Expression of Homeobox Genes in Oral Squamous Cell Carcinoma Cell Lines Treated With All-Trans Retinoic Acid

Oral squamous cell carcinoma (OSCC) may arise from potentially malignant oral lesions. All-trans retinoic acid (atRA), which plays a role in cell growth and differentiation, has been studied as a possible chemotherapeutic agent in the prevention of this progression. While the mechanism by which atRA suppresses cell growth has not been completely elucidated, it is known that homeobox genes are atRA targets. To determine if these genes are involved in the atRA-mediated OSCC growth inhibition, PCR array was performed to evaluate the expression of 84 homeobox genes in atRA-sensitive SCC-25 cells compared to atRA-resistant SCC-9 cells following 7 days with atRA treatment. Results showed that the expression of 8 homeobox genes was downregulated and expression of 4 was upregulated in SCC-25 cells but not in SCC-9 cells. Gene expression levels were confirmed for seven of these genes by RT-qPCR. Expression of three genes that showed threefold downregulation was evaluated in SCC-25 cells treated with atRA for 3, 5, and 7 days. Three different patterns of atRA-dependent gene expression were observed. ALX1 showed downregulation only on day 7. DLX3 showed reduced expression on day 3 and further reduced on clay 7. TLX1 showed downregulation only on days 5 and 7. Clearly the expression of homeobox genes is modulated by atRA in OSCC cell lines. However, the time course of this modulation suggests that these genes are not direct targets of atRA mediating OSCC growth suppression. Instead they appear to act as downstream effectors of atRA signaling. J. Cell. Biochem. 111: 1437-1444, 2010. (C) 2010 Wiley-Liss, Inc.NIH[DK070650]NIH[CA64945]CAPES (Brazil)[PDEE 0138-08-8

Acinus-S′ Represses Retinoic Acid Receptor (RAR)-Regulated Gene Expression through Interaction with the B Domains of RARs▿ †

The diverse biological actions of retinoic acid (RA) are mediated by RA receptors (RARs) and retinoid X receptors (RXRs). Modulation of transcription by RARs/RXRs is achieved through two activation functions, ligand-independent AF-1 and ligand-dependent AF-2, located in the A/B and E domains, respectively. While the coregulatory proteins that interact with the E domain are well studied, the A/B domain-interacting partners and their influence(s) on the function of RARs are poorly understood. Acinus-S′ is an ubiquitous nuclear protein that has been implicated in inducing apoptotic chromatin condensation and regulating mRNA processing. Our data demonstrate that Acinus-S′ can specifically repress ligand-independent and ligand-dependent expression of a DR5 RA response element(RARE)-dependent reporter gene and several endogenous RAR-regulated genes in a dose-dependent and gene-specific manner. Chromatin immunoprecipitation assays show that Acinus-S′ associates with RAREs within the promoters of endogenous genes independent of RA treatment. Furthermore, the C-terminal end of Acinus-S′ and the B domain of RARβ interact independently of ligand, and the C-terminal end of Acinus-S′ is sufficient for the repression of RAR-regulated gene expression. Finally, histone deacetylase activity only partially accounts for the repressive effect of Acinus-S′ on RAR-dependent gene expression. These findings identify Acinus-S′ as a novel RAR-interacting protein that regulates the expression of a subset of RAR-regulated genes through direct binding to the N-terminal B domains of RARs

Activation of a cell-cycle-regulated histone gene by the oncogenic transcription factor IRF-2

The human histone H4 gene FO108 is regulated during the cell cycle with a peak in transcription during early S phase. The cell-cycle element (CCE) required for H4 histone activation is a sequence of 11 base pairs that binds a protein factor in electrophoretic mobility shift assays that has been designated histone nuclear factor M (HiNF-M). Here we report the purification of HiNF-M, and show it to be a protein of relative molecular mass (M(r)) 48K that is identical to interferon (IFN) regulatory factor 2 (IRF-2), a negative transcriptional regulator of the IFN response. Recombinant IRF-2 (as well as the related protein IRF-1 (ref. 5)) binds the CCE specifically and activates transcription of this H4 histone gene. IRF-2 has been shown to have oncogenic potential, and our results demonstrate a link between IRF-2 and a gene that is functionally coupled to DNA replication and cell-cycle progression at the G1/S phase transition

Protein-DNA interactions at the H4-site III upstream transcriptional element of a cell cycle regulated histone H4 gene: differences in normal versus tumor cells

Upstream sequences of the H4 histone gene FO108 located between nt -418 to -213 are stimulatory for in vivo transcription. This domain contains one protein/DNA interaction site (H4-Site III) that binds factor H4UA-1. Based on methylation interference, copper-phenanthroline protection, and competition assays, we show that H4UA-1 interacts with sequences between nt -345 to -332 containing an element displaying sequence-similarity with the thyroid hormone response element (TRE). Using gel retardation assays, we also demonstrate that H4UA-1 binding activity is abolished at low concentrations of Zn2+ (0.75 mM), a characteristic shared with the thyroid hormone (TH) receptor DNA binding protein. Interestingly, phosphatase-treatment of nuclear proteins inhibits formation of the H4UA-1 protein/DNA complex, although a complex with higher mobility (H4UA-1b) can be detected; both complexes share identical protein-DNA contacts and competition behaviors. These findings suggest that phosphorylation may be involved in the regulation of H4-Site III protein/DNA interactions by directly altering protein/protein associations. H4-Site III interactions were examined in several cell culture systems during cell growth and differentiation. We find that H4UA-1 binding activity is present during the cell cycle of both normal diploid and transformed cells. However, during differentiation of normal diploid rat calvarial osteoblasts, we observe a selective loss of the H4UA-1/H4-Site III interaction, concomitant with an increase of the H4UA-1b/H4-Site III complex, indicating modifications in the heteromeric nature of protein/DNA interactions during downregulation of transcription at the cessation of proliferation. Transformed cells have elevated levels of H4UA-1, whereas H4UA-1b is predominantly present in normal diploid cells; this alteration in the ratio of H4UA-1 and H4UA-1b binding activities may reflect deregulation of H4-Site III interactions in transformed cells. We propose that H4-Site III interactions may contribute, together with protein/DNA interactions at proximal regulatory sequences, in determining the level of H4-FO108 histone gene transcription