Interactions of mitomycin C with mammalian DNA detected by alkaline elution

Robert T Dorr, G. T. Bowden, David S Alberts, J. D. Liddil

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Abstract

The antitumor antibiotic mitomycin C (MMC) was studied in vitro using L1210 leukemia and 8226 human myeloma cells. Cytotoxicity was evaluated by colony formation in soft agar, and DNA damage was analyzed using alkaline elution filter assays. The purposes of these studies were: (a) to characterize the time course of MMC-DNA damage; (b) to characterize the type of DNA damage [DNA-DNA interstrand cross-links (ISC), DNA-protein cross-links (DPC), single and double strand breaks (SSBs, DSBs)]; and (c) to correlate this damage with cytotoxicity in vitro. Colony-forming assays showed the D0 value for 1 h MMC to be 15.0 μM for L1210 cells and 17 μM for 8226 cells. Alkaline elution studies showed that dose-dependent ISCs and DPCs formed rapidly following MMC exposure. Removal of cross-links was delayed, with only 50% repaired 32 h after exposure. There was a good correlation between ISCs and cytotoxicity in dose-response studies in each cell line. ISCs appeared to comprise most of the MMC-DNA lesions in both cell lines. No DNA SSBs or DSBs were observed following MMC exposure. Nuclei isolated from both cell lines and exposed to MMC produced less MMC alkylation than whole cells but, again, no strand breaks were evident. These results demonstrate that MMC is principally an alkylating agent when used at pharmacological (cytotoxic) concentrations in vitro. The lack of evidence for DNA strand breaks discounts a significant role for putative quinone-generated oxygen free radicals in the production of MMC cytotoxicity.

Original languageEnglish (US)
Pages (from-to)3510-3516
Number of pages7
JournalCancer Research
Volume45
Issue number8
StatePublished - 1985

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Mitomycin
DNA
DNA Damage
Cell Line
Leukemia L1210
DNA Breaks
Alkylating Agents
Alkylation
Agar
Free Radicals
Reactive Oxygen Species
Pharmacology
Anti-Bacterial Agents

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Interactions of mitomycin C with mammalian DNA detected by alkaline elution. / Dorr, Robert T; Bowden, G. T.; Alberts, David S; Liddil, J. D.

In: Cancer Research, Vol. 45, No. 8, 1985, p. 3510-3516.

Research output: Contribution to journalArticle

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abstract = "The antitumor antibiotic mitomycin C (MMC) was studied in vitro using L1210 leukemia and 8226 human myeloma cells. Cytotoxicity was evaluated by colony formation in soft agar, and DNA damage was analyzed using alkaline elution filter assays. The purposes of these studies were: (a) to characterize the time course of MMC-DNA damage; (b) to characterize the type of DNA damage [DNA-DNA interstrand cross-links (ISC), DNA-protein cross-links (DPC), single and double strand breaks (SSBs, DSBs)]; and (c) to correlate this damage with cytotoxicity in vitro. Colony-forming assays showed the D0 value for 1 h MMC to be 15.0 μM for L1210 cells and 17 μM for 8226 cells. Alkaline elution studies showed that dose-dependent ISCs and DPCs formed rapidly following MMC exposure. Removal of cross-links was delayed, with only 50{\%} repaired 32 h after exposure. There was a good correlation between ISCs and cytotoxicity in dose-response studies in each cell line. ISCs appeared to comprise most of the MMC-DNA lesions in both cell lines. No DNA SSBs or DSBs were observed following MMC exposure. Nuclei isolated from both cell lines and exposed to MMC produced less MMC alkylation than whole cells but, again, no strand breaks were evident. These results demonstrate that MMC is principally an alkylating agent when used at pharmacological (cytotoxic) concentrations in vitro. The lack of evidence for DNA strand breaks discounts a significant role for putative quinone-generated oxygen free radicals in the production of MMC cytotoxicity.",
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