These results show that Cl amidine is effective in inhi biting the growth of luminal HER2 ERBB2 cell lines, BT 474 and SK BR 3, and agree with previously reported data on Cl amidine inhibition of growth in MCF7 cells. We wanted to test whether there would be any effect on a basal cell line, and maybe chose MDA MB 231 for comparison. Surprisingly, we see an effect on both cell growth and apoptosis, with the top 10 upregulated and downre gulated genes presented in Table 2. Importantly, previ ous studies have shown that increased expression of GADD45, the second most highly upregulated gene in our study, leads to cell cycle arrest and apoptosis in a range of cell types, including breast cancer cells. This observation suggested that, in addition to affecting cell cycle gene expression, Cl amidine might also alter MCF10DCIS cell growth by inducing apop tosis.

To test this hypothesis, we next treated MCF10A and MCF10DCIS cells with increasing concentrations of Cl amidine for 4 days. Cells were fixed and labeled with anti activated Caspase 3 antibody or DAPI, and then analyzed by flow cytometry. Results show that Cl amidine treatment significantly increased the percent of apoptotic MCF10DCIS cells in a dose dependent man ner. In contrast, the MCF10A cells were largely unaffected. Furthermore, we also show that treat ment of MCF10DCIS cells with Cl amidine appears to induce cell cycle arrest in S phase. Lastly, we wanted to see whether the increase in apoptosis occurs earlier after treatment, so we tested the cells again fol lowing 2 days of treatment, but were unable to see any effect.

However, this was not surprising, as the effects of Cl amidine are most pro nounced after 3 days of treatment. Taken together, it appears that Cl amidine treatment after 4 days leads to S phase coupled apoptosis, which is an intrinsic mechanism that prevents DNA replication and c albeit a smaller effect on apoptosis than we see in BT 474 and SK BR 3. While this is interesting, and perhaps suggests the expression of a different PADI fam ily member in this basal cell line, we have focused on PADI2 expressing cancers for this study, which are pre dominantly luminal and HER2 ERBB2 expressing. Taken together, these results suggest that Cl amidine blocks the growth of MCF10DCIS cells by inducing cell cycle arrest and apoptosis. This prediction is supported by our previous finding that Cl amidine can also drive apoptosis in lymphocytic cell lines in vitro. Importantly, the lack of an apoptotic effect in MCF10A cells suggests that Cl amidine may primarily target tumor cells Drug_discovery for killing. Consistent with this possibility is the fact that Cl amidine did not affect the growth of non tumorigenic NIH3T3 cells and HL60 granulocytes.