Mechanisms of suberoylanilide hydroxamic acid inhibition of mammary cell growth

Hybrid polar compounds (HPCs) have induced cell growth arrest, terminal differentiation and/or apoptosis in various transformed cell lines. We have previously reported that the prototype HPC (hexamethylene bisacetamide [HMBA]) was able to arrest the growth of transformed mammary (TM) 2H cells (p53 null), a highly tumorigenic mouse mammary epithelial cell line, by inhibiting G1 kinase activities, concomitant with an increase in the cyclin D2 protein level and hypophosphorylated isoforms of the three pRb pocket proteins, which led to the formation of stable cyclin D2/pRb complexes and G1 cell arrest. It has been reported that the second generation of HPCs (suberoylanilide hydroxamic acid [SAHA]), structurally related to but 2000-fold more potent than HMBA, was an inhibitor of histone deacetylase activity and caused accumulation of hyperacetylated histone H4 in murine erythroleukemia.To determine the mechanism of SAHA in cell growth inhibition in TM10 (p53 wt) and TM2H (p53 null) hyperplastic mouse mammary cell lines.TM10 and TM2H cells were examined in the presence or absence of 2.5 μM SAHA for cell growth rate by [3H]-thymidine uptake, DNA synthesis by flow cytometry after cells were labeled with BrdU, G1/S cyclin-dependent kinase (cdk) activities, phosphorylation levels of pRb pocket proteins, protein levels of E2F-1, PCNA and p21, pRb-2/p130 interaction, and nuclear localization with E2F-4 by western blot, immunoprecipitation and immunostaining assays.SAHA was able to arrest cell growth at G1, and inhibited DNA synthesis in both TM10 and TM2H cell lines. Cell growth arrest was accompanied by increases in histone H3 and H4 protein and acetylation levels, a profound increase in the interaction and nuclear localization of pRb-2/p130–E2F-4 complexes, significant reductions in E2F-1 and PCNA protein levels, inhibition in G1/S cdk activities and increases in the levels of hypophosphorylated isoforms of three pRb pocket proteins.A novel mechanism of SAHA mediated growth i