Antibodies elicited against cis-diamminedichloroplatinum(II)-modified DNA are specific for cis-diamminedichloroplatinum(II)-DNA adducts formed in vivo and in vitro

M. C. Poirier, S. J. Lippard, L. A. Zwelling, H. M. Ushay, D. Kerrigan, C. C. Thill, R. M. Santella, D. Grunberger, S. H. Yuspa

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Rabbit antiserum elicited against calf thymus DNA modified to 4.4% (Pt drug/nucleotide ratio = 0.044) with the antitumor drug cis-diamminedichloroplatinum (II) (cis-DDP) contains antibodies specific for the Pt-modified DNA immunogen as well as for Pt-DNA adducts formed in both cultured mouse leukemia L1210 cells and in L1210 cells from the ascites fluid of tumor-bearing mice exposed to cis-DDP. Pt-modified DNA was electrostatically complexed to methylated bovine serum albumin and injected into rabbits. Early bleedings of the derived antiserum were used to establish a competitive enzyme-linked immunosorbent assay (ELISA), which demonstrated specificity for the Pt-modified DNA but not for DNA or the Pt drug alone. In the ELISA, 50% inhibition occurred at a concentration of 0.5 nM Pt (on DNA) as determined by atomic absorption spectroscopy. This value corresponds to a lower limit of detectability of one adduct in 10 7 nucleotides, with 50 μg of sample DNA added per microtiter well. DNA isolated from cultured mouse L1210 cells exposed to increasing doses of the Pt drug was found by ELISA to contain from 0.2 to 10.0 fmol of Pt adduct per μg of DNA. These levels remained stable for up to 4 hr after a 1-hr drug treatment, during which time DNA interstrand crosslinks developed. Thus, the antiserum appears not to be specific for DNA interstrand crosslinks. DNAs from L1210 cells exposed to trans-diamminedichloroplatinum(II) and L-phenylalanine mustard were not recognized in the ELISA. DNA prepared from the ascites cells of mice bearing the L1210 tumor 5 hr after injection of cis-DDP was found to contain about 2 fmol of Pt per μg of DNA. This work establishes that cis-DDP-DNA adducts prepared in vitro are relevant to be in vivo binding of the Pt drug to its biological target, DNA, and opens new avenues for studying the mechanism of action of the Pt anticancer drugs.

Original languageEnglish (US)
Pages (from-to)6443-6447
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number21 I
Publication statusPublished - Dec 1 1982
Externally publishedYes


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