title: Immunocytochemical analysis of cisplatin-induced platinum-DNA adducts with double-fluorescence video microscopy.
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.
authors.affiliation: Department of Medical Oncology, University Hospital Groningen, The Netherlands.
authors.forename: C
authors.initials: C
authors.lastname: Meijer
authors.forename: E G
authors.initials: EG
authors.lastname: de Vries
authors.forename: W A
authors.initials: WA
authors.lastname: Dam
authors.forename: M H
authors.initials: MH
authors.lastname: Wilkinson
authors.forename: H
authors.initials: H
authors.lastname: Hollema
authors.forename: H J
authors.initials: HJ
authors.lastname: Hoekstra
authors.forename: N H
authors.initials: NH
authors.lastname: Mulder
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002945:Cisplatin / Q000494:pharmacology*; D018736:DNA Adducts / Q000032:analysis*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008214:Lymphocytes; D009061:Mouth Mucosa / Q000166:cytology; D010984:Platinum / Q000032:analysis; D011817:Rabbits; D014741:Video Recording
chemical_list: D018736:DNA Adducts; D010984:Platinum; D002945:Cisplatin