Oct4 and Nanog directly regulate Dnmt1 to maintain self-renewal and undifferentiated state in mesenchymal stem cells.

The roles of Oct4 and Nanog in maintaining self-renewal and undifferentiated status of adult stem cells are unclear. Here, increase in Oct4 and Nanog expression along with increased proliferation and differentiation potential but decreased spontaneous differentiation were observed in early-passage (E), hypoxic culture (H), and p21 knockdown (p21KD) mesenchymal stem cells (MSCs) compared to late-passage (L), normoxic culture (N), and scrambled shRNA-overexpressed (Scr) MSCs. Knockdown of Oct4 and Nanog in E, H, and p21KD MSCs decreased proliferation and differentiation potential and enhanced spontaneous differentiation, whereas overexpression of Oct4 and Nanog in L, N, and Scr MSCs increased proliferation and differentiation potential and suppressed spontaneous differentiation. Oct4 and Nanog upregulate Dnmt1 through direct binding to its promoter, thereby leading to the repressed expression of p16 and p21 and genes associated with development and lineage differentiation. These data demonstrate the important roles of Oct4 and Nanog in maintaining MSC properties.

Dioscorea phytocompounds enhance murine splenocyte proliferation ex vivo and improve regeneration of bone marrow cells in vivo.

Specific cytokines have been tested clinically for immunotherapy of cancers; however, cytotoxicity has often impaired their usefulness. Consequently, alternative approaches are increasingly desirable. Dioscorea spp. tuber is a widely used traditional Chinese medicinal herb claimed to confer immunostimulatory activity. In this study, we evaluated Dioscorea as an adjuvant therapy for use alongside chemotherapy for cancer. Phytocompounds from Dioscorea tubers were ethanol fractioned and used for ex vivo splenocyte proliferation assay or in vivo force-feeding of mice pre-treated with the chemotherapy agent 5-fluorouracil. Co-treatment with a 50-75% ethanol-partitioned fraction of the tuber extract of D. batatas (DsCE-II) and interleukin (IL)-2 resulted in a significantly higher rate of murine splenocyte cell proliferation ex vivo than treatment with DsCE-II or IL-2 alone. This DsCE-II fraction, which contains a polysaccharide with a high proportion of ß-1,4-linkage mannose (≥64%), also promoted the regeneration of specific progenitor cell populations in damaged bone marrow tissues of 5-fluorouracil-treated mice. Colony-forming unit (CFU) analyses demonstrated that the population of CFU-GM cells, but not CFU-GEMM or BFU-E cells, preferentially recovered to ~67% in the bone marrow of immune-suppressed mice fed with DsCE-II. DsCE-II efficacy level was ~85% of that obtained by subcutaneous administration of recombinant G-CSF proteins (5 µg kg(-1)) in mice tested in parallel. This study suggests that the DsCE-II fraction of D. batatas extract may be considered for further development as a dietary supplement for use alongside chemotherapy during cancer treatment.

Ectopic expression of interleukin-1 receptor type II enhances cell migration through activation of the pre-interleukin 1alpha pathway.

Expression of interleukin-1 receptor type II (IL1R2), a decoy receptor for pro-inflammatory interleukin 1 (IL-1), is enhanced by chronic exposure of the human uroepithelial cell line HUC-1 to arsenite. To explore the function of IL1R2, we ectopically expressed IL1R2 in HUC-1 cells. IL1R2 overexpression results in changes in cell morphology, actin rearrangement, and promoted cell migration. Ectopic expression of IL1R2 specifically blocked exogenous IL-1beta signaling but increased expression of the precursor form of IL-1alpha (pIL-1alpha) and its downstream targets, including interleukin 6 (IL-6), interleukin 8 (IL-8), and type I collagen alpha1 (COL1A1). However, depleting gene expression using small RNA interference specific to either pIL-1alpha or COL1A1, but not IL-6 or IL-8, significantly attenuated the migration of IL1R2-overexpressing cells. Furthermore, IL1R2 overexpression was associated with enhanced expression of Smad-interacting protein 1 (SIP-1) and reduced expression of E-cadherin. Because SIP-1 is a repressor of COL1A1-induced E-cadherin expression, the present results suggest that IL1R2 overexpression is likely through activation of the pIL-1alpha pathway to enhance cell migration.

Immunomodulatory effects of phytocompounds characterized by in vivo transgenic human GM-CSF promoter activity in skin tissues.

To investigate the immunomodulatory activities of phytocompounds for potential therapeutics, we devised an in vivo, transgenic, human cytokine gene promoter assay using defined epidermal skin cells as test tissue. Test compounds were topically applied to mouse skin before or after gene gun transfection, using a cytokine gene promoter-driven luciferase reporter. Croton oil, an inflammation inducer, induced transgenic GM-CSF and TNF-alpha promoter activities in skin epidermis 6-fold and 3.4-fold, respectively; however, it produced a less than 1.5-fold and 1.7-fold change in IL-1beta and IL-18 promoter activity, respectively. The phytocompound shikonin drastically inhibited inducible GM-CSF promoter activity. However, a fraction of Dioscorea batatas extract significantly increased the GM-CSF promoter activity in normal and inflamed skin. Shikonin suppressed the transcriptional activity of GM-CSF promoter by inhibiting the binding of TFIID protein complex (TBP) to TATA box. Our results demonstrate that this in vivo transgenic promoter activity assay system is cytokine gene-specific, and highly responsive to pro-inflammatory or anti-inflammatory stimuli. Currently it is difficult to profile the expression and cross-talk of various types of cytokines in vivo. This investigation has established a bona fide in vivo, in situ, immune tissue system for research into cytokine response to inflammation.

Distinct gene expression profiles in immortalized human urothelial cells exposed to inorganic arsenite and its methylated trivalent metabolites.

Inorganic arsenic is an environmental carcinogen. The generation of toxic trivalent methylated metabolites complicates the study of arsenic-mediated carcinogenesis. This study systematically evaluated the effect of chronic treatment with sodium arsenite (iAs(III)), monomethylarsonous acid (MMA(III)), and dimethylarsinous acid (DMA(III)) on immortalized human uroepithelial cells (SV-HUC-1 cells) using cDNA microarray. After exposure for 25 passages to iAs(III) (0.5 microM), MMA(III) (0.05, 0.1, or 0.2 microM), or DMA(III) (0.2 or 0.5 microM), significant compound-specific morphologic changes were observed. A set of 114 genes (5.7% of the examined genes) was differentially expressed in one or more sets of arsenical-treated cells compared with untreated controls. Expression analysis showed that exposure of cells to DMA(III) resulted in a gene profile different from that in cells exposed to iAs(III) or MMA(III), and that the iAs(III)-induced gene profile was closest to that in the tumorigenic HUC-1-derived 3-methylcholanthrene-induced tumorigenic cell line MC-SV-HUC T2, which was derived from SV-HUC-1 cells by methylcholanthrene treatment. Of the genes affected by all three arsenicals, only one, that coding for interleukin-1 receptor, type II, showed enhanced expression, a finding confirmed by the reduced increase in NF-kappaB (nuclear factor kappa B) activity seen in response to interleukin-1beta in iAs(III)-exposed cells. The expression of 11 genes was suppressed by all three arsenicals. 5-Aza-deoxycytidine partially restored the transcription of several suppressed genes, showing that epigenetic DNA methylation was probably involved in arsenical-induced gene repression. Our data demonstrate that chronic exposure to iAs(III), MMA(III), or DMA(III) has different epigenetic effects on urothelial cells and represses NF-kappaB activity.

Enhanced filopodium formation and stem-like phenotypes in a novel metastatic head and neck cancer cell model.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world, and metastasis is the major cause of cancer-related mortality. Prevention or elimination of metastasis may improve the survival of cancer patients; however, the availability of systemic HNSCC cell model with which to investigate the mechanisms of metastasis is limited. In the present study, we established a set of metastatic cell lines from HNSCC cells. In combination with their low-tumorigenic and high-tumorigenic ancestor cell lines, a cell model containing cell lines with varying malignant characteristics was established. Transcriptome analysis revealed distinct signatures among the metastatic cell lines, in comparison to the ancestor cell lines. Signaling of GTPase RhoA and mammalian embryonic stem cell pluripotency were identified in the metastatic cells. Moreover, we examined the expression of genes related to epithelial-mesenchymal transition (EMT) (Snail, Slug, Twist, vimentin and fibronectin) and stemness (Oct4, Nanog and Bmi1). The capabilities of the cells for migration, invasion, spheroid formation and pulmonary colonization in nude mice were determined. Together, we demonstrated gain of Slug expression, filopodium formation and stem-like properties in metastatic HNSCC cells, rendering this model a powerful tool for the development of diagnostic biomarkers and identification of therapeutic targets for HNSCC.

p16(INK4A) promoter hypermethylation is associated with invasiveness and prognosis of oral squamous cell carcinoma in an age-dependent manner.

Oral squamous cell carcinoma (OSCC) is a common cancer worldwide that is highly lethal due to its recurrence and metastasis. Our candidate-based study aimed to link promoter CpG island hypermethylation to OSCC risk assessment. We examined the promoter hypermethylation status of p16(INK4A) (p16), glutathione S-transferase pi (GSTP1), O(6)-methylguanine-DNA methyltransferase (MGMT), death-associated protein kinase 1 (DAPK1), RAS-association domain family 1, isoform A (RASSF1A), and E-cadherin (CDH1) genes in OSCC tumors. Quantitative methylation-specific PCR analysis showed a significant increase in promoter hypermethylation of p16 and CDH1 in OSCC tumors relative to paired non-tumorous tissues. The mean age of patients with hypermethylated p16 was lesser than those without (P=0.027). Multiple logistic regression predicted patients with hypermethylated p16 have higher risks of lymph node invasion (adjusted OR=6.21, P=0.030) in young patients and distant metastasis (adjusted OR=19.23, P=0.007) in older patients. Moreover, p16 promoter hypermethylation was significantly associated with shortened disease-free survival (P=0.034) in older patients. Our results suggested that p16 hypermethylation was associated with early incidence of OSCC, increased lymph node invasion in young patients, and poor prognosis in older patients. Further, p16 hypermethylation may also be implicated in age-related tumor invasion in carcinogenesis.

Differential DNA methylation associated with hepatitis B virus infection in hepatocellular carcinoma.

Gene inactivation through DNA hypermethylation plays a pivotal role in carcinogenesis. This study aimed to profile aberrant DNA methylation in different stages of liver disease, namely noncirrhosis, cirrhosis and hepatocellular carcinoma (HCC), and also to clarify the influence of hepatitis B virus (HBV) infection on the aberrant DNA methylation in HCCs. Promoter methylation in p14(ARF), p16(INK4a), O(6)-methylguanine-DNA methyltransferase (MGMT), glutathione S-transferase pi (GSTP1) and E-cadherin (E-Cad) genes of 58 HCCs paired with adjacent nontumorous tissues was assayed by methylation-specific PCR. HBV infection was determined using a hepatitis B virus surface antigen (HBsAg) serological assay. The frequency of p16(INK4a) promoter methylation increased from noncirrhotic, cirrhotic, to HCC tissues (noncirrhotic vs. HCC, p < 0.001), while that of GSTP1 promoter methylation increased in cirrhotic tissues compared to noncirrhotic ones (p = 0.029). The frequency of GSTP1 promoter hypermethylation is significantly higher in HCC than in nontumorous tissues (p = 0.022) from HBsAg-positive patients, but not the HBsAg-negative controls (p = 0.289). While the frequency of E-Cad promoter hypermethylation remained high in both nontumorous tissues and HCCs from HBsAg-positive patients (p = 0.438), it was lower in HCCs than in nontumorous tissues from HBsAg-negative patients (p = 0.002). In contrast, the frequency of p16(INK4a), MGMT and p14(ARF) promoter hypermethylation in HCCs was unrelated to HBsAg status. In conclusion, aberrant DNA methylation may begin at different stages of liver disease in a gene-dependent manner. Moreover, HBV infection may enhance or maintain GSTP1 and E-Cad promoter methylation and thereby affect hepatocarcinogenesis.

Arsenite pretreatment enhances the cytotoxicity of mitomycin C in human cancer cell lines via increased NAD(P)H quinone oxidoreductase 1 expression.

Arsenic is an effective therapeutic agent for the treatment of patients with refractory or relapsed acute promyelocytic leukemia. The use of arsenic for treating solid tumors, particularly in combination with other chemotherapeutic agents, has been extensively studied. Here, we report that arsenite-resistant human lung cancer CL3R15 cells constitutively overexpress NAD(P)H quinone oxidoreductase 1 (NQO1), an enzyme responsible for activation of mitomycin C (MMC), and are more susceptible to MMC cytotoxicity than parental CL3 cells. The effects of arsenite pretreatment on NQO1 induction were examined in CL3, H1299, H460, and MC-T2 cells. Arsenite pretreatment significantly enhanced the expression of NQO1 and susceptibility to MMC in CL3, H1299, and MC-T2 cells, but not in H460 cells that express high endogenous levels of NQO1. Alternatively, arsenic pretreatment reduced adriamycin sensitivity of CL3 cells. Arsenite-mediated MMC susceptibility was abrogated by dicumarol (DIC), an NQO1 inhibitor, indicating that NQO1 is one of the key regulators of arsenite-mediated MMC susceptibility. Various cancer cell lines showed different basal levels of NQO1 activity and a different capacity for NQO1 induction in response to arsenite treatment. However, overall, there was a positive correlation between induced NQO1 activity and MMC susceptibility in cells pretreated with various doses of arsenite. These results suggest that arsenite may increase NQO1 activity and thus enhance the antineoplastic activity of MMC. In addition, our results also showed that inhibition of NQO1 activity by DIC reversed the arsenite resistance of CL3R15 cells.