HDAC inhibitor sodium butyrate sensitizes E1A+Ras-transformed cells to DNA damaging agents by facilitating formation and persistence of γH2AX foci.

HDAC inhibitors (HDACi) suppress the growth of tumor cells due to induction of cell cycle arrest, senescence or apoptosis. Recent data demonstrate that HDACi can interfere with DNA Damage Response (DDR) thereby sensitizing the cells to DNA damaging agents. Here, we show that HDACi sodium butyrate (NaBut) potentiates the formation of γH2AX foci predominantly in S-phase E1A+Ras cells. Accumulation of γH2AX foci sensitizes the cells toward such DNA damaging agents as irradiation (IR) and adriamycin. In fact, NaBut potentiates the persistence of γH2AX foci induced by genotoxic agents. The synergizing effects depend on DNA damaging factors and on the order of NaBut treatment. Indeed, NaBut treatment for 24 h leads to an accumulation of G 1-phase cells and a lack of S-phase cells, therefore, adriamycin, a powerful S-phase-specific inhibitor, when added to NaBut-treated cells, is unable to substantially add γH2AX foci. In contrast, IR produces both single- and double-strand DNA breaks at any stage of the cell cycle and was shown to increase γH2AX foci in NaBut-treated cells. Further, a lifetime of IR-induced γH2AX foci depends on the subsequent presence of HDACi. Correspondingly, NaBut withdrawal leads to the extinction of IR-induced γH2AX foci. This necessitates HDACi to hold the IR-induced γH2AX foci unrepaired. However, the IR-induced γH2AX foci persist after long-term NaBut treatment (72 h) even after washing the drug. Thus, although signaling pathways regulating H2AX phosphorylation in NaBut-treated cells remain to be investigated, the obtained results show that NaBut potentiates effects of DNA damaging agents by facilitating formation and persistence of γH2AX foci.

e2f1 Gene is a new member of Wnt/beta-catenin/Tcf-regulated genes.

HDAC inhibitors induce cell cycle arrest of E1A+Ras-transformed cells accompanied by e2f1 gene down-regulation and activation of Wnt pathway. Here we show that e2f1 expression is regulated through the Wnt/Tcf-pathway: e2f1 promoter activity is inhibited by sodium butyrate (NaB) and by overexpression of beta-catenin/Tcf. The e2f1 promoter was found to contain two putative Tcf-binding elements: the proximal one competes well with canonical Tcf element in DNA-binding assay. Being inserted into luciferase reporter vector, the identified element provides positive transcriptional regulation in response to beta-catenin/Tcf co-transfection and NaB treatment. Thus we have firstly demonstrated that e2f1 belongs to genes regulated through Wnt/beta-catenin/Tcf pathway.

G1/S arrest induced by histone deacetylase inhibitor sodium butyrate in E1A + Ras-transformed cells is mediated through down-regulation of E2F activity and stabilization of beta-catenin.

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 beta-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 beta-catenin.

Downregulation of c-fos gene transcription in cells transformed by E1A and cHa-ras oncogenes: a role of sustained activation of MAP/ERK kinase cascade and of inactive chromatin structure at c-fos promoter.

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-specific antibodies to MAP kinases and in vitro kinase assay with specific 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 non-transformed 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 effect, 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 confirming 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.