Zhini He

Aberrant Expression of miR-638 Contributes to Benzo(a)pyrene-Induced Human Cell Transformation

Identification of aberrant microRNA (miRNA) expression during chemical carcinogen-induced cell transformation will lead to a better understanding of the substantial role of miRNAs in cancer development. To explore whether aberrant miRNAs expression can be used as biomarkers of chemical exposure in risk assessment of chemical carcinogenesis, we analyzed miRNA expression profiles of human bronchial epithelial cells expressing an oncogenic allele of H-Ras (HBER) at different stages of transformation induced by benzo(a)pyrene (BaP) by miRNA array. It revealed 12 miRNAs differentially expressed in HBER cells at both pretransformed and transformed stages. Differentially expressed miRNAs were confirmed in transformed cells and examined in 50 pairs of primary human non-small-cell lung cancer (NSCLC) tissues using real-time PCR. Among these miRNAs, downregulation of miR-638 was found in 68% (34/50) of NSCLC tissues. However, the expression of miR-638 in HBER cells increased upon treatment of BaP in a dose-dependent manner. The expression of miR-638 was also examined in peripheral lymphocytes from 86 polycyclic aromatic hydrocarbons (PAHs)-exposed (PE) workers. We found that the average expression level of miR-638 in peripheral lymphocytes from 86 PE workers increased by 72% compared with control group. The levels of miR-638 were correlated with the concentration of urinary 1-hydroxypyrene (1-OHP) and external levels of PAHs. Overexpression of miR-638 aggravated cell DNA damage induced by BaP, which might be mediated by suppression of breast cancer 1 (BRCA1), one of the target genes of miR-638. In summary, we suggest that miR-638 is involved in the BaP-induced carcinogenesis by targeting BRCA1.

CpG Site–Specific Hypermethylation of p16INK4α in Peripheral Blood Lymphocytes of PAH-Exposed Workers

Background: Sufficient epidemiologic evidence shows an etiologic link between polycyclic aromatic hydrocarbons (PAH) exposure and lung cancer risk. While the genetic modifications have been found in PAH-exposed population, it is unclear whether gene-specific methylation involves in the process of PAH-associated biologic consequence. Methods: Sixty-nine PAH-exposed workers and 59 control subjects were recruited. Using bisulfite sequencing, we examined the methylation status of p16INK4α promoter in peripheral blood lymphocytes (PBL) from PAH-exposed workers and in benzo(a)pyrene (BaP)-transformed human bronchial epithelial (HBE) cells. The relationships between p16INK4α methylation and the level of urinary 1-hydroxypyrene (1-OHP) or the frequency of cytokinesis block micronucleus (CBMN) were analyzed. Results: Compared with the control group, PAH-exposed workers exhibited higher levels of urinary 1-OHP (10.62 vs. 2.52 μg/L), p16INK4α methylation (7.95% vs. 1.14% for 22 “hot” CpG sites), and CBMN (7.28% vs. 2.92%) in PBLs. p16INK4α hypermethylation in PAH-exposed workers exhibited CpG site specificity. Among the 35 CpG sites we analyzed, 22 were significantly hypermethylated. These 22 hypermethylated CpG sites were positively correlated to levels of urinary 1-OHP and CBMN in PBLs. Moreover, the hypermethylation and suppression of p16 expression was also found in BaP-transformed HBER cells. Conclusion: PAH exposure induced CpG site–specific hypermethylation of p16INK4α gene. The degree of p16INK4α methylation was associated with the levels of DNA damage and internal exposure. Impact: p16INK4α hypermethylation might be an essential biomarker for the exposure to PAHs and for early diagnosis of cancer. Cancer Epidemiol Biomarkers Prev; 21(1); 182–90. ©2011 AACR.

Investigation of microstructure of textured YBCO with addition of nanopowder SnO2

Abstract By using top seeding melt-textured growth (TSMTG) process, large single domain YBa2Cu3O7−δ/Y2BaCuO5 (YBCO) samples were prepared with addition of nanometric SnO2 and normal CeO2. Polarized light microscope studies at each step of the TSMTG process showed that before YBa2Cu3O7−δ (123) melting, the Sn-based reacted products were uniformly distributed in micrometer scale; after solidification, the trapped Y2BaCuO5 (211) and Sn-based inclusions were more homogeneously distributed and with mean sizes of about one micrometer. Composition images of scanning electronic microscope for the textured samples showed that, on the whole, the Sn-based inclusions were smaller than the 211. They must be on average below one micrometer in size. Although evidence was found that very fine Sn-based inclusions were uniformly trapped in 123 matrix, the fraction of them was small in comparison to that of 211. No significant enhancement of superconducting critical current density (Jc) was evidenced for the nanometric SnO2-doped samples at a low magnetic field ( B T ). However, values of Jc were decreased at intermediate magnetic field ( 1 T ).

PP2A-AMPKα-HSF1 axis regulates the metal-inducible expression of HSPs and ROS clearance.

Metals such as cadmium and arsenic are ubiquitous toxicants that cause a variety of adverse health effects. Heat shock proteins (HSPs) response to metal-induced stress and protect cells from further damage. However, the intracellular signalling pathways responsible for activation of HSPs expression are not fully understood. Here, we demonstrate that protein phosphatase 2A (PP2A) regulates expression of HSP70 and HSP27 via dephosphorylation of an AMP-activated protein kinase α subunit (AMPKα) at Thr172. Dephosphorylated AMPKα phosphorylates heat shock factor 1 (HSF1) at Ser303, leading to significant transcriptional suppression of HSP70 and HSP27 in CdCl2- or NaAsO2-treated cells. Suppression of PP2A regulatory B56δ subunit resulted in the sustained phosphorylation of AMPKα upon CdCl2 treatment, subsequent reduction in expression of HSP70 and HSP27, and thereby dramatic reduction of reactive oxygen species (ROS) clearance. We further revealed that PP2A B56δ physically interacted with AMPKα, providing evidence that PP2A B56δ-AMPKα-HSF1 signalling pathway participated in regulating the inducible expression of HSPs and ROS clearance. Taken together, we identified a novel PP2A-dependent signalling pathway involved in regulation of HSPs expression in response to metal stress.

Specific histone modification responds to arsenic-induced oxidative stress

To explore whether specific histone modifications are associated with arsenic-induced oxidative damage, we recruited 138 arsenic-exposed and arsenicosis subjects from Jiaole Village, Xinren County of Guizhou province, China where the residents were exposed to arsenic from indoor coal burning. 77 villagers from Shang Batian Village that were not exposed to high arsenic coal served as the control group. The concentrations of urine and hair arsenic in the arsenic-exposure group were 2.4-fold and 2.1-fold (all P<0.001) higher, respectively, than those of the control group. Global histone modifications in human peripheral lymphocytes (PBLCs) were examined by ELISA. The results showed that altered global levels of H3K18ac, H3K9me2, and H3K36me3 correlated with both urinary and hair-arsenic levels of the subjects. Notably, H3K36me3 and H3K18ac modifications were associated with urinary 8-OHdG (H3K36me3: β=0.16; P=0.042, H3K18ac: β=-0.24; P=0.001). We also found that the modifications of H3K18ac and H3K36me3 were enriched in the promoters of oxidative stress response (OSR) genes in human embryonic kidney (HEK) cells and HaCaT cells, providing evidence that H3K18ac and H3K36me3 modifications mediate transcriptional regulation of OSR genes in response to NaAsO2 treatment. Particularly, we found that reduced H3K18ac modification correlated with suppressed expression of OSR genes in HEK cells with long term arsenic treatment and in PBLCs of all the subjects. Taken together, we reveal a critical role for specific histone modification in response to arsenic-induced oxidative damage.

Heavy Metal-Induced Metallothionein Expression is Regulated by Specific Protein Phosphatase 2A Complexes

Background: The molecular mechanism and key signaling pathways underlying MT expression in response to metal stress remains elusive. Results: Upon metal stress, PP2A PR110 complexes bind to and dephosphorylate MTF-1 at Thr-254, leading to the transactivation of MTs. Conclusion: Specific PP2A complexes regulate metal-induced MTs expression. Significance: Delineate a novel pathway regulating metal-induced cytotoxicity and clarify the role of PP2A in cellular stress response. Induction of metallothionein (MT) expression is involved in metal homeostasis and detoxification. To identify the key pathways that regulate metal-induced cytotoxicity, we investigate how phosphorylated metal-responsive transcription factor-1 (MTF-1) contributed to induction of MT expression. Immortal human embryonic kidney cells (HEK cells) were treated with seven kinds of metals including cadmium chloride (CdCl2), zinc sulfate (ZnSO4), copper sulfate(CuSO4), lead acetate (PbAc), nickel sulfate (NiSO4), sodium arsenite (NaAsO2), and potassium bichromate (K2Cr2O7). The MT expression was induced in a dose-response and time-dependent manner upon various metal treatments. A cycle of phosphorylation and dephosphorylation was required for translocation of MTF-1 from cytoplasm to nucleus, leading to the up-regulation of MTs expression. Protein phosphatase 2A (PP2A) participated in regulating MT expression through dephosphorylation of MTF-1. A loss-of-function screen revealed that the specific PP2A complexes containing PR110 were involved in metal-induced MT expression. Suppression of PP2A PR110 in HEK cells resulted in the persistent MTF-1 phosphorylation and the disturbance of MTF-1 nuclear translocation, which was concomitant with a significant decrease of MT expression and enhanced cytotoxicity in HEK cells. Notably, MTF-1 was found in complex with specific PP2A complexes containing the PR110 subunit upon metal exposure. Furthermore, we identify that the dephosphorylation of MTF-1 at residue Thr-254 is directly regulated by PP2A PR110 complexes and responsible for MTF-1 activation. Taken together, these findings delineate a novel pathway that determines cytotoxicity in response to metal treatments and provide new insight into the role of PP2A in cellular stress response.

Specific long non-coding RNAs response to occupational PAHs exposure in coke oven workers

Highlights • HOTAIR and MALAT1 were upregulated in PBLCs of PAHs-exposed workers.• HOTAIR and MALAT1 expression was positively associated with the degree of DNA damage induced by PAHs.• H3K27me3 modification was positively correlated with the degree of genetic damage and the increase of HOTAIR expression.

Y2Ba5(Sn3−y−zCuyPtz)Ox in textured YBCO superconductors

Abstract Ultrafine SnO 2 powder was made with grain size down to 20 nm. Such powder was introduced into the textured growth of Y 1+ x Ba 2 Cu 3 O 7+ δ (YBCO) superconductors. Crystalline inclusions, which were cubic in shape and from micrometer to submicrometer in size, were found trapped inside such grown YBCO superconductors. Determined by energy dispersive spectroscopy (EDS), and further confirmed by X-ray diffraction, the composition of the inclusions was Y 2 Ba 5 (Sn 3− y − z Cu y Pt z )O x . This Sn-based phase belongs to double perovskite structures. They were not trapped uniformly inside the YBa 2 Cu 3 O 7− δ (123), possibly caused by the agglomeration of the SnO 2 powder at the very beginning. Nevertheless, it is possible for fine Sn-based inclusions down to a few hundred nanometers to be trapped inside the 123. The addition of the ultrafine SnO 2 to YBCO textured superconductors without Pt did not seem to affect the size of Y 2 BaCuO 5 (211) particles. For comparison, Y 2 Sn 2 O 7 was also introduced to YBCO textured superconductor. In this case, the so-called φ phase YBa 3 Sn 2− x Cu x O 9− δ was found.

Aberrant methylation of RUNX3 is present in Aflatoxin B1-induced transformation of the L02R cell line

Chronic exposure to aflatoxin B1 (AFB1) is linked to the development of hepatocellular carcinoma (HCC). To identify differentially methylated genes involved in AFB1-induced cell transformation, we analyzed DNA methylation patterns in immortal human hepatocyte L02 cells expressing an oncogenic H-Ras allele (L02R cells) and AFB1-transformed L02R (L02RT-AFB1) cells by performing genome-wide methylation profiling. We treated L02R cells with 0.3μM AFB1 weekly and observed a transformed phenotype at the 17th week post-treatment. The transformed cells (L02RT-AFB1) could grow in an anchorage independent fashion and form tumors in immunodeficient mice. qRT-PCR was performed to examine whether gene methylation led to a reduction in gene expression of methylated candidate genes. As a result, the expression of the following seven genes including JUNB, RUNX3, NAV1, CXCR4, RARRES1, INTS1, and POLL was down-regulated in transformed L02RT-AFB1 cells. The reduction of gene expression of these genes could be reversed by treatment of 5-azadeoxycytidine. The methylated CpG sites of RUNX3 genes were verified using bisulfite sequencing PCR (BSP) assay. Furthermore, a dynamic change in RUNX3 methylation was observed over the course of AFB1-induced cell transformation, which was corresponded to the alteration of gene expression and the extent of DNA damage. In vitro study showed that methylation of RUNX3 tended to abate in L02R cells treated with AFB1 for a short-term period of time. Notably, hypermethylation of RUNX3 appeared in 70% (14/20) of human hepatocellular carcinomas. Moreover, LINE-1 hypomethylation and dynamic changes of DNMTs, TETs and MeCP2 expression were also observed during AFB1-induced transformation. Taken together, these observations suggest that aberrant methylation of RUNX3 and LINE-1 might be involved in AFB1-induced carcinogenesis.

Persistent phosphorylation at specific H3 serine residues involved in chemical carcinogen-induced cell transformation

Identification of aberrant histone H3 phosphorylation during chemical carcinogenesis will lead to a better understanding of the substantial roles of histone modifications in cancer development. To explore whether aberrant H3 phosphorylation contributes to chemical carcinogenesis, we examined the dynamic changes of H3 phosphorylation at various residues in chemical carcinogen‐induced transformed human cells and human cancers. We found that histone H3 phosphorylation at Ser10 (p‐H3S10) and Ser28 (p‐H3S28) was upregulated by 1.5‐4.8 folds and 2.1‐4.3 folds, respectively in aflatoxin B1‐transformed hepatocytes L02 cells (L02RT‐AFB1), benzo(a)pyrene‐transformed HBE cells (HBERT‐BaP), and coke oven emissions‐transformed HBE cells (HBERT‐COE). The ectopic expression of histone H3 mutant (H3S10A or H3S28A) in L02 cells led to the suppression of an anchorage‐independent cell growth as well as tumor formation in immunodeficient mice. In addition, an enhanced p‐H3S10 was found in 70.6% (24/34) of hepatocellular carcinoma (HCC), and 70.0% (21/30) of primary lung cancer, respectively. Notably, we found that expression of H3 carrying a mutant H3S10A or H3S28A conferred to cells the ability to maintain a denser chromatin and resistance to induction of DNA damage and carcinogen‐induced cell transformation. Particularly, we showed that introduction of a mutant H3S10A abolished the bindings of p‐H3S10 to the promoter of DNA repair genes, PARP1 and MLH1 upon AFB1 treatment. Furthermore, we revealed that PP2A was responsible for dephosphorylation of p‐H3S10. Taken together, these results reveal a key role of persistent H3S10 or H3S28 phosphorylation in chemical carcinogenesis through regulating gene transcription of DNA damage response (DDR) genes.