9 nM, 4. 4 ?M, 0. 52 nM and 8. 8 ?M. Analyses of cell prolifera tion curves showed strong inhibitory effects at low concentrations of R115,777 when associated with each of the three anti estrogens and there was a suggestion of synergy for each of the combined pairs. To construct isobolograms according to the method described by Steel and Peckham we carried out another set of experiments using combina tions of the two drugs at concentrations resulting in 50% cell growth inhibition. Additive effects close to synergistic were observed between Tam and R115,777, con firming our earlier results using another FTI from a different chemical class in association with Tam.

Although it can be argued that there is only a tenuous difference between additivity and synergy for this combination of an FTI with Tam, and it is accepted that this type of analysis is not really precise enough to definitively establish additivity between two agents, the methodology does identify clear additivity with two different FTIs with diverse molecular struc tures. To extend this observation to the evaluation of other anti estrogens further, isobolograms were constructed. Isobolo gram analyses revealed a synergistic inhibi tion of MCF 7 growth with combinations of R115,777 with both ICI182,780 or PBPE. Because the main high affinity tar gets of Tam are ERs and AEBS and because of the synergistic effects between R115,777 and the ER ligand, results in agreement with data from Ellis et al,together with the additive or synergistic effects observed with Tam, we had expected a negative effect with the combination that includes the selective AEBS ligand.

Surprisingly though, PBPE also synergizes with R115,777, suggesting that cross talk between FTIs and Tam is likely to occur via at least two different pathways. To determine at what level this cross talk occurs, we analysed the combined effects of R115,777 and Tam on various mark ers of cellular apoptosis. The rationale for this study Drug_discovery was pro vided by an earlier publication by Ellis et al. who proposed that hydroxy tamoxifen and ICI182,780 MCF 7 cell proliferationanti estrogens and R115,777 on the inhibition of enhanced the number of cells with condensed nuclei. Apoptosis was further assessed using the monoclonal anti body M30 CytoDeath, which is specific for the neo epitope in cytokeratin 18 that becomes available after an early caspase cleavage during apoptosis.

The specific caspase cleavage site within cytokeratin 18 was assessed either immuncytochemically or was analysed by flow cyto moetry. We used M30 CytoDeath to selectively stain apoptotic cells, and M30 positive cells were scored. Of the cells treated by Tam alone, 19. 6% were positive. This result confirmed that Tam induced cleavage of the specific caspase cleavage site within cytokeratin 18 in breast cancer cells. R115,777 treatment induced 4. 1% to 8. 4% M30 positive cells, as pre viously reported for other FTIs with mammalian cells.