Jae Cheol Lee

Histone deacetylase inhibitor apicidin downregulates DNA methyltransferase 1 expression and induces repressive histone modifications via recruitment of corepressor complex to promoter region in human cervix cancer cells.

Dysregulation of DNA methyltransferase (DNMT)1 expression is associated with cellular transformation, and inhibition of DNMT1 exerts antitumorigenic effects. Here, we report that DNMT1 abnormally expressed in HeLa cells is downregulated by a histone deacetylase (HDAC) inhibitor apicidin, which is correlated with induction of repressive histone modifications on the promoter site. Apicidin selectively represses the expression of DNMT1 among DNMTs in HeLa cells, independent of cell cycle arrest at G0/G1. Furthermore, apicidin causes a significant reduction in the recruitment of RNA polymerase II into the promoter. Chromatin immunoprecipitation analysis shows that even though apicidin causes global hyperacetylation of histone H3 and H4, localized deacetylation of histone H3 and H4 occurs at the E2F binding site, which is accompanied by the recruitment of pRB and the replacement of P/CAF with HDAC1 into the sites. In addition, K4-trimethylated H3 on nucleosomes associated with the transcriptional start site is depleted following apicidin treatment, whereas repressive markers, K9- and K27-trimethylation of H3 are enriched on the site. The downregulation of DNMT1 expression seems to require de novo protein synthesis, because the apicidin effect is antagonized by cycloheximide treatment. Moreover, knock down of DNMT1 with siRNA induces the apoptosis of HeLa cells, indicating that downregulation of DNMT1 might be a good strategy for therapeutics of human cervix cancer. Collectively, our findings will provide a mechanistic rationale for the use of HDAC inhibitors in cancer therapeutics.

Depletion of Embryonic Stem Cell Signature by Histone Deacetylase Inhibitor in NCCIT Cells: Involvement of Nanog Suppression

The embryonic stem cell-like gene expression signature has been shown to be associated with poorly differentiated aggressive human tumors and has attracted great attention as a potential target for future cancer therapies. Here, we investigate the potential of the embryonic stem cell signature as molecular target for the therapy and the strategy to suppress the embryonic stem cell signature. The core stemness gene Nanog is abnormally overexpressed in human embryonic carcinoma NCCIT cells showing gene expression profiles similar to embryonic stem cells. Down-regulation of the gene by either small interfering RNAs targeting Nanog or histone deacetylase inhibitor apicidin causes reversion of expression pattern of embryonic stem cell signature including Oct4, Sox2, and their target genes, leading to cell cycle arrest, inhibition of colony formation in soft agar, and induction of differentiation into all three germ layers. These effects are antagonized by reintroduction of Nanog. Interestingly, embryonic carcinoma cells (NCCIT, NTERA2, and P19) exhibit a higher sensitivity to apicidin in down-regulation of Nanog compared with embryonic stem cells. Furthermore, the down-regulation of Nanog expression by apicidin is mediated by a coordinated change in recruitment of epigenetic modulators and transcription factors to the promoter region. These findings indicate that overexpression of stemness gene Nanog in NCCIT cells is associated with maintaining stem cell-like phenotype and suggest that targeting Nanog might be an approach for improved therapy of poorly differentiated tumors.

Reversine Increases the Plasticity of Lineage-committed Cells toward Neuroectodermal Lineage

Functional dedifferentiation of lineage-committed cells toward pluripotency may have a great potential in regenerative medicine. Reversine has been shown to induce dedifferentiation of multiple terminally differentiated mesodermal origin cells, which are capable of being directed to differentiate into other cell types within mesodermal lineages. However, the possibilities of these cells to give rise to other lineages have not been examined. Here we show that large scale gene expression profiling of reversine-treated C2C12 myoblasts identifies a subset of up-regulated genes involved in specification of neuroectodermal as well as mesodermal lineages. Reversine treatment leads to up-regulation of priming genes of neuroectodermal lineages, such as Ngn2, Nts, Irx3, Pax7, Hes1, and Hes6, through active histone modifications in the promoter regions of these genes. Additionally, reversine increases the expression of markers for other cell types of mesodermal lineages, Ogn and apoE, via inducing active histone modifications, while down-regulating the myogenic basic helix-loop-helix factor, MyoD, via repressive histone modifications. Consistent with up-regulation of these genes, reversine-treated C2C12 myoblasts redifferentiate into neural as well as mesodermal lineages, under appropriate stimuli. Taken together, these results indicate that reversine induces a multipotency of C2C12 myoblasts via inducing a specific combination of active histone modifications. Collectively, our findings provide a mechanistic rationale for the application of reversine to dedifferentiation of somatic cells.

Protein L -isoaspartyl methyltransferase regulates p53 activity

Protein methylation plays important roles in most, if not all, cellular processes. Lysine and arginine methyltransferases are known to regulate the function of histones and non-histone proteins through the methylation of specific sites. However, the role of the carboxyl-methyltransferase protein L-isoaspartyl methyltransferase (PIMT) in the regulation of protein functions is relatively less understood. Here we show that PIMT negatively regulates the tumour suppressor protein p53 by reducing p53 protein levels, thereby suppressing the p53-mediated transcription of target genes. In addition, PIMT depletion upregulates the proapoptotic and checkpoint activation functions of p53. Moreover, PIMT destabilizes p53 by enhancing the p53–HDM2 interaction. These PIMT effects on p53 stability and activity are attributed to the PIMT-mediated methylation of p53 at isoaspartate residues 29 and 30. Our study provides new insight into the molecular mechanisms by which PIMT suppresses the p53 activity through carboxyl methylation, and suggests a therapeutic target for cancers.

Suppression of the NF‐kB signalling pathway by ergolide, sesquiterpene lactone, in HeLa cells

We have previously reported that ergolide, a sesquiterpene lactone isolated from Inula britannica, suppresses inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) expression by inhibiting nuclear factor‐kB (NF‐kB) in RAW 264.7 macrophages. In this study, we show that ergolide suppresses the DNA binding activity of NF‐kB and nuclear translocation of NF‐kB p65 subunit, leading to the inhibition of NF‐kB‐dependent gene transcription in 12‐O‐tetradecanoylphorbol 13‐acetate (TPA)‐stimulated HeLa cells. We also show that ergolide decreases the degradation and phosphorylation of IkB, an inhibitory protein of NF‐kB, and this effect is accompanied by a simultaneous reduction of IkB kinase (IKK) activity. However, ergolide does not inhibit in‐vitro IKK activity directly, suggesting the possible involvement of upstream IKK kinases in the regulation of NF‐kB activation. Furthermore, ergolide‐mediated protein kinase Cα (PKCα) inhibition is involved in reduction of NF‐kB inhibition, as demonstrated by the observation that dominant negative PKCα, but not p44/42 MAPK and p38 MAPK, inhibits TPA‐stimulated reporter gene expression. Taken together, our results suggest that ergolide suppresses NF‐kB activation through the inhibition of PKCα‐IKK activity, providing insight for PKCα as a molecular target for anti‐inflammatory drugs.

Requirement of protein l-isoaspartyl O-methyltransferase for transcriptional activation of trefoil factor 1 (TFF1) gene by estrogen receptor alpha

Lysine- and arginine-specific methyltransferases have been shown to act as either direct or secondary transcriptional co-activator of the estrogen receptor (ERα). However, little is known about the role of protein l-isoaspartyl O-methyltransferase (PIMT) on transcriptional regulation. Here, we show that PIMT acts as a co-activator for ERα-mediated transcription. Activation of the estrogen response element (ERE) promoter by β-estradiol (E(2)) was suppressed by knockdown of PIMT, and enhanced by overexpression of wild-type PIMT. However, the ERE promoter activity was resistant to E(2) stimulation in cells overexpressing a catalytically inactive PIMT mutant, G88A. Consistent with these results, the expression of the endogenous ERα response gene trefoil factor 1 (TFF1) by E(2) was completely abrogated by PIMT depletion and decreased to approximately 50% when PIMT mutant G88A was expressed. In addition, over-expression of PIMT significantly increased the levels of TFF1 mRNA in the presence or absence of E(2). Interestingly, PIMT interacted with ERα and was distributed to the cytosol and the nucleus. The chromatin immunoprecipitation analysis revealed that PIMT was recruited to the promoter of TFF1 gene together with ERα in an E(2)-dependent manner, which was accompanied by uploading of RNA polymerase II on the promoter. Taken together, the results suggest that PIMT may act as a co-activator in ERα-mediated transcription through its recruitment to the promoter via interacting with ERα.

Destabilization of TNF-α mRNA by Rapamycin.

Stimulation of mast cells through the high affinity IgE receptor (FcεRI) induces degranulation, lipid mediator release, and cytokine secretion leading to allergic reactions. Although various signaling pathways have been characterized to be involved in the FcεRI-mediated responses, little is known about the precious mechanism for the expression of tumor necrosis factor-α (TNF-α) in mast cells. Here, we report that rapamycin, a specific inhibitor of mammalian target of rapamycin (mTOR), reduces the expression of TNF-α in rat basophilic leukemia (RBL-2H3) cells. IgE or specific antigen stimulation of RBL-2H3 cells increases the expression of TNF-α and activates various signaling molecules including S6K1, Akt and p38 MAPK. Rapamycin specifically inhibits antigen-induced TNF-α mRNA level, while other kinase inhibitors have no effect on TNF-α mRNA level. These data indicate that mTOR signaling pathway is the main regulation mechanism for antigen-induced TNF-α expression. TNF-α mRNA stability analysis using reporter construct containing TNF-α adenylate/uridylate-rich elements (AREs) shows that rapamycin destabilizes TNF-α mRNA via regulating the AU-rich element of TNF-α mRNA. The antigen-induced activation of S6K1 is inhibited by specific kinase inhibitors including mTOR, PI3K, PKC and Ca2+chelator inhibitor, while TNF-α mRNA level is reduced only by rapamycin treatment. These data suggest that the effects of rapamycin on the expression of TNF-α mRNA are not mediated by S6K1 but regulated by mTOR. Taken together, our results reveal that mTOR signaling pathway is a novel regulation mechanism for antigen-induced TNF-α expression in RBL-2H3 cells.

Abstract 4870: A Novel gamma-Lactam-Based Histone Deacetylase Inhibitor Potently Inhibits the Growth of Human Breast and Renal Cancer Cells

We evaluated the novel gamma-lactam-based analogue, KBH-A145, for its anticancer activities. KBH-A145 markedly inhibited histone deacetylase (HDAC) activity in vitro and in vivo to an extent comparable to suberoylanilide hydroxamic acid (SAHA). The proliferation of various types of cancers was significantly suppressed by KBH-A145, among which MDA-MB-231 and MCF, human breast cancer cells and ACHN human renal cancer cells, were most sensitive. This was accompanied by induction of p21 WAF1/Cip1 through compromised recruitment of HDAC1, which leads to hyperacetylation of its promoter region and thus arrested both cells in the G 2 /M phase. Interestingly, this compound induced apoptosis of MDA-MB-231 cells, but not ACHN cells, through cleavage of poly(ADP-ribose) polymerase (PARP). Taken together, these results show that this novel gamma -lactam-based HDAC inhibitor potently inhibits the growth of human breast and renal cancer cells. Thus KBH-A145 is a potential therapeutic agent for the treatment of these types of cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4870.