Faculty Bibliography

Mesenchymal stromal cells (MSC) have attracted the attention of scientists and clinicians due to their self-renewal, capacity for multipotent differentiation, and immunomodulatory properties. Some essential problems remain to be solved before the clinical application of MSC. Platelet lysate (PL) has recently been used as a substitute for FBS in MSC amplification in vitro to achieve clinically applicable numbers of MSC. In addition to promising trials in regenerative medicine, such as in the treatment of major bone defects and myocardial infarction, MSC have shown therapeutic effect other than direct hematopoiesis support in hematopoietic reconstruction. It has been confirmed that MSC promote hematopoietic cell engraftment and immune recovery after allogeneic hematopoietic stem cell transplantation, probably through the provision of cytokines, matrix proteins, and cell-to-cell contacts. Their suppressive effects on immune cells, including T cells, B cells, NK cells and DC cells, suggest MSCs as a novel therapy for GVHD and other autoimmune disorders. These cells thus present as promising candidates for cellular therapy in the fields of regenerative medicine, allogeneic hematopoietic stem cell transplantation, and autoimmune disorders

BACKGROUND: Hexavalent chromium [Cr(VI)] is a potent human carcinogen. Occupational exposure has been associated with increased risk of respiratory cancer. Multiple mechanisms have been shown to contribute to Cr(VI) induced carcinogenesis, including DNA damage, genomic instability, and epigenetic modulation, however, the molecular mechanism and downstream genes mediating chromium's carcinogenicity remain to be elucidated. METHODS/RESULTS: We established chromate transformed cell lines by chronic exposure of normal human bronchial epithelial BEAS-2B cells to low doses of Cr(VI) followed by anchorage-independent growth. These transformed cell lines not only exhibited consistent morphological changes but also acquired altered and distinct gene expression patterns compared with normal BEAS-2B cells and control cell lines (untreated) that arose spontaneously in soft agar. Interestingly, the gene expression profiles of six Cr(VI) transformed cell lines were remarkably similar to each other yet differed significantly from that of either control cell lines or normal BEAS-2B cells. A total of 409 differentially expressed genes were identified in Cr(VI) transformed cells compared to control cells. Genes related to cell-to-cell junction were upregulated in all Cr(VI) transformed cells, while genes associated with the interaction between cells and their extracellular matrices were down-regulated. Additionally, expression of genes involved in cell proliferation and apoptosis were also changed. CONCLUSION: This study is the first to report gene expression profiling of Cr(VI) transformed cells. The gene expression changes across individual chromate exposed clones were remarkably similar to each other but differed significantly from the gene expression found in anchorage-independent clones that arose spontaneously. Our analysis identified many novel gene expression changes that may contribute to chromate induced cell transformation, and collectively this type of information will provide a better understanding of the mechanism underlying chromate carcinogenicity

Liprin-alpha4 was strongly induced following nickel (II) chloride exposure in a variety of cell types including BEAS-2B, A549, BEP2D and BL41 cells. Liprin-alpha4, a member of the Liprin alpha family, has seven isoforms but only three of these variants were detected in BEAS-2B cells (004, 201 and 202). The level of Liprin-alpha4 variants 201 and 004 were highly increased in BEAS-2B cells in response to nickel. We showed that Liprin-alpha4 bound directly to the cytoplasmic region of RPTP-LAR (receptor protein tyrosine phosphatase-leukocyte antigen-related receptor F). The cytoplasmic region of RPTP-LAR contains two phosphatase domains but only the first domain shows activity. The second domain interacts with other proteins. The phosphatase activity was increased both following nickel treatment and also in the presence of nickel ions in cell extracts. Liprin-alpha4 knock-down lines with decreased expression of Liprin-alpha4 variants 004 and 201 exhibited greater nickel toxicity compared to controls. The RPTP-LAR phosphatase activity was only slightly increased in a Liprin-alpha4 knock-down line. Liprin-alpha4 appeared necessary for the nickel induced tyrosine phosphatase activity. The presence of Liprin-alpha4 and nickel increased tyrosine phosphatase activity that reduced the global levels of tyrosine phosphorylation in the cell

Histone H3 lysine 4 (H3K4) trimethylation (H3K4me3) at the promoter region of genes has been linked to transcriptional activation. In the present study, we found that hypoxia (1% oxygen) increased H3K4me3 in both normal human bronchial epithelial Beas-2B cells and human lung carcinoma A549 cells. The increase of H3K4me3 from hypoxia was likely caused by the inhibition of H3K4 demethylating activity, as hypoxia still increased H3K4me3 in methionine-deficient medium. Furthermore, an in vitro histone demethylation assay showed that 1% oxygen decreased the activity of H3K4 demethylases in Beas-2B nuclear extracts because ambient oxygen tensions were required for the demethylation reaction to proceed. Hypoxia only minimally increased H3K4me3 in the BEAS-2B cells with knockdown of JARID1A, which is the major histone H3K4 demethylase in this cell line. However, the mRNA and protein levels of JARID1A were not affected by hypoxia. GeneChip and pathway analysis in JARID1A knockdown Beas-2B cells revealed that JARID1A regulates the expression of hundreds of genes involved in different cellular functions, including tumorigenesis. Knocking down of JARID1A increased H3K4me3 at the promoters of HMOX1 and DAF genes. Thus, these results indicate that hypoxia might target JARID1A activity, which in turn increases H3K4me3 at both the global and gene-specific levels, leading to the altered programs of gene expression and tumor progression

Nickel (Ni) compounds have been found to cause cancer in humans and animal models and to transform cells in culture. At least part of this effect is mediated by stabilization of hypoxia inducible factor (HIF1a) and activating its downstream signaling. Recent studies reported that hypoxia signaling might either antagonize or enhance c-myc activity depending on cell context. We investigated the effect of nickel on c-myc levels, and demonstrated that nickel, hypoxia, and other hypoxia mimetics degraded c-myc protein in a number of cancer cells (A549, MCF-7, MDA-453, and BT-474). The degradation of the c-Myc protein was mediated by the 26S proteosome. Interestingly, knockdown of both HIF-1alpha and HIF-2alpha attenuated c-Myc degradation induced by Nickel and hypoxia, suggesting the functional HIF-1alpha and HIF-2alpha was required for c-myc degradation. Further studies revealed two potential pathways mediated nickel and hypoxia induced c-myc degradation. Phosphorylation of c-myc at T58 was significantly increased in cells exposed to nickel or hypoxia, leading to increased ubiquitination through Fbw7 ubiquitin ligase. In addition, nickel and hypoxia exposure decreased USP28, a c-myc de-ubiquitinating enzyme, contributing to a higher steady state level of c-myc ubiquitination and promoting c-myc degradation. Furthermore, the reduction of USP28 protein by hypoxia signaling is due to both protein degradation and transcriptional repression. Nickel and hypoxia exposure significantly increased the levels of dimethylated H3 lysine 9 at the USP28 promoter and repressed its expression. Our study demonstrated that Nickel and hypoxia exposure increased c-myc T58 phosphorylation and decreased USP28 protein levels in cancer cells, which both lead to enhanced c-myc ubiquitination and proteasomal degradation

Hexavalent chromium [Cr(VI)] is a mutagen and carcinogen, and occupational exposure can lead to lung cancers and other adverse health effects. Genetic changes resulting from DNA damage have been proposed as an important mechanism that mediates chromate's carcinogenicity. Here we show that chromate exposure of human lung A549 cells increased global levels of di- and tri-methylated histone H3 lysine 9 (H3K9) and lysine 4 (H3K4) but decreased the levels of tri-methylated histone H3 lysine 27 (H3K27) and di-methylated histone H3 arginine 2 (H3R2). Most interestingly, H3K9 dimethylation was enriched in the human MLH1 gene promoter following chromate exposure and this was correlated with decreased MLH1 mRNA expression. Chromate exposure increased the protein as well as mRNA levels of G9a a histone methyltransferase that specifically methylates H3K9. This Cr(VI)-induced increase in G9a may account for the global elevation of H3K9 dimethylation. Furthermore, supplementation with ascorbate, the primary reductant of Cr(VI) and also an essential cofactor for the histone demethylase activity, partially reversed the H3K9 dimethylation induced by chromate. Thus our studies suggest that Cr(VI) may target histone methyltransferases and demethylases, which in turn affect both global and gene promoter specific histone methylation, leading to the silencing of specific tumor suppressor genes such as MLH1

Occupational exposure to nickel (Ni), chromium (Cr), and arsenic (As) containing compounds has been associated with lung cancer and other adverse health effects. Their carcinogenic properties may be attributable in part, to activation and/or repression of gene expression induced by changes in the DNA methylation status and histone tail post-translational modifications. Here we show that individual treatment with nickel, chromate, and arsenite all affect the gene activating mark H3K4 methylation. We found that nickel (1 mM), chromate (10 microM), and arsenite (1 microM) significantly increase tri-methyl H3K4 after 24 h exposure in human lung carcinoma A549 cells. Seven days of exposure to lower levels of nickel (50 and 100 microM), chromate (0.5 and 1 microM) or arsenite (0.1, 0.5 and 1 microM) also increased tri-methylated H3K4 in A549 cells. This mark still remained elevated and inherited through cell division 7 days following removal of 1 microM arsenite. We also demonstrate by dual staining immunofluorescence microscopy that both H3K4 tri-methyl and H3K9 di-methyl marks increase globally after 24 h exposure to each metal treatment in A549 cells. However, the tri-methyl H3K4 and di-methyl H3K9 marks localize in different regions in the nucleus of the cell. Thus, our study provides further evidence that a mechanism(s) of carcinogenicity of nickel, chromate, and arsenite metal compounds may involve alterations of various histone tail modifications that may in turn affect the expression of genes that may cause transformation

Nickel compounds are carcinogenic to humans and have been shown to alter epigenetic homeostasis. The c-Myc protein controls 15% of human genes and it has been shown that fluctuations of c-Myc protein alter global epigenetic marks. Therefore, the regulation of c-Myc by nickel ions in immortalized but not tumorigenic human bronchial epithelial Beas-2B cells was examined in this study. It was found that c-Myc protein expression was increased by nickel ions in non-tumorigenic Beas-2B and human keratinocyte HaCaT cells. The results also indicated that nickel ions induced apoptosis in Beas-2B cells. Knockout of c-Myc and its restoration in a rat cell system confirmed the essential role of c-Myc in nickel ion-induced apoptosis. Further studies in Beas-2B cells showed that nickel ion increased the c-Myc mRNA level and c-Myc promoter activity, but did not increase c-Myc mRNA and protein stability. Moreover, nickel ion upregulated c-Myc in Beas-2B cells through the MEK/ERK pathway. Collectively, the results demonstrate that c-Myc induction by nickel ions occurs via an ERK-dependent pathway and plays a crucial role in nickel-induced apoptosis in Beas-2B cells

Arsenic (As) is a well-characterized human carcinogen but is generally not mutagenic. The evidence that As induces both loss of global DNA methylation and gene promoter DNA hypermethylation has suggested that epigenetic mechanisms may play an important role in As-induced carcinogenesis. In the present study, we examined the change in histone methylation by As exposure. In human lung carcinoma A549 cells, exposure to inorganic trivalent As (arsenite) increased H3K9 dimethylation (H3K9me2) and decreased H3K27 trimethylation (H3K27me3), both of which represent gene silencing marks, while increasing the global levels of the H3K4 trimethylation (H3K4me3), a gene-activating mark. The increase in H3K9me2 was mediated by an increase in the histone methyltransferase G9a protein and messenger RNA levels. We also observed strikingly significant altered histone modifications induced by very low-dose (0.1 microM) arsenite. Taken together, these results suggest a potential mechanism by which As induces carcinogenesis through the alteration of specific histone methylations that represent both gene silencing and activating marks. Furthermore, these marks are known to affect DNA methylation, and it is likely that arsenic's effect is not limited to histone modifications alone, but extends, perhaps by them, to DNA methylations as well. Future studies in our laboratory will address the genomic location of these silencing and activating marks using ChIP-on-chip technology

Nickel (Ni) is a known carcinogen, although the mechanism of its carcinogenicity is not clear. Here, we provide evidence that Ni can induce phosphorylation of histone H3 at its serine 10 residue in a c-jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK)-dependent manner. Ni induces the phosphorylation of JNK, with no effect on the phosphorylation states of the extracellular signal-regulated kinase (ERK) or p38 mitogen-activated protein kinases. An inhibitor of JNK eliminated the Ni-initiated JNK-mediated induction of histone H3 phosphorylation at serine 10, whereas inhibitors specific for ERK or p38 kinases had no effect on the phosphorylation levels of histone H3 at serine 10 (P-H3S10) in Ni-treated cells. A complete loss of Ni ion-induced phosphorylation of H3S10 was observed when JNK was specifically knocked down with RNAi. These results are the first to show the specific JNK-mediated phosphorylation of histone H3 at its serine 10 residue. We show that addition of Ni to an in vitro P-H3S10 dephosphorylation reaction does not change the loss of phosphorylation in the reaction, supporting the notion that Ni causes H3S10 phosphorylation via the JNK/SAPK pathway. It is likely that modification of H3S10 is one of a growing number of epigenetic changes believed to be involved in the carcinogenesis caused by Ni