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

Abstract

SummaryTo 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 CDDPsensitive 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.Keywords: cisplatin; platinum-DNA adducts; immunocytochemistry; histochemistry; polyclonal antibody; image analysis CDDP and its analogues are among the most important chemotherapeutic drugs in clinical practice with a broad spectrum of activity. Almost all patients treated for testicular cancer with CDDP-containing combinations respond, and approximately 80% are cured. In contrast, no cures can be obtained in the case of colon cancer and most other solid tumours, even when initially sensitive to this drug, for example in the case of small-cell lung cancer (Loehrer and Einhorn, 1984). In all of these settings, a number of patients will not respond to therapy, although side-effects will be similar to those of responders. It would be desirable to be able to predict response to therapy as, for non-responders, either dose intensification or other forms of treatment would be preferable to standard CDDP treatment.In the past, rather unsuccessful attempts have been made to predict responses to therapy. Studies were performed with detection methods for cell kill (Hamburger and Salmon, 1977) or proliferation inhibition (Mosman, 1983; Weisenthal et al, 1983) in solid tumour samples exposed to drugs in vitro. Attempts to predict the effects of treatment of childhood lymphoblastic leukaemia by exposure of (non-proliferating) peripheral blood blast cells in vitro to drugs before systemic treatment have been more successful (Pieters et al, 1991). For (Poirier et al, 1985(Poirier et al, , 1992Reed et...

New insights in the cellular processing of platinum antitumor compounds, using fluorophore-labeled platinum complexes and digital fluorescence microscopy

“…Studies reported so far utilize fluorescence microscopy to visualize natively fluorescing drugs, such as daunorubicin [27], or ± in the case of non-fluorescing drugs such as cisplatin ± use for instance an immunocytochemical detection method to visualize cisplatin-DNA adducts in cells. For the latter technique, antibodies against Pt-DNA adducts have been generated [28,29].…”

Personalized Medicine for Targeted and Platinum-Based Chemotherapy of Lung and Bladder Cancer

“…Terheggen et al , developed a peroxidase-based immunoassay assay that was relatively insensitive, with an LOD of two adducts per 10 6 nucleotides, but compatible with quantitative determination of cisplatin–DNA adducts in tissue sections or cells from animals and cancer patients [145–149]. Meijer et al developed an immunohistochemistry assay that allowed the detection of cisplatin–DNA adducts at a single-cell level in buccal and tumor cells of patients [150]. Tilby et al [151] developed an ELISA method based upon a monoclonal antibody that was used to quantify carboplatin–DNA and cisplatin–DNA adducts formed in patients during therapy [120,121,152].…”

Cytometric assessment of DNA damage in relation to cell cycle phase and apoptosis

“…The list of different DNA adducts which can be detected immunocytochemically is extensive (den Engelse et al . 1990; Meijer et al . 1997; Mistry et al .…”

“…Also, the methylated bases O6-methylguanine and 7-methylguanine were immunocytochemically identified in rodent tissues after exposure to the tobacco-specific nitrosamine-4(methylnitrosamino)-1-(3-pyridyl)-1-butanone (Van Benthem et al 1994). The list of different DNA adducts which can be detected immunocytochemically is extensive (den Engelse et al 1990;Meijer et al 1997;Mistry et al 2003), but it is not in the scope of this review to cover this topic. Potentially, all these adducts that can be immnunocytochemically identified can be studied by multiparameter cytometry with respect to cell cycle phase specificity and induction of apoptosis.…”