Tumor cells are often characterized by a high and growth factor-independent proliferation rate. We have previously shown that REF cells transformed with oncogenes E1A and c-Ha-Ras do not undergo G 1 /S arrest of the cell cycle after treatment with genotoxic factors. In this work, we used sodium butyrate, a histone deacetylase inhibitor, to show that E1A ؉ Ras transformants were able to stop proliferation and undergo G 1 /S arrest. Apart from inducing G 1 /S arrest, sodium butyrate was shown to change expression of a number of cell cycle regulatory genes. It down-regulated cyclins D1, E, and A as well as c-myc and cdc25A and up-regulated the cyclin-kinase inhibitor p21 waf1 . Accordingly, activities of cyclin E-Cdk2 and cyclin A-Cdk2 complexes in sodium butyrate-treated cells were decreased substantially. Strikingly, E2F1 expression was also down-modulated at the levels of gene transcription, the protein content, and the E2F transactivating capability. To further study the role of p21 waf1 in the sodium butyrate-induced G 1 /S arrest and the E2F1 down-modulation, we established E1A ؉ Ras transformants from mouse embryo fibroblast cells with deletion of the cdkn1a (p21 waf1 ) gene. Despite the absence of p21 waf1 , sodium butyrate-treated mERas transformants reveal a slightly delayed G 1 /S arrest as well as down-modulation of E2F1 activity, implying that the observed effects are mediated through an alternative p21 waf1 -independent signaling pathway. Subsequent analysis showed that sodium butyrate induced accumulation of ␤-catenin, a downstream component of the Wnt signaling. The results obtained indicate that the antiproliferative effect of histone deacetylase inhibitors on E1A ؉ Ras-transformed cells can be mediated, alongside other mechanisms, through down-regulation of E2F activity and stabilization of ␤-catenin.Chromatin structure and eventually gene transcription are regulated at the level of histone acetylation and histone deacetylation (1-4). Higher levels of histone acetylation have been associated with transcriptional activation; correspondingly, an inactive chromatin structure correlates with histone deacetylation mediated by histone deacetylases (HDACs). 3Both of these processes, acetylation and deacetylation of histones, are involved in control of cell growth, differentiation, and apoptosis. Deregulation of these pathways can trigger development of human diseases (e.g. p300 histone acetylase gene alteration is linked with development of tumors such as colorectal and gastric carcinomas) (1, 5).Increasing evidence has been reported that HDAC inhibitors are able to suppress cell proliferation and to induce G 1 /S and/or G 2 /M block of the cell cycle in various tumor cells (2, 3, 6, 7). Moreover, it has been reported that HDAC inhibitors can cause apoptotic cell death in a number of tumor cells (3,8,9). Due to their marked antiproliferation effect, inhibitors of HDAC activity are currently being tested in cancer therapeutics clinical trials (2, 6).In the majority of tumor cells, there are various dysfun...

REF cells transformed by oncogenes E1A and cHa-ras reveal high and constitutive DNA-binding activity of AP-1 factor lacking in c-Fos protein. Consistently, the transcription of c-fos gene has been found to be downregulated. To elucidate the mechanisms of c-fos downregulation in E1A+cHa-ras transformants, we studied the levels of activity of ERK, JNK/SAPK and p38 kinases and phosphorylation state of Elk-1 transcription factor involved in regulation of c-fos gene. Using two approaches, Western blot analysis with phospho-speci®c antibodies to MAP kinases and in vitro kinase assay with speci®c substrates, we show here that ectopic expression of E1A and ras oncogenes leads to a sustained activation of ERK and p38 kinases, whereas JNK/SAPK kinase activity is similar to that in nontransformed REF52 cells. Due to sustained activity of the MAP kinase cascades, Elk-1 transcription factor is being phosphorylated even in serum-starved E1A+cHa-ras cells; moreover, serum does not additionally increase phosphorylation of Elk-1, which is predominant TCF protein bound to SRE region of c-fos gene promoter in these cells. Although the amount of ternary complexes SRE/SRF/TCF estimated by EMSA was similar both in serum-starved and serum-stimulated transformed cells, serum addition still caused a modest activation of c-fos gene transcription at the level of 20% to normal REF cells. In attempt to determine how serum caused the stimulatory eect, we found that PD98059, an inhibitor of MEK/ERK kinase cascade, completely suppressed serum-induced c-fos transcription both in REF and E1A+cHa-ras cells, implicating the ERK as primary kinase for c-fos transcription in these cells. In contrast, SB203580, an inhibitor of p38 kinase, augmented noticeably serum-stimulated transcription of c-fos gene in REF cells, implying the involvement of p38 kinase in negative regulation of c-fos. Furthermore, sodium butyrate, an inhibitor of histone deacetylase activity, was capable of activating c-fos transcription both in serum-stimulated and even in serum-starved E1A+cHa-ras cells. Conversely, serum-starved REF cells fail to respond to sodium butyrate treatment by c-fos activation con®rming necessity of prior Elk-1 phosphorylation. Taken together, these data suggest that downregulation of c-fos in E1A+cHa-ras cells seems to occur due to a maintenance of a refractory state that arises in normal REF cells after serum-stimulation. The refractory state of c-fos in E1A+cHa-ras cells is likely a consequence of Ras-induced sustained activation of MAPK (ERK) cascade and persistent phosphorylation of TCF (Elk-1) bound to SRE. Combination of these events eventually does contribute to formation of an inactive chromatin structure at c-fos promoter mediated through recruitment of histone deacetylase activity.

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