Inhibition Of Histone Deacetylation Research Articles

TGF-beta and IL-6 signals modulate chromatin binding and promoter occupancy by acetylated FOXP3.

Expression of FOXP3, a potent gene-specific transcriptional repressor, in regulatory T cells is required to suppress autoreactive and alloreactive effector T cell function. Recent studies have shown that FOXP3 is an acetylated protein in a large nuclear complex and FOXP3 actively represses transcription by recruiting enzymatic corepressors, including histone modification enzymes. The mechanism by which extracellular stimuli regulate the FOXP3 complex ensemble is currently unknown. Although TGF-beta is known to induce murine FOXP3(+) Treg cells, TGF-beta in combination with IL-6 attenuates the induction of FOXP3 functional activities. Here we show that TCR stimuli and TGF-beta signals modulate the disposition of FOXP3 into different subnuclear compartments, leading to enhanced chromatin binding in human CD4(+)CD25(+) regulatory T cells. TGF-beta treatment increases the level of acetylated FOXP3 on chromatin and site-specific recruitment of FOXP3 on the human IL-2 promoter. However, the proinflammatory cytokine IL-6 down-regulates FOXP3 binding to chromatin in the presence of TGF-beta. Moreover, histone deacetylation inhibitor (HDACi) treatment abrogates the down-regulating effects of IL-6 and TGF-beta. These studies indicate that HDACi can enhance regulatory T cell function via promoting FOXP3 binding to chromatin even in a proinflammatory cellular microenvironment. Collectively, our data provide a framework of how different signals affect intranuclear redistribution, posttranslational modifications, and chromatin binding patterns of FOXP3.

Altered epigenetic events in docetaxel-resistant human breast cancer cells

Introduction: Docetaxel is an effective chemotherapy drug for breast cancer treatment. The mechanisms of resistance to docetaxel are poorly understood, however, epigenetic modifications, DNA methylation and histone modification, may be important. DNA methyltransferase (DNMT) 1, 3a and 3b are involved in DNA methylation whereas histone deacetylases control histone deacetylation. Epigenetic changes can be reversed using DNA methylation and histone deacetylation inhibitors. We hypothesise that inhibiting epigenetic events can reverse docetaxel resistance.

Epigenetic modifications alter telomere length during preimplantation embryo development

OBJECTIVE: To study epigenetic effects on telomere length during preimplantation development and in embryonic stem cells (ESC's). DESIGN: Laboratory study of embryos and ESC's. MATERIALS AND METHODS: Telomere length affects the development, differentiation and longevity of cells. In oocytes telomeres shorten with age and reset during early embryo development (NatureCell Biol 9:1436-41, 2007). Epigenetic changes regulate telomere length in somatic cells, but their role in telomere length regulation during preimplantation embryo development and in ESC's have not been studied. We examined the effects of a histone deacetylase inhibitor, trichostatin A (TSA) on telomere length in embryos and ESC's. Eggs were collected from super ovulated C57BL6 mice and activated by strontium. Embryos were cultured in KSOM for 24 hours in the presence or absence of 0.3 μM TSA. After 24 hours, treated (N=62) and untreated (N=58) morphologically normal 2 cell embryos were frozen at – 20°C for later telomere length measurement by Real Time PCR as described (Nature Cell Biol 9:1436-41, 2007). Histone deacetylase inhibition was verified by immunostaining. ESC's, isolated from blastocysts of young C57BL6 mice, were cultured for 24 hours in the presence or absence of 0.3 μM TSA. Stem cell markers verified ESC's. Telomere length was measured in 0.3-0.6 ng DNA from embryos and 40 ng of stem cells. Experiments were run in duplicate for embryos and triplicate for stem cells. For each PCR reaction, a standard curve was made using serial dilutions of known concentration of genomic DNA. Data analysis was by the 2-ΔΔ Ct method. RESULTS: Inhibition of histone deactylation shortened telomeres over five fold in embryos (mean Ct 19.55±1.55 for TSA group and 17.5 ± 2.90 for controls). TSA did not alter telomere length in ESC's (mean Ct 9.85±0.29 for controls group and 9.76±0.35 for TSA treated). CONCLUSIONS: Epigenetic regulation has been shown to underlie the global changes in transcription during development. Here we show that inhibition of a key epigenetic modification, histone deacetylation, alters telomere length in preimplantation embryos. Telomere length in ESC's was not affected by inhibition of histone deacetylation, perhaps because of a different role for histone deacetylation in these cells. We do not know whether the epigenetic changes associated with in vitro culture also can alter telomere length in embryos.

Sodium-Coupled Transport of the Short Chain Fatty Acid Butyrate by SLC5A8 and Its Relevance to Colon Cancer

IntroductionSLC5A8, expressed predominantly in the colon, is a Na+-coupled transporter for short-chain fatty acids. In this paper, we report on the characterization of butyrate transport by SLC5A8 and the relevance of SLC5A8-mediated butyrate transport to colon cancer. ResultsSLC5A8 transports butyrate via a Na+-dependent electrogenic process. Na+ activation of the transport process exhibits sigmoidal kinetics, indicating involvement of more than one Na+ in the activation process. SLC5A8 is silenced in colon cancer in humans, in a mouse model of intestinal/colon cancer, and in colon cancer cell lines. The tumor-associated silencing of SLC5A8 involves DNA methylation by DNA methyltransferase 1. Reexpression of SLC5A8 in colon cancer cells leads to apoptosis but only in the presence of butyrate. SLC5A8-mediated entry of butyrate into cancer cells is associated with inhibition of histone deacetylation. The changes in gene expression in SLC5A8/butyrate-induced apoptosis include upregulation of pro-apoptotic genes and downregulation of anti-apoptotic genes. In addition, the expression of phosphatidylinositol-3-kinase subunits is affected differentially, with downregulation of p85α and upregulation of p55α and p50α. ConclusionThese studies show that SLC5A8 mediates the tumor-suppressive effects of the bacterial fermentation product butyrate in the colon.

Deoxyribonucleic Acid Methyltransferase 3B Promotes Epigenetic Silencing through Histone 3 Chromatin Modifications in Pituitary Cells

Epigenetic dysregulation is implicated in pituitary neoplasia as the cause of silencing of several tumor suppressor genes. However, the upstream mediators of such events remain unknown. We examined the three members of the DNA methyltransferase (DNMT) enzyme family in normal and neoplastic human and mouse pituitary cells. This study was performed at a university-affiliated cancer research institute. Gene expression, promoter DNA methylation, histone modifications, and cell proliferation were determined. In contrast to DNMT1 and DNMT3a, DNMT3b was expressed at relatively higher levels in neoplastic pituitary cells. However, examination of the human DNMT3b 5' region showed uniformly low DNA methylation levels with little difference between normal and tumor samples. Through pharmacological methylation inhibition or histone deacetylation inhibition, we identified that DNMT3b gene expression is subject to histone modifications. Down-regulation of DNMT3b resulted in induction of retinoblastoma, p21, and p27, and reduction in cell proliferation. These targeted effects were associated with enhanced histone 3 acetylation and diminished histone methylation. Our findings identify DNMT3b as a putative mediator of epigenetic control through histone modifications of gene expression in pituitary cells.

Modulation of transcription parameters in glucocorticoid receptor-mediated repression

Glucocorticoid receptors (GRs) affect both gene induction and gene repression. The disparities of receptor binding to DNA and increased vs. decreased gene expression have suggested significant mechanistic differences between GR-mediated induction and repression. Numerous transcription factors are known to modulate three parameters of gene induction: the total activity (Vmax) and position of the dose-response curve with glucocorticoids (EC50) and the percent partial agonist activity with antiglucocorticoids. We have examined the effects on GR-mediated repression of five modulators (coactivators TIF2 [GRIP1, SRC-2] and SRC-1, corepressor SMRT, and comodulators STAMP and Ubc9), a glucocorticoid steroid (deacylcortivazol [DAC]) of very different structure, and an inhibitor of histone deacetylation (trichostatin A [TSA]). These factors interact with different domains of GR and thus are sensitive topological probes of GR action. These agents altered the Vmax, EC50, and percent partial agonist activity of endogenous and exogenous repressed genes similarly to that previously observed for GR-regulated gene induction. Collectively, these results suggest that GR-mediated induction and repression share many of the same molecular interactions and that the causes for different levels of gene transcription arise from more distal downstream steps.

Epigenetic regulation of the human mucin gene MUC4 in epithelial cancer cell lines involves both DNA methylation and histone modifications mediated by DNA methyltransferases and histone deacetylases

The human gene MUC4 encodes a transmembrane mucin, ligand of ErbB2, that is associated with pancreatic tumor progression. In the normal pancreas, MUC4 is not expressed, whereas activation of its expression is observed in the early steps of pancreatic carcinogenesis. The molecular mechanisms responsible for MUC4 gene activation are however still unknown. The MUC4 5'-flanking region being GC-rich and including two CpG islands, we hypothesized that epigenetic regulation may be involved and undertook to decipher the molecular phenomenons implied. By treating cancer cell lines with 5-aza-2'-deoxycytidine (5-aza) and trichostatin A (TSA), we were able to restore MUC4 expression in a cell-specific manner. We showed by bisulfite-treated genomic DNA sequencing and chromatin immunoprecipitation that methylation of five CpG sites and establishment of a repressive histone code at the 5'-untranslated region were associated with MUC4 silencing and impaired its activation by Sp1. Direct involvement of DNMT3A, DNMT3B, HDAC1, and HDAC3 was demonstrated by RNA interference and chromatin immunoprecipitation. Moreover, inhibition of histone deacetylation by TSA was associated with strong MUC4 repression in high-expressing cells. In conclusion, this work shows for the first time the importance of epigenetics in regulating MUC4 expression and may represent a new strategy to inhibit its expression in epithelial tumors.

Hereditary Diffuse Gastric Cancer and Lost Cell Polarity: A Short Path To Cancer

The mechanisms that underlie the initiation of human cancer are poorly understood. Here, we describe the development of hereditary diffuse gastric cancer and argue that it arises from the disruption of the regenerative processes that are inherent to all epithelial tissues. This model supports the cancer stem cell hypothesis, in which tumors contain a subpopulation of cells with the key stem cell characteristics of capacity for self renewal, differentiation and limitless replication. We argue that epigenetic modifications induced by common environmental and physiological pressures are able to initiate this disruption. The carcinogenic effects of these modifications are potentially reversible through the use of epigenetic therapies such as DNA demethylating agents and histone deacetylation inhibitors.

Promoter-targeted siRNAs Induce Gene Silencing of Simian Immunodeficiency Virus (SIV) Infection In Vitro

RNA interference is a conserved process by which sequence-specific double-stranded RNA is converted into small interfering double-stranded RNAs (siRNAs) that can induce gene silencing via two pathways: post-transcriptional gene silencing and transcriptional gene silencing (TGS). We previously reported TGS of human immunodeficiency virus-1 (HIV-1) could be induced by siRNAs targeting regions within its 5'-long-terminal repeat (5'LTR) promoter region. Here we show that promoter-targeted siRNAs can also induce silencing of simian immunodeficiency virus (SIV) replication by similar mechanisms. Suppression of productive infection was achieved in two different cell lines: a CD4, CCR5, CXCR4 expressing HeLa cell line (MAGIC-5) and in a human lymphoid cell line (CEMx174). HpaII digestion demonstrated induction of methylation at a CpG site within the SIV promoter region following siRNA-induced suppression. Both 5-azacytidine (5-AzaC) and trichostatin A (TSA), inhibitors of DNA methyltransferases (DNMTs) and histone deacetylation, respectively, partially reversed the silencing effect. Furthermore, using chromatin immunoprecipitation (ChIP) assays we found enrichment in the region of the LTR of heterochromatin markers dimethylated histone 3 lysine 9 (H3K9) and trimethylated histone 3 lysine 27 (H3K27) in the siRNA silenced cultures. Together, these results strongly suggest certain siRNAs targeting the promoter region of SIV can effect viral silencing through the induction of epigenetic changes.

Supportive care, growth factors, and new therapies in myelodysplastic syndromes

Treatment of myelodysplastic syndromes (MDS) has evolved to encompass a broad spectrum of therapies aiming to inhibit apoptosis, promote hemopoiesis, and reduce proliferation of clonal immature cells. A small but expanding cohort of patients with MDS may be cured, but for the majority the aim of treatment is to prolong survival and to improve quality of life. Patients with low-risk MDS mainly suffer from the effects of severe anemia and an important therapeutic goal is to maintain acceptable hemoglobin levels by optimal transfusion regimens or by erythropoietin+/-granulocyte-colony-stimulating factor, which normalizes hemoglobin levels or abolish transfusion need in around 40% of patients. Lenalidomide has emerged as a drug of choice for patients with low-risk MDS and a 5q deletion, leading to complete erythroid response and cytogenetic remission in 2/3 of patients. A small cohort of younger patients may show excellent responses to anti-thymocyte globulin. Patients with more advanced disease may respond to treatment with the hypomethylating agents azacytidine and decitabine, who both have been shown to prolong time to leukemic transformation / death in MDS. In addition, there are several new agents under clinical investigation targeted to potential mechanisms of disease and progression in MDS. New therapeutic drug include inhibitors of angiogenesis, histone deacetylation, tyrosine kinases and farnesylation, as well as drugs interacting with apoptotic mechanisms. The role of these, alone and in combination with more established therapies will be discussed.

Gene silencing of TSPYL5 mediated by aberrant promoter methylation in gastric cancers

DNA methylation is crucial for normal development, but gene expression altered by DNA hypermethylation is often associated with human diseases, especially cancers. The gene TSPYL5, encoding testis-specific Y-like protein, was previously identified in microarray screens for genes induced by the inhibition of DNA methylation and histone deacetylation in glioma cell lines. The TSPYL5 showed a high frequency of DNA methylation-mediated silencing in both glioma cell lines and primary glial tumors. We now report that TSPYL5 is also inactivated by DNA methylation and could be a putative epigenetic target gene in gastric cancers. We found that the expression of TSPYL5 mRNA was frequently downregulated and inversely correlated with DNA methylation in seven out of nine gastric cancer cell lines. TSPYL5 mRNA expression was also restored after treating with a DNA methyltransferase inhibitor. In primary gastric tumors, methylation-specific PCR results in 23 of the 36 (63.9%) cases revealed that the hypermethylation at CpG islands of the TSPYL5 was detectable at a high frequency. Furthermore, TSPYL5 suppressed the growth of gastric cancer cells as demonstrated by a colony formation assay. Thus, strong associations between TSPYL5 expression and hypermethylation were observed, and aberrant methylation at a CpG island of TSPYL5 may play an important role in development of gastric cancers.

DNA demethylation and histone deacetylation inhibition co‐operate to re‐express estrogen receptor beta and induce apoptosis in prostate cancer cell‐lines

Epigenetic silencing mechanisms are increasingly thought to play a major role in the development of human cancers, including prostate cancer. Promoter CpG island hypermethylation and histone hypoacetylation, catalyzed by DNA methyltransferase (DNMT) and histone deacetylase (HDAC), respectively, are associated with transcriptional repression in a number of cancers. Evidence is accumulating the two mechanisms are dynamically linked, yet few studies have examined a potential interaction in prostate cancer. LNCaP, DU-145, and PC-3 prostate cancer cells were co-treated with a DNMT inhibitor, 5'-aza-2'-deoxycytidine (5-AZAC), and an HDAC inhibitor, trichostatin A (TSA). Following treatment cells were processed for cell proliferation/apoptosis assays, or harvested for real-time RT-PCR. Assessed target genes were estrogen receptor beta (ERbeta), estrogen receptor alpha (ERalpha), androgen receptor (AR), progesterone receptor (PGR), and prostate specific antigen (PSA). In all cell-lines, co-treatment was associated with reduced cell proliferation compared with control groups (P<0.05). A reciprocal rise in caspase activation was identified, indicating apoptosis was the major mechanism of cell death. Most marked effects were seen in the androgen-dependent, AR-positive LNCaP cell-line. In all cell-lines, an additive re-expression of ERbeta was identified in the co-treatment group, a finding not seen for either AR or PSA. At concentrations associated with gene re-expression, the DNA demethylating agent 5-AZAC and the HDAC inhibitor TSA co-operate to induce apoptosis in prostate cancer cell-lines. Increased apoptosis in the co-treatment group was associated with marked re-expression of ERbeta, raising the possibility of further targeting of prostate cancer cells with ERbeta-selective agents.

Epigenetic Lesions in Malignant Melanoma

Malignant melanoma arises through a series of genetic and epigenetic events. A more profound understanding of the biology of metastatic melanoma should greatly aid in the development of new and effective treatments. Currently, avenues being pursued to improve treatment of metastatic melanoma include dendritic cell vaccines and other vaccination strategies, tyrosine kinase inhibitors, adoptive transfer of ex vivo stimulated T cells, and, as reviewed here, epigenetic approaches. The "methylator phenotype", with inactivation by promoter hypermethylation of numerous genes in malignant melanoma cell lines and primary tumors (p16, PTEN, RASSF1, estrogen receptor, retinoic acid receptor beta, SOCS1 and -2, MGMT etc.) offers a strong rationale for treatment approaches based on the use of DNA demethylating agents. The clinical literature on treatment of metastasized malignant melanoma with either 5-azacytidine or 5-aza-2'-deoxycytidine (decitabine) is reviewed. Future trials in malignant melanoma with these compounds might profit from prolonged low-dose exposure, since they unfold their full effects not immediately but with a certain delay, which may be associated with their DNA demethylating activity. Combinations of DNA demethylation agents with either histone deacetylase inhibitors, interleukin-2, chemotherapy or tamoxifen have been embarked on both in in vitro models of melanoma and recent clinical trials. The in vitro synergism between inhibitors of DNA methylation and histone deacetylation strongly invites a systematic study of combinations of both groups of agents. Up-regulation of cancer testis antigens by epigenetic therapy in melanoma also offers a very strong rationale to place these drugs and schedules within a larger treatment concept of immunotherapy which may include also T cell activation e.g. by interleukin-2, and vaccination strategies. In conclusion, the epigenome of malignant melanoma, with a well-established in vitro reversal potential, holds promise as a novel molecular target.

Sodium butyrate stimulates monoclonal antibody over‐expression in CHO cells by improving gene accessibility

Sodium butyrate treatment can increase the specific productivity of recombinant proteins in mammalian cells; however, it dramatically decreases cell growth and frequently leads to apoptosis. We have studied the responses of several Chinese hamster ovary (CHO) cells lines with different specific productivities (qP) to sodium butyrate treatment. Cell clones with lower productivities exhibited greater enhancement from butyrate treatment than cells with higher productivities. As we observed previously in cell clone characterization (Jiang et al., 2006. Biotechnol Prog 22: 313-318), heavy chain (HC) mRNA levels correlate very well with specific productivity and are amplified by butyrate treatment, indicating that sodium butyrate regulates the HC transcription. Sodium butyrate is an inhibitor of histone deacetylation, and possibly, increases gene transcription by enhancing gene accessibility to transcription factors. In this study, we applied DNase I footprinting to probe the HC and LC gene accessibility. We determined that more HC and LC gene copies are accessible by DNase I in sodium butyrate-treated CHO cells than in untreated controls, demonstrating that sodium butyrate regulates gene transcription by improving gene accessibility. However, the increase in accessibility did not correlate with the increase in transcript abundance, suggesting that butyrate enhances transcription by other mechanisms as well.

Phase I Study of Suberoylanilide Hydroxamic Acid (SAHA) and Decitabine in Patients with Relapsed, Refractory or Poor Prognosis Leukemia.

Few effective treatment strategies are available for patients with relapsed and refractory leukemias and their prognosis remains poor. Epigenetic therapy by targeting epigenetic pathways is currently under investigation as a potential strategy to reverse transcriptional silencing of tumor suppressor genes leading to the reversal of the neoplastic phenotype. 5-Aza-2′-deoxycytidine (DAC, Decitabine) and suberoylanilide hydroxamic acid (SAHA, Vorinostat) can affect epigenetic change in leukemic cells through promoter CpG island hypomethylation and inhibition of histone deacetylation, respectively. We have conducted a phase I study of combination of DAC plus SAHA (using a sequential dosing schedule) in patients with relapsed, refractory, or high risk leukemias. Five cohorts (Dose levels 0 – 4) of 6 patients each received escalating doses of decitabine (10, 10, 15, 20 and 25 mg/m2 IV daily x 5) followed by SAHA (100 mg PO tid x 14 in the first cohort and 200 mg PO tid x 14 in all subsequent cohorts). From 7/06 to 8/07, 31 patients (21 M, 10 F) have been accrued with the last cohort receiving DAC 25 mg/m2 daily x 5 followed by SAHA 200 mg tid x 14. Median age was 62 years (range, 22 – 82). Twenty one patients (68%) had refractory AML (5 with prior MDS), 1 (3%) refractory acute biphenotypic leukemia, 3 (10%) relapsed MDS, 3 (10%) refractory ALL, 1 (3%) untreated high risk MDS, and 2 (6%) Philadelphia chromosome-negative myeloproliferative disease. Median number of prior regimens was 2 (range, 0 – 8). One patient did not receive therapy due to rapid disease progression and 30 were evaluable for assessment of toxicity. Median number of cycles of DAC + SAHA administered was 2 (range, 0 – 6). One patient had pulmonary embolism and one grade 3 diarrhea, both considered to be dose limiting. Other adverse events included syncope, neutropenic fever, diarrhea, fatigue, renal failure, rash, nausea, thrombosis, and angioedema. Of the 30 evaluable pts, 1 patient achieved complete remission lasting 5.5 weeks, 4 had significant reductions in the bone marrow blasts, 4 had stable disease, 7 are too early for response evaluation, and 14 had no response/disease progression. We conclude that the sequential combination of DAC and SAHA is safe and has activity in advanced leukemia. Correlative studies evaluating DNA promoter methylation and histone acetylation will be presented.

Investigation of the Efficacy of PXD101 (Belinostat) on Primary Leukaemic Cells and Cell Lines as a Novel Agent for Childhood Acute Lymphoblastic Leukaemia (ALL).

PXD101 (belinostat) is a novel hydroxamate-type of histone deacetylation inhibitor that has been reported to inhibit the growth of a panel of adult carcinomas and melanoma tumour cell lines both in vitro and as xenografts. Activity was associated with a rapid and sustained increase in acetylation of histone proteins H3 and H4 in vitro, and in some cell lines with the induction of apoptosis as measured by PARP cleavage. We have evaluated the in vitro and ex vivo activity of belinostat in a panel of leukaemic cell lines and in primary childhood acute lymphoblastic leukaemia (ALL) to determine the potential for taking this compound forward into early clinical trials in childhood leukaemia. The cell lines used in this study were CCRF-CEM (T-ALL), including steroid sensitive and resistant derivatives, MOLT4 and Jurkat (T-ALL), NALM6 (B-ALL), Daudi (Burkitt's lymphoma) and Kasumi (AML). In vitro cytotoxicity was measured using the Sulphorodamine B assay and cell viability and apoptosis was assessed by flow cytometry using annexin V and propidium iodide staining respectively. The effects of belinostat as a single agent and in combination with dexamethasone, vincristine and doxorubicin were evaluated. Belinostat was also studied in primary leukaemia cells from children with ALL and mononuclear cells from normal bone marrow. Western blotting measuring the acetylation of histones H3 and H4 was used to assess belinostat HDAC inhibitory activity. The time course for onset and duration of HDAC inhibition was investigated. The leukaemia cell lines demonstrated a wide range of in vitro sensitivity to belinostat, with LC50s ranging from 0.3 μM in the Jurkat cells to 10 μM in MOLT4 cells. Normal bone marrow was shown to be refractory to belinostat compared to primary leukaemia cells and the majority of leukaemia cells lines. Primary childhood ALL samples were notably more sensitive to belinostat than the cell lines studied. Interestingly, primary ALL samples observed to be resistant dexamethasone were sensitive to belinostat. Inhibition of histone deacetylation (H3 and H4) was demonstrated within 30 minutes of exposure to belinostat in all cell lines and persisted throughout continuous 24 hour exposure. Histone acetylation was restored between 4–8 hours following removal of belinostat. The induction of histone acetylation did not always correlate with induction of cell death. Rapid inhibition of histone deacetylation was observed in the AML cell line Kasumi, which was refractory to belinostat. The cytotoxic activity of belinostat was associated with markers of apoptosis, as demonstrated by PARP cleavage and Annexin V staining In conclusion, belinostat demonstrated dose dependent in vitro cytotoxicity against a panel of leukaemia cell lines and primary childhood ALL samples. Notably, belinostat was effective in steroid resistant cell lines and primary ALL samples. No synergy was observed between belinostat and drugs used in the treatment of ALL. Further investigation of the scheduling of belinostat when combined with conventional cytotoxic agents is on-going. In addition, the in vivo efficacy of belinostat in a xenograft model of childhood leukaemia is currently being evaluated.

The Epigenetic Effect of Phenylhexyl Isothiocyanate on Histone Acetylation in Patients with Acute Leukemia and Non-Leukemic Disorders.

Phenyhexyl isothiocyanate (PHI) has been shown to inhibit histone deacetylation in leukemia cell lines in vitro and in animal models. In this study we examined the histone acetylation status in leukemia cells from patients with acute leukemia as well as in non-leukemia cells. From January to September 2006, 19 patients with newly diagnosed acute leukemia were enrolled. 12 patients had AML, 7 had ALL. The age ranged between 2 months to 65 years old. There were 12 males and 7 females among them. 5 non- leukemia patients were also enrolled, with 1 anemia, 1 infection disease, 2 ITP, and 1 healthy volunteer. Mononuclear cells were isolated from patients' specimen. Apoptosis of the cells and histone acetylation of H3 and H4 were characterized pre- and post- PHI treatment. The expression of acetylated H3 and H4 in acute leukemia cells was decreased, as compared to those in non-leukemia cells. Increased apoptosis was seen in the acute leukemia cells after PHI treatment of the primary culture of the leukemia cells for 3 and 7 hours. The effect of PHI on the leukemia cells from patients was time- and dose-dependent. There was a significant increase of PHI effect on leukemia cells versus non-leukemia cells (p<0.05), suggesting that PHI may have preferential activity against leukemia cells. This study presents the first evidence of PHI with preferential activity against leukemia cells over non-leukemia cells from human subjects. Clinical trial is warranted to study PHI in leukemia patients.

Extended treatment with physiologic concentrations of dietary phytochemicals results in altered gene expression, reduced growth, and apoptosis of cancer cells

Dietary phytochemicals exhibit chemopreventive potential in vivo through persistent low-dose exposures, whereas mechanistic in vitro studies with these agents generally use a high-dose single treatment. Because the latter approach is not representative of an in vivo steady state, we investigated antitumor activity of curcumin, 3,3'-diindolylmethane (DIM), epigallocatechin gallate (EGCG), genistein, or indole-3-carbinol (I3C) in breast cancer MDA-MB-231 cells, exposed in long-term culture to low concentrations, achievable in vivo. Curcumin and EGCG increased cell doubling time. Curcumin, EGCG, and I3C inhibited clonogenic growth by 55% to 60% and induced 1.5- to 2-fold higher levels of the basal caspase-3/7 activity. No changes in expression of cell cycle-related proteins or survivin were found; however, I3C reduced epidermal growth factor receptor expression, contributing to apoptosis. Because some phytochemicals are shown to inhibit DNA and histone modification, modulation of expression by the agents in a set of genes (cadherin-11, p21Cip1, urokinase-type plasminogen activator, and interleukin-6) was compared with changes induced by inhibitors of DNA methylation or histone deacetylation. The phytochemicals modified protein and/or RNA expression of these genes, with EGCG eliciting the least and DIM the most changes in gene expression. DIM and curcumin decreased cadherin-11 and increased urokinase-type plasminogen activator levels correlated with increased cell motility. Curcumin, DIM, EGCG, and genistein reduced cell sensitivity to radiation-induced DNA damage without affecting DNA repair. This model has revealed that apoptosis and not arrest is likely to be responsible for growth inhibition. It also implicated new molecular targets and activities of the agents under conditions relevant to human exposure.

Role of a consensus AP-2 regulatory sequence within the Epstein-Barr Virus LMP1 promoter in EBNA2 mediated transactivation

The Epstein-Barr virus (EBV) tumor-associated latent membrane protein 1 (LMP1) gene expression is transactivated by EBV nuclear antigen 2 (EBNA2) in human B cells. We previously reported that an E-box element at the LMP1 regulatory sequence (LRS) represses transcription of the LMP1 gene through the recruitment of a Max-Mad1-mSin3A complex. In the present study, using deletion/mutation analysis, and electrophoretic mobility shift assays, we show that the promoter region adjacent to the E-box (-59/-67) is required for the full repression conferred by E-box binding proteins. The repressive effect of these factors was overcome by an inhibitor of histone deacetylation, Trichostatin A (TSA), concurring with the reports that histone deacetylation plays an important role in repression mediated by Max-Mad1-mSin3A complex. Furthermore, ChIP analyses showed that histones at the transcriptionally active LMP1 promoter were hyperacetylated, whereas in the absence of transcription they were hypoacetylated. EBNA2 activation of the promoter required a consensus AP-2 sequence in the -103/-95 LRS region. While EMSA results and the low level of AP-2 factors expression in B cells argue against known AP-2 factors binding to this site, several pieces of evidence point to a similar mechanism of promoter activation as seen by AP-2 factors. We conclude that an AP-2 site-binding factor and EBNA2 act in concert to overcome the repression of the LMP1 promoter via the consensus AP-2 site. This activation showed strong correlation with histone hyperacetylation at the promoter, indicating this to be a major mechanism for the EBNA2 mediated LMP1 transactivation.

The Epigenetics of Mantle Cell Lymphoma

There is no consensus treatment for newly diagnosed mantle cell lymphoma. The CHOP + rituximab and hyperCVAD + rituximab regimens are most commonly used. The former is limited by relatively lower rates of complete remission (CR) and frequent relapses. The latter is limited by toxicities, especially in older patients, and relapses that occur later than those usually seen with CHOP + rituximab. Thus, improved therapies are needed. The purine analog cladribine (2-cda) + rituximab has been studied as an alternative frontline regimen in MCL and is quite active with minimal toxicity. Cladribine has epigenetic activity in that it inhibits DNA methylation. Cladribine + rituximab should be further studied in newly diagnosed mantle cell lymphoma in combination with new agents such as inhibitors of histone deacetylation, the mTOR pathway, and the proteasome.