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Isolation and characterization of a mitomycin C-resistant variant of human colon carcinoma HT-29 cells

  • Original Articles
  • Drug Resistance, Mitomycin C, DT-Diaphorase, Human Colon Carcinoma
  • Published:
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Abstract

To investigate the resistant mechanisms against MMC in human tumor cells, we isolated an MMC-resistant variant (HT-29/MMC) of HT-29 human colon carcinoma cells. HT-29/MMC cells showed 5-fold resistance to MMC as compared with the parental cell line but did not show cross-resistance to Adriamycin, vincristine, ACNU, bleomycin, or cisplatin. Treatment of the cells with dicoumarol, an inhibitor of DT-diaphorase, reduced the cytotoxicity of MMC in DT-diaphorase proficient HT-29 cells but not in HT-29/MMC cells. HT-29/MMC cells were 5 times more sensitive than HT-29 cells to menadione, which is detoxified by DT-diaphorase. DT-diaphorase was deficient in HT-29/MMC cells as determined by the enzyme activity and immunoblot analysis of the cytoplasmic proteins. Levels of cytochrome P-450 reductase and glutathione S-transferase, however, were comparable in both cell lines. The amount of [3H]-MMC found covalently bound to chromosomal DNA in HT-29/MMC cells was one-fourth that detected in HT-29 cells. Treatment with dicoumarol reduced the DNA-bound MMC in HT-29 cells but not in HT-29/MMC cells. These results indicate that the deficiency in DT-diaphorase, an activating enzyme of MMC, is one of the mechanisms of resistance in HT-29/MMC cells.

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Abbreviations

MMC:

mitomycin C

DCPIP:

2,6-dichlorophenolindophenol

TEMPOL:

4-hydroxytetramethyl piperidine-1-oxyl

ACNU:

1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea

SDS-PAGE:

sodium dodecyl sulfate polyacrylamide gel electrophoresis

PMSF:

phenyl methyl sulfonyl fluoride

PBS.(−):

phosphatebuffered saline without calcium or magnesium

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Authors and Affiliations

  1. Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi, Bunkyo-ku, 113, Tokyo, Japan

    Jeong-Hyung Lee, Mikihiko Naito, Motowo Nakajima & Takashi Tsuruo

  2. Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Kami-Ikebukuro, Toshima-ku, 170, Tokyo, Japan

    Takashi Tsuruo

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  1. Jeong-Hyung Lee
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  2. Mikihiko Naito
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  3. Motowo Nakajima
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  4. Takashi Tsuruo
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This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan

Correspondence to: Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan

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Lee, JH., Naito, M., Nakajima, M. et al. Isolation and characterization of a mitomycin C-resistant variant of human colon carcinoma HT-29 cells. Cancer Chemother. Pharmacol. 33, 215–220 (1993). https://doi.org/10.1007/BF00686219

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  • DOI: https://doi.org/10.1007/BF00686219

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