Xiaonian Zhu

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.

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.

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.

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.

The role of specific PP2A complexes in the dephosphorylation of γH2AX

Retraction of: J. Cell Sci. doi: [10.1242/jcs.147223][1]. The authors wish to retract the above online paper. After it was brought to our attention by Journal of Cell Science , we identified that the data presented in Fig. 1C, Fig. 2D and Fig. 4A in the manuscript were inappropriately modified with

CpG site-specific RASSF1a hypermethylation is associated with occupational PAH exposure and genomic instability

Previous studies have shown an etiologic link between exposure to polycyclic aromatic hydrocarbons (PAHs) and lung cancer development. While the tumor suppressor gene RASSF1a is mostly silenced by DNA methylation in various tumors, it is unclear whether aberrant methylation of RASSF1a is involved in the process of PAH-induced biological consequences. To address this issue, 69 coke-oven workers (exposure group) and 46 steel rolling workers (control group) were recruited in this study. Bisulfite sequencing (BSP) was performed to examine the methylation status of RASSF1a promoter in peripheral blood lymphocytes (PBLs) from PAH-exposed and control workers. The DNA fragment examined lies across −282 bp to +638 bp from the transcription start site, containing 966 bp and 87 CpG sites across the RASSF1a promoter. Of the 87 CpG sites we analyzed, 5 were significantly hypermethylated in the PAH-exposed workers compared to the control (2.5% vs. 0%, P < 0.001). We defined these 5 CpG sites as “Hot CpG sites”. The levels of methylation from Hot CpG sites were positively correlated with the concentration of urinary 1-OHP (β = 0.98, P = 0.001) and the frequency of cytokinesis-block micronucleus (CBMN) (β = 1.29, P = 0.019) in PBLs, indicating that PAH exposure induced CpG site-specific hypermethylation of RASSF1a was associated with the levels of internal exposure and the degree of DNA damage. Moreover, the Hot CpG site hypermethylation and the corresponding down-regulation of RASSF1a expression were also found in COE (coke-oven emission)-treated human primary lymphocytes and HBE cells. Taken together, these observations revealed that RASSF1a Hot CpG site hypermethylation could be a promising biomarker for the PAH exposure and DNA damage.

Application of human cell transformation assay on assessment of carcinogenic potential of river organic pollutants

Approaches for assessing the carcinogenic potential of complex mixtures of environmental pollutants are still under development. In this study, a human cell transformation assay was used to evaluate the carcinogenic potential of organic extracts of water pollutants collected from the Jialu River (S1), one of the main tributaries of the Huaihe River in Henan province, China. The incidence of digestive cancers in the basin has risen dramatically within the past three decades. In addition, we collected water samples from the local wells in two liver cancer patients’ homes. The distance of these two wells is 1 km (S2) and 20 km (S3) from the site of S1. Organic chemicals were extracted using hydrophilic–lipophilic balance solid phase cartridges and the fraction was dissolved in DMSO. Human hepatic immortal cells (HL-7702) were treated with each extract and the cytotoxicity was measured. The cells were treated with each extract and the efficiency of cell transformation was examined periodically. The subcutaneous injection of treated cells in immune-deficient mice was performed to confirm the malignant cell transformation. The latency of malignant transformation for samples S1, S2, and S3 was 14, 14, and 16 weeks, respectively, at lowest concentrations of 1.0, 0.5, and 2.0 microlitres (μL) of extract per millilitre medium (mL), enriched from 10, 50, and 200 mL source water, respectively. Moreover, we analyzed the organic extracts for 16 polycyclic aromatic hydrocarbons (PAHs) using GC-MS analysis and found 13 components appearing in all water samples. Our study indicates that human cell transformation assays can potentially be used for assessing the carcinogenic potential of mixtures of environmental pollutants.