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Characterization of the DNA damage recognized by an antiserum elicited against cis-diamminedichloroplatinum
E Reed1, S Gupta-Burt, C L Litterst
1Medicine Branch, National Cancer Institute, NIH, Bethesda, MD 20892.
Abstract:
A series of in vitro and in vivo studies 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 enzyme-linked 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. (1990) Proc. Natl. Acad. Sci., 84; 5024, and 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 (AAS) to assess absolute platinum content, and by ELISA to determine the antiserum specificity. The antiserum recognizes native cisplatin-modified calf thymus DNA, and native oligonucleotides containing intrastrand cis-Pt (NH3)2-d(pGpG) adducts (Pt-GG) and intrastrand cis-Pt (NH3)2-d(pApG) adducts (Pt-AG). Modified plasmid DNA fragments of varying sizes (down to 309 base pairs) are recognized 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 in kidney DNA samples. Values obtained by ELISA were substantially lower than those measured by AAS, and the 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, which 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.
Insights
This study characterizes DNA damage from cisplatin chemotherapy using a specific assay. While the cisplatin-DNA ELISA correlates with treatment outcomes in cancer patients, its relationship with total platinum DNA adducts varies between rodents and humans.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Cisplatin is a platinum-based chemotherapy drug widely used for various cancers.
- Platinum-DNA adducts are the primary mechanism of cisplatin's cytotoxic and antitumor effects.
- Accurate quantification of DNA adducts is crucial for understanding cisplatin efficacy and toxicity.
Purpose of the Study:
- To characterize the DNA damage recognized by an antiserum against cisplatin-modified DNA.
- To evaluate the specificity and utility of a cisplatin-DNA enzyme-linked immunosorbent assay (ELISA).
- To compare ELISA-measured adducts with total platinum DNA adducts determined by atomic absorption spectrometry (AAS) in both in vitro and in vivo models.
Main Methods:
- In vitro modification of various DNA sources (calf thymus, salmon sperm, plasmid, oligonucleotides) with cisplatin.
- Assay of modified DNA using atomic absorption spectrometry (AAS) for total platinum content.
- Assay of modified DNA using ELISA with a specific antiserum to detect cisplatin-DNA adducts.
- In vivo studies involving cisplatin administration to Sprague-Dawley rats and analysis of kidney DNA.
- Analysis of human blood cell DNA samples from cancer patients.
Main Results:
- The antiserum specifically recognizes native cisplatin-modified DNA, including intrastrand cis-Pt(NH3)2-d(pGpG) (Pt-GG) and cis-Pt(NH3)2-d(pApG) (Pt-AG) adducts.
- The ELISA method detects cisplatin-modified DNA fragments as small as 309 base pairs.
- In rats, ELISA-measured adducts were consistently lower than AAS-measured platinum but showed a strong direct correlation (0.91).
- In human blood DNA, the ELISA measured a highly variable fraction (0.2-33.0%) of the total platinum detected by AAS, indicating significant interindividual variability.
Conclusions:
- The cisplatin-DNA ELISA quantifies a specific three-dimensional DNA lesion formed by cisplatin.
- In rodent models, the ELISA provides a reliable measure of platinum-DNA adducts proportional to total platinum.
- In humans, interindividual variability in DNA repair or adduct formation complicates a direct mathematical relationship between ELISA and total platinum adducts.
- The findings highlight the importance of considering biological variability when using immunoassays for quantifying drug-induced DNA damage in clinical settings.
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