Immunocytochemical analysis of cisplatin-induced platinum-DNA adducts with double-fluorescence video microscopy

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Abstract

To detect low-level DNA platination, a sensitive immunocyto- and histochemical technique was developed using a polyclonal antibody. The antibody GPt, derived after immunization of rabbits with highly platinated DNA and purified with affinity chromatography, detected the main platinum (Pt)-containing intrastrand and interstrand adducts. Double-fluorescence microscopy image analysis was used to quantify Pt-DNA adducts with Hoechst 33258 fluorescence to locate the nuclei and with fluorescein isothiocyanate fluorescence to measure the immunosignal. A two- to five-fold dose-dependent difference in the level of cisplatin (CDDP)-induced Pt-DNA adducts between a CDDP-sensitive and -resistant human tumour cell line was detected. Large differences in Pt-DNA adduct levels after in vitro CDDP incubation between human buccal cells, lymphocytes and biopsies of different tumour types were observed. Pt-DNA adduct levels were fivefold higher in human testicular tumours than in colon tumours, representing CDDP-sensitive and -resistant tumours, respectively, in the clinic. These data suggest the possibility of predictive testing by measuring Pt-DNA adduct levels. Pt-DNA adducts in patients after treatment with CDDP were shown in normal buccal cells and in imprints of fresh tumour biopsies as well as in paraffin-embedded tumour cells. The analysis of Pt-DNA adducts at a single-cell level in small samples of normal and tumour cells during and/or after treatment is feasible with GPt and will hopefully enable more selective treatment of patients.
Original languageEnglish
Pages (from-to)290-298
Number of pages9
JournalBritish Jounal of Cancer
Volume76
Issue number3
Publication statusPublished - Aug-1997

Keywords

  • cisplatin
  • platinum-DNA adducts
  • immunocytochemistry
  • histochemistry
  • polyclonal antibody
  • image analysis
  • OVARIAN-CANCER PATIENTS
  • PATIENTS RECEIVING CARBOPLATIN
  • CELL-LINES
  • LEUKOCYTE DNA
  • ACQUIRED-RESISTANCE
  • MONOCLONAL-ANTIBODY
  • DISEASE RESPONSE
  • HUMAN-TUMORS
  • BLOOD-CELLS
  • INVITRO

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