W. Maksymiuk, D. A. Ross, and R. P. Bird A NAD(P)H:Quinone Oxidoreductase 1 Polymorphism Is a Risk Factor for Human Colon Cancer Cancer Epidemiol. Biomarkers Prev., December 1, 2006; 15(12): 2422 - 2426. [Abstract] [Full Text] [PDF]

				Y. Han, H. Shen, B. I. Carr, P. Wipf, J. S. Lazo, and S.-s. Pan NAD(P)H:Quinone Oxidoreductase-1-Dependent and -Independent Cytotoxicity of Potent Quinone Cdc25 Phosphatase Inhibitors J. Pharmacol. Exp. Ther., April 1, 2004; 309(1): 64 - 70. [Abstract] [Full Text]

				M. Krajinovic, D. Labuda, G. Mathonnet, M. Labuda, A. Moghrabi, J. Champagne, and D. Sinnett Polymorphisms in Genes Encoding Drugs and Xenobiotic Metabolizing Enzymes, DNA Repair Enzymes, and Response to Treatment of Childhood Acute Lymphoblastic Leukemia Clin. Cancer Res., March 1, 2002; 8(3): 802 - 810. [Abstract] [Full Text] [PDF]

				W. Lang, J. Mao, T. W. Doyle, and B. Almassian Isolation and Identification of Urinary Metabolites of Porfiromycin in Dogs and Humans Drug Metab. Dispos., August 1, 2000; 28(8): 899 - 904. [Abstract] [Full Text]

				R. J. Knox, T. C. Jenkins, S. M. Hobbs, S. Chen, R. G. Melton, and P. J. Burke Bioactivation of 5-(Aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) by Human NAD(P)H Quinone Oxidoreductase 2: A Novel Co-substrate-mediated Antitumor Prodrug Therapy Cancer Res., August 1, 2000; 60(15): 4179 - 4186. [Abstract] [Full Text]

				J. L. Wiemels, A. Pagnamenta, G. M. Taylor, O. B. Eden, F. E. Alexander, and M. F. Greaves A Lack of a Functional NAD(P)H:Quinone Oxidoreductase Allele Is Selectively Associated with Pediatric Leukemias That Have MLL Fusions Cancer Res., August 1, 1999; 59(16): 4095 - 4099. [Abstract] [Full Text] [PDF]

				J. J. Reiners Jr., J.-Y. Lee, R. E. Clift, D. T. Dudley, and S. P. Myrand PD98059 Is an Equipotent Antagonist of the Aryl Hydrocarbon Receptor and Inhibitor of Mitogen-Activated Protein Kinase Kinase Mol. Pharmacol., March 1, 1998; 53(3): 438 - 445. [Abstract] [Full Text]