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Characterization of the DNA damage recognized by the antiserum elicited against cis-diamminedichloroplatinum (II)-modified DNA
Medicine Branch, Bldg 10, Rm 6N119, National Cancer Institute NIH, Bethesda MD 20892
1Laboratroy of Cellular Carcinogenesis and Tumor Promotion, Division of Cancer Etiology, Bld 37, Rm 3B25, National Cancer Institute NIH, Bethesda, MD 20892
2Developmental Therapeutics Branch, AIDS Program, National Institute of Allergy and Infectious Disease NIH, 6000 Executive Blvd, Bethesda, MD 20892, USA
3To whom correspondence should be addressed
A series of in vitro and in vivo were performed to characterize DNA damage recognized by an antiserum elicited against DNA modified with cis-diamminedichloroplatinum (II) (cisplatin). Adducts determined by the cisplatin-DNA enzymelinked immunosorbent assay (ELISA) in human blood cell DNA have been shown to correlate well with positive clinical outcome in testicular and ovarian cancer patients receiving platinum drug-based chemotherapy (Reed et al. (1988) Carcinogenesis, 9, 1909). DNAs from calf thymus, salmon sperm, pBR322 and synthetic oligonucleotides were modified with cisplatin in vitro before or after specific DNA digestion steps to yield adducted samples of known size and /or chemical composition. These cisplatin modified DNAs were assayed by atomic absorption spectrometry (ASS) tp assess absolute platinum content, and by ELISA to determine the antiserum specificity. The antiserum recognzes native cisplatin-modified calf thymus DNA, and native oligonucleotides containing intrastrand cis-Pt (NH3)2- d(pGpG) adducts (Pt-GG) and intrastrand cis-pit(NH3)2-d(pApG) adducts (Pt-AG). Modified plasmid DNA fragments similarly to cisplatin-modified calf thymus DNA. The antiserum does not cross-react with individual Pt-GG or Pt-AG adducts not bound to DNA. in experiments designed to assess the relationship between adduct measured by ELISA and total platinum bound to DNA as measured by AAS, male and female Sprague-Dawley rats were injected i.p. with cisplatin and a dose response for adduct formation was determined inkidney DNA samples. Values obtained by ELISA were substantially lower than those measured by AAS, the and two were directly related in DNA from kidney tissues of rodents but not in DNA from human nucleated blood cells. In rodent samples the ELISA measured a consistent 0.2% of the total DNA-bound platinum determined by AAS, with a correlation coefficient of 0.91. Among 54 blood cell DNA samples from human patients, whtich gave measurable adduct values in both ELISA and AAS, the ELISA measured a variable fraction (0.2–33.0%) of the total DNA-bound platinum measured by AAS. We conclude that the cisplatin-DNA ELISA measures a three dimensional lesion in DNA that is formed in direct proportion to total DNA-bound platinum in rat kidney, but that in human biological samples, interindividual variability precludes a relationship that conforms to simple mathematical algorithms.
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