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 β-Catenin

Abstract

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...

p21Waf1is required for cellular senescence but not for cell cycle arrest induced by the HDAC inhibitor sodium butyrate

“…6F and 7B). Thus, in line with our data that NaB inhibits clonability of mERas cells in a soft-agar assay and formation of clones on a plastic surface, 5 here we show that NaB effectively inhibits migration and invasion through a porous of membrane, while rapamycin cancels those NaB effects that are connected with migration. Importantly, these effects of rapamycin are p21 Waf1 -dependent.…”

“…17 Taken together, these data serve as evidence that accumulation of senescence-associated γH2AX foci can be a trigger of hyperactivation of pathways involved in cell senescence, as was recently proposed on a mouse model, 18 or its consequence. 9,19 Thus, although HDACI exert antiproliferative effect due in part to activation of the p21 Waf1 gene transcription, 5 nevertheless, p21…”

“…-independent and Wnt/ Tcf-dependent downregulation of S-phase transcription factor E2F1. 5 Transformation of normal cells induced by oncogene activation is restrained by triggering the program of cellular senescence. 6,7 The block of cell cycle in case of premature cell senescence has many common features with cell cycle arrest, which is induced by DNA damaging and stress factors and is often based on activation of p53/p21…”

“…meRas-Waf1 +/+ and meRas-Waf1 -/-cell lines were cultured as described previously. 5 Flow cytometry was peformed as described. Cells were washed with pBS and permeabilized for 30 min with 0.01% saponin.…”

Role of aquaporins in cell proliferation: What else beyond water permeability?

“…We also performed cell cycle analysis in the AQP1-overexpressing cells after incubation with the synchronization drug, sodium butyrate, which synchronizes cells in G1 by inhibiting cyclin E activity. 92 The cell cycle profile in the presence of butyrate showed that cells that overexpressed AQP1 are more resistant to being synchronized by this drug. The higher expression in these cells of cyclin E1, which regulates the checkpoint for G1-S transition, [89][90][91] could support this response to butyrate treatment.…”