Fig. 1. Structure of trans-[PtCl₂(NH₃)(thiazole)] (trans-amminedichloro(thiazole)platinum(II[TV1]) (ATZ) and cisplatin.

Fig. 2. Sensitivity of A2780 ovarian (•) and MCF-7 breast () tumor cells to ATZ and cisplatin. Cells were treated continuously for 3 days with ATZ (A) or cisplatin (B) and cell growth was assessed by MTT conversion. (C) Exponentially growing MCF-7 and A2780 cells were treated with ATZ for 12 h and clonogenic survival was assessed. Each point represents the mean ± S.E.M. for three to four determinations from at least two independent experiments.

Fig. 3. Cell death and growth arrest in A2780 (□) and MCF-7 (▪) cells. Cells were continuously exposed to 10 μM ATZ and cell viability was monitored by trypan blue exclusion over a period of 72 h. Each point represents the mean ± S.E.M. for three independent experiments.

Fig. 4. Detection of apoptosis in MCF-7 and A2780 cells by the TUNEL assay. A2780 and MCF-7 cells were treated with 10 μM ATZ for the indicated times and processed for the TUNEL assay. Cells were examined by fluorescence microscopy. All photomicrographs were taken at equal magnification and exposure times.

Fig. 5. Detection of senescence in MCF-7 breast tumor cells by β-galactosidase staining. MCF-7 cells were continuously exposed to 10 μM ATZ, 10 μM cisplatin or to 1 μM adriamycin (ADR) for 2 h. β-Galactosidase staining was assessed at the indicated times after initiation of drug exposure.

Fig. 6. ATZ binding to DNA. A2780 (•) or MCF-7 () cells were treated with ATZ for 5–24 h. One culture was treated for 24 h and then drug was removed (arrow) and the culture incubated in the absence of ATZ for an additional 24 h. Points represent the mean ± S.E.M. of three independent experiments.

Fig. 7. DNA–protein crosslink induction. The formation of crosslinks between DNA and cell protein was assessed as the percentage of cellular [³H]thymidine associated with protein precipitated by SDS/K. Values are presented as the percentage of total ³H in the precipitate for samples exposed to drug for 12 h, and represent the means and range for two replicate experiments.

Fig. 8. Detection of topoisomerase I-DNA complexes in MCF-7 cells by ICE bioassay. MCF-7 cells were treated with the indicated concentrations of ATZ, cisplatin, or camptothecin for the times indicated. Cesium chloride fractions were collected from the bottom of the gradients and subjected to topoisomerase I immunoblotting using C21 topoisomerase I monoclonal antibody. Fractions 6–10, encompassing the peak of cellular DNA, are shown. (Previous studies have established that fractions one to five contain neither DNA nor DNA-topoisomerase complexes.)

Table 1. Rad-equivalent DNA damage induced by ATZ in MCF-7 and A2780 tumor cell lines

Cells were exposed to drug for 12 h and strand breaks were determined using the alkaline unwinding assay, as described in Section 2. Data represent mean and standard error for three to four separate experiments.