Yiqun Huang

Effect of phenylhexyl isothiocyanate on aberrant histone H3 methylation in primary human acute leukemia

BackgroundWe have previously studied the histone acetylation in primary human leukemia cells. However, histone H3 methylation in these cells has not been characterized.MethodsThis study examined the methylation status at histone H3 lysine 4 (H3K4) and histone H3 lysine 9 (H3K9) in primary acute leukemia cells obtained from patients and compared with those in the non-leukemia and healthy cells. We further characterized the effect of phenylhexyl isothiocyanate (PHI), Trichostatin A (TSA), and 5-aza-2’-deoxycytidine (5-Aza) on the cells.ResultsWe found that methylation of histone H3K4 was virtually undetectable, while methylation at H3K9 was significantly higher in primary human leukemia cells. The histone H3K9 hypermethylation and histone H3K4 hypomethylation were observed in both myeloid and lymphoid leukemia cells. PHI was found to be able to normalize the methylation level in the primary leukemia cells. We further showed that PHI was able to enhance the methyltransferase activity of H3K4 and decrease the activity of H3K9 methyltransferase. 5-Aza had similar effect on H3K4, but minimal effect on H3K9, whereas TSA had no effect on H3K4 and H3K9 methyltransferases.ConclusionsThis study revealed opposite methylation level of H3K4 and H3K9 in primary human leukemia cells and demonstrated for the first time that PHI has different effects on the methyltransferases for H3K4 and H3K9.

Reactivating aberrantly hypermethylated p15 gene in leukemic T cells by a phenylhexyl isothiocyanate mediated inter-active mechanism on DNA and chromatin

BackgroundWe have previously demonstrated that phenylhexyl isothiocyanate (PHI), a synthetic isothiocyanate, inhibits histone deacetylases and remodels chromatins to induce growth arrest in HL-60 myeloid leukemia cells in a concentration-dependent manner.MethodsTo investigate the effect of PHI, a novel histone deacetylases inhibitor (HDACi), on demethylation and activation of transcription of p15 in acute lymphoid leukemia cell line Molt-4, and to further decipher the potential mechanism of demethylation, DNA sequencing and modified methylation specific PCR (MSP) were used to screen p15-M and p15-U mRNA after Molt-4 cells were treated with PHI, 5-Aza and TSA. DNA methyltransferase 1 (DNMT1), 3A (DNMT3A), 3B (DNMT3B) and p15 mRNA were measured by RT-PCR. P15 protein, acetylated histone H3 and histone H4 were detected by Western Blot.ResultsThe gene p15 in Molt-4 cells was hypermethylated and inactive. Hypermethylation of gene p15 was attenuated and p15 gene was activated de novo after 5 days exposure to PHI in a concentration-dependent manner. DNMT1 and DNMT3B were inhibited by PHI (P < 0.05). Alteration of DNMT3A was not significant at those concentrations. Acetylated histone H3 and histone H4 were accumulated markedly after exposure to PHI.ConclusionPHI could induce both DNA demethylation and acetylated H3 and H4 accumulation in Molt-4 cells. Hypermethylation of gene p15 was reversed and p15 transcription could be reactivated de novo by PHI.

Silencing of LSD1 gene modulates histone methylation and acetylation and induces the apoptosis of JeKo-1 and MOLT-4 cells

Lysine-specific demethylase 1 (LSD1) has been identified and biochemically characterized in epigenetics; however, the pathological roles of its dysfunction in mantle cell lymphoma (MCL) and T-cell acute lymphoblastic leukemia remain to be elucidated. In this study, we evaluated LSD1, and histone H3 lysine 4 (H3K4)me1 and H3K4me2 expression in patients with MCL and silenced LSD1 in JeKo-1 and MOLT-4 cells, in order to define its role in JeKo-1 and MOLT-4 cell proliferation and apoptosis. We retrospectively analyzed the protein expression of LSD1, and mono- and dimethylated H3K4 (H3K4me1 and H3K4me2), and cyclin D1 and Ki67 in 30 cases of MCL by immunohistochemistry. The correlation of LSD1, H3K4me1 and H3K4me2 with Ki67 was determined by statistical analysis. LSD1 was silenced by small interfering RNA (siRNA). Cell apoptosis and cell proliferation were detected by flow cytometry and 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay. The protein expression levels of LSD1, histone methylated H3K4, histone acetylated H3, cyclin D1, apoptotic proteins, p15 and DNA methyltransferase 1 (DNMT1) were examined by western blot analysis. We demonstrated that LSD1 was upregulated, and that H3K4me1 and H3K4me2 were downregulated in the cases with MCL, compared to those with proliferative lymphadenitis (p<0.05). LSD1 positively correlated with Ki67 in MCL [Cohens kappa (κ)=0.667, p<0.01]. There was no significant correlation between H3K4me1 and H3K4me2, and Ki67 (κ=−0.182, p>0.05, κ=−0.200, p>0.05). The silencing of LSD1 decreased the levels of the apoptosis-related proteins, Bcl-2, pro-caspase-3 and C-myc, and decreased those of DNMT1 and increased p15, and resulted in the loss of cell viability and the induction apoptosis. The silencing of LSD1 increased the expression of H3K4me1 and H3K4me2, and histone acetylated H3 in the JeKo-1 and MOLT-4 cells. LSD1 siRNA also decreased cyclin D1 expression in the JeKo-1 cells. On the whole, our findings demonstrate that the overexpression of LSD1 may be associated with the pathogenesis in MCL. We demonstrated that the silencing of LSD1 is capable of removing the mono- and dimethyl groups from H3K4, and upregulating the histone acetylation of H3 in JeKo-1 and MOLT-4 cells. The silencing of LSD1 inhibited cell growth and induced cell apoptosis. Of note, in JeKo-1 cells, the silencing of LSD1 decreased cyclin D1 expression, which is one of the genes that contribute to the pathogenesis of MCL. LSD1 may thus be a possible therapeutic target in MCL and acute lymphoblastic leukemia MOLT-4 cells.

Depletion of G9a gene induces cell apoptosis in human gastric carcinoma.

G9a is a mammalian histone methyltransferase that contributes to the epigenetic silencing of tumor suppressor genes. Evidence suggests that G9a is required to maintain the malignant phenotype, but little documentation show the role of G9a function in mediating tumor growth. We retrospectively analyzed the protein of G9a and monomethylated histone H3 lysine 9 (H3K9 me1), and dimethylated histone H3 lysine 9 (H3K9 me2) in 175 cases of gastric carcinoma by immunohistochemistry. RNAi-based inhibition of G9a in MGC803 cancer cell line was studied. G9a depletion was done by transient transfection using Lipofectamine 2000. Depletion efficiency of G9a was tested using real-time PCR and western blot analysis. Cell apoptosis and proliferation were detected by TUNEL assay and MTT, respectively. The proteins of H3K9 me1, me2, trimethylation of H3K9 (H3K9 me3), monomethylated histone H3 lysine 27 (H3K27 me1), dimethylated histone H3 lysine 27 (H3K27 me2) and histone acetylated H3, apoptotic proteins were studied by western blot analysis. G9a and H3K9 me2 expression was higher in gastric cancer cells compared to the control (p<0.05). Both G9a and H3K9 me2 were positively correlated with the degree of differentiation, depth of infiltration, lymphatic invasions and tumor-node-metastasis stage in gastric carcinoma, (p<0.05). RNAi-mediated knockdown of G9a induced cell apoptosis and inhibited cell proliferation. Depletion of G9a reduced the levels of H3K9 me1 and me2, H3K27 me1 and me2. Nonetheless, it did not activate acetylation of H3 and H3K9 me3. These data suggest that G9a is required in tumorigenesis, and correlated with prognosis. Furthermore, G9a plays a critical role in regulating epigenetics. Depletion of G9a inhibits cell growth and induces cells apoptosis in gastric cancer. It might be of therapeutic benefit in gastric cancers.

Effect of BIX-01294 on proliferation, apoptosis and histone methylation of acute T lymphoblastic leukemia cells

OBJECTIVE To determine effect of G9a inhibitor BIX-01294 on proliferation, apoptosis and histone methylation of acute T lymphoblastic leukemia cells (MOLT-4 and Jurkat) and to explore the underlying mechanism. METHODS Cell proliferation was detected by MTT assay and apoptosis and cell cycle were measured by flow cytometry. Western blot was performed to determine expression of caspase-3, Bcl-2, Bax, P21, P15 and DNMT1 as well as levels of histone H3 acetylation, histone H3K9 mono- di- and tri-methylation. RESULTS BIX-01294 inhibits expression of Bcl-2, upregulates expression of Bax and caspase-3 and induces cell apoptosis. BIX-01294 upregulates cell cycle inhibitor P21 expression and induces cell cycle arrest in the phase G0/G1. Furthermore, BIX-01294 suppresses expression of DNA demethylase DNMT1 and promotes expression of tumor suppressor protein P15, thereby inhibiting proliferation of MOLT-4 and Jurkat cells. BIX-01294 downregulates histone H3K9 mono- and di-methylation levels and has no effect on H3K9 trimethylation and histone H3 acetylation. CONCLUSION Taken together, our results indicate that by regulating H3K9 methylation and cell cycle, BIX-01294 inhibits the proliferation and induces apoptosis of acute T lymphoblastic leukemia cells.

Synergistic effects of phenylhexyl isothiocyanate and LY294002 on the PI3K/Akt signaling pathway in HL‑60 cells

The aim of the present study was to investigate the synergistic effect of phenylhexyl isothiocyanate (PHI) and LY294002 [an inhibitor of phosphoinositide 3-kinase (PI3K)] on the PI3K/protein kinase B (Akt) signaling pathway, modulating histone acetylation, inhibiting cell viability and inducing apoptosis in HL-60 cells. The inhibition of HL-60 cell viability was monitored using an MTT assay. Cell apoptosis was measured using flow cytometry. Expression of acetylated histone H3 and histone H4, and the Akt signaling pathway proteins phosphorylated Akt (p-Akt), phosphorylated mammalian target of rapamycin (p-mTOR) and phosphorylated ribosomal protein S6 kinase (p-p70S6K) was detected using western blotting. The results of the present study identified that PHI and LY294002 were able to inhibit cell viability and induce cell apoptosis in HL-60 cells. The combination exhibited a synergistic effect on cell viability and apoptosis. PHI treatment led to an accumulation of acetylated histone H3 and histone H4, but LY294002 treatment had no effect on histone acetylation. However, LY294002 was identified to enhance the effect of PHI on histone acetylation in HL-60 cells. PHI and/or LY294002 were identified to dephosphorylate proteins in the PI3K/Akt signaling pathway, with a synergistic effect observed when used in combination. The results of the present study indicated that the combination of PHI and LY294002 may offer a novel therapeutic strategy for acute myeloid leukemia.

Notch 1 is a valuable therapeutic target against cell survival and proliferation in clear cell renal cell carcinoma

Notch 1 is a key component of the Notch pathway, which performs a crucial role in clear cell renal cell carcinoma (CCRCC) development. The present study aimed to investigate whether Notch 1 could serve as a potential target for CCRCC treatment. Firstly, an association analysis was performed using 52 CCRCC cases and 30 normal controls. The results indicated that Notch 1 protein expression in renal tissues was closely associated with the incidence of CCRCC. In addition, higher Notch 1 expression in CCRCC tissues was positively associated with higher tumor-node-metastasis stage and Fuhrman grade, in addition to larger tumor size. Subsequently, an in vitro study was conducted to examine the biological functions of Notch 1 in CCRCC 786-O cells through inhibiting the Notch 1 expression with Notch 1-specific small interfering RNA (siRNA). As a result, the inhibition of Notch 1 expression by increasing concentrations of Notch 1-specific siRNA dose-dependently decreased cell proliferation and increased cell apoptosis in 786-O cells. Furthermore, B-cell lymphoma-2 and procaspase-3 expression exhibited a dose-dependent decrease accompanied with a dose-dependent inactivation of the Akt/mammalian target of rapamycin (mTOR) signaling pathway in Notch 1 siRNA-treated 786-O cells. These findings demonstrated that Notch 1 was associated with CCRCC carcinogenesis and progression, the underlying mechanism of which was that Notch 1 acted as an activator for cell proliferation and a suppressor for cell apoptosis through the Akt/mTOR signaling-dependent pathway in CCRCC. In conclusion, the present study confirmed that Notch 1 is a valuable target against cell survival and proliferation in CCRCC treatment.

[Antiproliferative effect of silencing mTOR gene on MCL Jeko-1 cell line and its mechanism].

目的 探讨RNA干扰沉默mTOR基因后对套细胞淋巴瘤Jeko-1细胞株增殖、凋亡的影响及其作用机制。 方法 设计针对mTOR基因短发夹RNA，将其连入pGPU6/GFP/Neo质粒中，构建mTOR shRNA真核表达载体，经脂质体转染入Jeko-1细胞，采用实时定量PCR及Western blot方法鉴定其干扰效果；用MTT法绘制细胞生长曲线，经流式细胞术检测细胞凋亡的变化，用Western blot方法检测凋亡相关蛋白Bcl-2、Bax、procaspase-3、procaspase-9及mTOR下游底物激酶P70S6K、p-P70S6K的表达。 结果 mTOR shRNA转染Jeko-1细胞后，mTOR基因的mRNA及蛋白表达均明显下降（P< 0.05）；转染组增殖率明显低于Neg-shRNA组和空白对照组（P<0.05），mTOR shRNA转染48 h后，凋亡率为（36.62±3.24）%，而Neg-shRNA组和空白对照组分别为（2.58±1.04）%、（1.24±0.30）%，差异有统计学意义（P<0.05）；凋亡相关蛋白Bcl-2、procaspase-3、procaspase-9的表达下降，而Bax表达上升，mTOR下游底物激酶P70S6K未见明显变化，而其活性形式p-P70S6K的表达下降。 结论 干扰沉默mTOR基因后可通过抑制mTOR信号通路的活性，抑制套细胞淋巴瘤Jeko-1细胞增殖，激活凋亡相关蛋白诱导细胞凋亡。OBJECTIVE To investigate the effect of silencing mTOR gene by RNA interference on proliferation and apoptosis, and its mechanism on mantle cell lymphoma Jeko-1 cell Line. METHODS The hairpin-like oligonucleotide sequences targeting mTOR gene was designed and transfected into Jeko-1 cells by lipofectamine TM 2000. The mTOR mRNA and protein were detected by RQ-PCR and Western blot. Cell growth was determined by MTT. Cell apoptosis was analyzed by flow cytometry. The expressions of Bcl-2, Bax, procaspase-3, procaspase-9, P70S6K,and p-P70S6K were detected by Western blot. RESULTS mTOR mRNA was markedly suppressed by shRNA targeting mTOR. mTOR shRNA suppressed proliferation and induced cells apoptosis of Jeko-1 cells. The cell apoptotic rates were (36.62 ± 3.24)%, (2.58 ± 1.04)%, (1.24 ± 0.30)% respectively, in mTOR shRNA, Neg-shRNA and Blank with statistically significant difference among them (P<0.05). mTOR shRNA down-regulated the expressions of Bcl-2, proCaspase3, proCaspase9 and p-70S6K, up-regulated the expression of Bax. CONCLUSION Deplete of mTOR gene may be realized through inhibiting the Akt/mTOR signaling pathway to promote the cell apoptosis and inhibit cell growth in Jeko-1 cell line.

Epigenetics and Targeted Therapy in Acute Leukemia

Chromatin is a highly ordered structure consisting of repeats of nucleosomes connected by linker DNA. It consists of DNA, histone, and nonhistone proteins condensed into nucleoprotein complexes and it functions as the physiological template of all eukaryotic genetic information. Histones are small basic proteins containing a globular domain and a flexible charged NH2 terminus known as the histone tail, which protrudes from the nucleosome. Epigenetic codes are set up by modifications on the DNA (methylation) or on the histones (acetylation, methylation, phosphorylation, ubiquitination, and ADP ribosylation, etc.), by different classes of enzymes in a precise and targeted manner. Posttranslational modification to histones affects chromatin structure and function resulting in altered gene expression and changes in cell behavior. These modifications do not alter the primary sequence of DNA but have an impact on gene expression regulation, most frequently gene suppression. They lead to pathological states in hematopoietic system resulting in acute leukemia. DNA methylation is catalyzed by DNA methyltransferases (DNMTs), of which three active enzymes have been identified in mammals, namely DNMT1, DNMT3A and DNMT3B. DNMT1 is responsible for maintaining pre-existing methylation patterns during DNA replication, while DNMT3A and DNMT3B are required for initiation of de novo methylation. Acetylation is a reversible process. The balance between acetylation (transcriptional activation) and deacetylation (transcriptional repression) is regulated by histone acetyltransferase (HATs) and histone deacetylases (HDACs) in specific lysine residues in the N-termini of histone tails and/or in transcription factors (eg, p53, E2F1, GATA1, RelA, YY1, and Mad/Max) without directly binding to the DNA (Minucci et al., 2006, Gallinare et al., 2007), and is critical in regulating gene expression. Mammalian HDACs are classified into three classes based on their homology to yeast HDACs. Class I HDACs (HDAC1, 2, 3, 8, and 11) are homologues of Sacharomyces cerevisiae histone deacetylase Rpd 3 (reduced potassium dependency 3) and those with greater similarity to yeast Hda1, are class II HDACs (Gray & Ekstrom, 2001; Gao et al., 2002; Kao et al., 2002). Class III HDACs are called Sirtuins, which are homologoues of yeast sir2 (silence information regulator). Histones can be mono-, di-, or tri-methylated at lysine and arginine residues by HMTs, and the recent identification of histone lysine demethylases such as KDM1/LSD1 and the Jumonji-domain (JMJD)-containing protein family shows that histone