wnregulation of several genes, most notably c myc , hTERT and Bcl XL . Vorinostat PLX-4720 Raf inhibitor downregulated another anti apoptotic gene, Mcl 1, while Bcl 2 levels changed very little . Since vorinostat downregulated message levels of c myc, we assayed levels of the Myc antagonist, Mxd1 , and found it was simultaneously upregulated . Such inverse patterns of expression of Myc and Mxd genes have been seen in multiple cell types studied, often in cells exiting the cell cycle and/or undergoing differentiation . In contrast to downregulation of anti apoptotic Bcl XL and Mcl 1, vorinostat upregulated the proapoptotic genes Bad, Bid and Noxa . Most gene expression changes were apparent within four hours of vorinostat addition and were still variably present at 24 hours for Myc and Mxd1 , hTERT and Bcl XL and Noxa .
However Bad and Bid message level increases were an early event, seen only at the 4 hour time point . By 24 hours their expression levels were at baseline or somewhat repressed . Immunoblotting experiments confirmed qPCR results and assessed post translational changes in L540 cell proteins . Figure BIIB021 848695-25-0 4A, top frame, shows vorinostat concentration dependent increases in acetylation at the histone H3 lysine nine residue , which were unchanged by addition of MK 0457. Acetylation of p53 seemed less sensitive to vorinostat than was H3 K9, becoming apparent only at higher concentrations. Acetylation of p53 was also seen in response to MK 0457, with greater response when combined with 3 μM vorinostat , acetylation of p53 is known to lead to stabilization .
MK 0457 mediated increased p53 acetylation was associated with Kretzner et al. Page 5 Cancer Res. Author manuscript, available in PMC 2012 June 1. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript increased protein levels of p53 target p21Waf1/Cip1, as well as the mRNA levels of p53 target Noxa . While the amount of p21 and p27 proteins increased in response to vorinostat or MK 0457 alone and in response to MK 0457 in combination with the lowest dose of vorinostat, the levels of these proteins subsequently decreased in response to MK 0457 in combination with the highest dose of vorinostat. This is in agreement with other studies showing that downregulation of p21 or p27 makes cells more prone to apoptosis and is also consistent with accumulation of cells in sub G1 .
The Western blot data in Figure 4A confirmed at the protein level the downregulation of c myc and FOXO3A genes detected by qPCR . Similarly, Bcl XL and Mcl 1 protein levels were also reduced . Cell cycle block experiments using the microtubule poison nocodazole allowed us to enrich for protein isoforms transiently present during the G2/M phase that are difficult to detect in nonsynchronized cells . Utilizing synchronized cell populations we were able to visualize the phosphorylated forms of three aurora kinase targets by Western blot assay. p53 is normally phosphorylated at Ser315 by AK A, leading to its association with the ubiquitin ligase MDM2 and proteosome destruction . Phosphorylation of p53at Ser46 is strongly associated with pro apoptotic activity of this tumor suppressor .
Histone H3 is a known substrate for AK B phosphorylation at Serine 10 resulting in dissociation of heterochromatin protein 1 during mitosis. . To assess the effects of Aurora Kinase treatment on these substrates, we treated L540 cells with nocodazole, with or without MK 0457, and compared them to cells treated with MK 0457 alone and to control cycling cells. Cell cycle analyses indicated MK 0457 and nocodazole both blocked cycling, the nocodazole treated +/�?MK 0457, were similarly enriched for G2/M phase cells . All drug treated cells also had similar viability All three phospho proteins analyzed were expressed at low levels in cycling cells but accumulated at detectable levels in the presence of nocodazole. MK 0457 inhibited the phosphorylation of histone H3 in the presence of nocodazole. p53 phosphorylation at both Se