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Antibodies against cisplatin-modified DNA and cisplatin-modified (di) nucleotides

  • Original Articles
  • Cisplatin-Modified DNA, Nucleotides, Antibodies
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Summary

Cytotoxic effects ofcis-diamminedichloroplatinum-(II) (cis-DDP) are thought to be mediated by binding to DNA. Studies on binding ofcis-DDP to cellular DNA rely heavily on the availability of specific antibodies. We therefore, raised and characterized four rabbit antisera: one againstcis-DDP-modified DNA (antiserum NKI-A59) and three others against thecis-DDP-modified (di)nucleotidescis-Pt(NH3)2d(pApG) (NKI-A68),cis-Pt(NH3)2d(GMP)2(NKI-A10), and Pt(NH3)3dGMP (NKI-A39). Reactivities to platinum compounds were determined in an enzyme-linked immunosorbent assay (ELISA) and in a quantitative immunocytochemical assay. In the ELISA, NKI-A59 showed a high affinity for DNA heavily substituted with eithercis-DDP or CBDCA [cis-diammine (1,1-cyclobutanedicarboxylato)platinum(II)]; amounts of platinum per well giving 50% inhibition (IA50) were as low as 15 and 76 fmol, respectively. NKI-A59 also showed affinity tocis-DDP-modified poly[d(G-C)] poly[d(G-C)], poly(dC), and poly(dG). No affinity was found fortrans-DDP [trans-diamminedichloro-platinum(II)]-modified DNA, enzymatically digestedcis-DDP-DNA, orcis-DDP-modified poly(dA)., poly(dT), oligo(dA) −15 , oligo(dT) −15 , oligo(dG)21, oligo(dG)42, or oligo(dAAAG)10. The efficiency of binding tocis-DDP-DNA decreased with decreasing DNA modification levels. Although othercis-DDP-DNA- andcis-DDP-(di)nuclcotide-specific antisera have been identified, NKI-A59 is the first antiserum described that is suitable for the in situ detection ofcis-DDP-DNA adducts at clinically relevant platinum levels. Adduct-specific immunostaining signals in cultured RIF-1 cells or rat liver paralleled platinum-DNA binding as measured by atomic absorption spectroscopy. The antisera NKI-A68, NKI-A10, and NKI-A39 showed high affinity for their corresponding haptens and varying affinity for non-haptencis-DDP-DNA adducts. Their affinity for digestedcis-DDP-modified DNA was up to 30 times that for intactcis-DDP-DNA. Neither NKI-A68 nor NKI-A10 resulted in specific immunocytochemical staining ofcis-DDP-DNA adducts. We conclude that NKI-A68, NKI-A10, and NKI-A39 are suitable for platinum-DNA adduct analysis of digested DNA in ELISA and that NKI-A59 is suitable for platinum-DNA adduct detection at the single-cell level using immunocytochemical methods.

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Abbreviations

cis-DDP:

cis-diamminedichloroplatinum(II)

trans-DDP:

trans-diamminedichloroplatinum(II)

CBDCA:

cis-diammine (1,1-cyclobutanedicarboxylato)platinum(II)

DACH-PT:

[sp-4-2-(1R,2R)]-(1,2-diaminecyclohexane)dichloroplatinum(II)

Pt-GG:

cis-Pt(NH3)2]d(pGpG)-N7(1),N7(2)]

Pt-AG:

cis-Pt(NH3)2[d(pApG)-N7(1), N7(2)]

G-Pt-G:

cis-Pt(NH3)2 [5′-dGMP-N7]2

Pt-G:

Pt(NH3)3-[5′-dGMP-N7]

CT-DNA:

ealf thymus DNA

Rb :

platinum-nucleotide ratio; bp, base pairs

IA50 :

amount of inhibitor per microtiter well giving 50% inhibition in a competitive ELISA

BSA:

bovine serum albumin; ELISA, enzyme-linked immunosorbent assay

HPLC:

high-pressure liquid chromatography

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Authors and Affiliations

  1. Division of Chemical Carcinogenesis, The Netherlands Cancer Institute (Antoni van Leeuwenhoek Huis), Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands

    P. M. A. B. Terheggen, B. G. J. Floot & L. den Engelse

  2. Division of Clinical Chemistry, The Netherlands Cancer Institute (Antoni van Leeuwenhoek Huis), Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands

    O. van Tellingen

  3. Division of Experimental Therapy, The Netherlands Cancer Institute (Antoni van Leeuwenhoek Huis), Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands

    A. C. Begg

  4. Gorlaeus Laboratories, State University of Leiden, 2300 RA, Leiden, The Netherlands

    E. L. M. Lempers

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  1. P. M. A. B. Terheggen
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  3. E. L. M. Lempers
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  6. L. den Engelse
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Terheggen, P.M.A.B., Floot, B.G.J., Lempers, E.L.M. et al. Antibodies against cisplatin-modified DNA and cisplatin-modified (di) nucleotides. Cancer Chemother. Pharmacol. 28, 185–191 (1991). https://doi.org/10.1007/BF00685507

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  • DOI: https://doi.org/10.1007/BF00685507

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