Microfluorometric size measurements of human and phage DNA correlate with the degree of in vitro cisplatin treatment.
The anticancer drug cisplatin is shown to inhibit the proofreading activity of the enzyme T4 polymerase. A microfluorometric assay that can measure the relative sizes of cisplatin treated human and phage DNA, after exposure to T4 polymerase, is described. Using phage DNA size standards, it is shown that the mean integrated fluorescent density (IFD) of fluorophore labeled DNA is linearly correlated with molecular size. Exonuclease digestion by T4 polymerase of cisplatin treated lambda DNA gave fragment sizes whose mean IFD was proportional to the degree of platination. This method of size measurement was then applied to DNA from human carcinoma cells that had been cultured untreated, cisplatin and/or 5-fluorouracil treated. Platination resulted in exonuclease digestion fragments whose mean IFD value was significantly larger than controls (p less than 0.0006). This technique of relative size measurement appears potentially promising for the clinical analysis of altered DNA in cell populations after treatment with cytotoxic agents.
Macromolecular Analysis Section, LTPB, NICHD National Institutes of Health, Bethesda, MD 20892.
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NJ
Rampino
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PG
Johnston
D002945:Cisplatin / Q000494:pharmacology*; D003110:Colonic Neoplasms; D004273:DNA, Neoplasm / Q000187:drug effects* / Q000302:isolation & purification / Q000648:ultrastructure; D004279:DNA, Viral / Q000187:drug effects* / Q000648:ultrastructure; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D010957:Plasmids; D013604:T-Phages / Q000235:genetics*; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D004279:DNA, Viral; D002945:Cisplatin