IMMUNOCYTOCHEMICAL ANALYSIS OF CISPLATIN-INDUCED PLATINUM-DNA ADDUCTSWITH DOUBLE-FLUORESCENCE VIDEO MICROSCOPY

To detect low-level DNA platination, a sensitive immunocyto- and histo chemical technique was developed using a polyclonal antibody. The anti body GPt, derived after immunization of rabbits with highly platinated DNA and purified with affinity chromatography, detected the main plat inum (Pt)-containing intrastrand and interstrand adducts. Double-fluor escence microscopy image analysis was used to quantify Pt-DNA adducts with Hoechst 33258 fluorescence to locate the nuclei and with fluoresc ein 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 t umour cell line was detected. Large differences in Pt-DNA adduct level s after in vitro CDDP incubation between human buccal cells, lymphocyt es 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, respecti vely, 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 imp rints 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 fe asible with GPt and will hopefully enable more selective treatment of patients.