Yen Hua Huang

Effects of Cadmium on Structure and Enzymatic Activity of Cu,Zn-SOD and Oxidative Status in Neural Cells

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder disease. Ten percent of the ALS patients are congenital (familial ALS), and the other 90% are sporadic ALS (SALS). It has been shown that mutations found in the Cu,Zn‐SOD cause 20% of the familial ALS due to its low enzyme activity. We hypothesized that heavy metals may interfere the structure of Cu,Zn‐SOD protein to suppress its activity in some of the SALS. In this study, we expressed and characterized the recombinant human Cu,Zn‐SOD under various concentrations of Cu2+, Zn2+, and Cd2+. By atomic absorption spectrophotometry, we demonstrated that adding of cadmium significantly increased the content of cadmium ion, but reduced its Zn2+ content and enzyme activity of the Cu,Zn‐SOD protein. The data of circular dichroism spectra demonstrated that the secondary structure of Cu,Zn‐SOD/Cd is different from Cu,Zn‐SOD, but close to apo‐SOD. In addition to the effect of cadmium on Cu,Zn‐SOD, cadmium was also shown to induce neural cell apoptosis. To further investigate the mechanism of neural cell apoptosis induced by cadmium, we used proteomics to analyze the altered protein expressions in neural cells treated with cadmium. The altered proteins include cellular structural proteins, stress‐related and chaperone proteins, proteins involved in reactive oxygen species (ROS), enzyme proteins, and proteins that mediated cell death and survival signaling. Taken together, in this paper, we demonstrate that cadmium decreases the content of Zn2+, changes the conformation of Cu,Zn‐SOD protein to decrease its enzyme activity, and causes oxidative stress‐induced neural cell apoptosis. J. Cell. Biochem. 98: 577–589, 2006.

A Seminal Vesicle Autoantigen of Mouse Is Able to Suppress Sperm Capacitation-Related Events Stimulated by Serum Albumin

Abstract We studied the effect of a mouse seminal vesicle autoantigen (SVA) on BSA-stimulated functions of mouse sperm. Uncapacitated, capacitated, and acrosome-reacted stages of sperm were morphologically scored, and the cellular zinc content was examined cytologically in a modified Tyrode solution at 37°C for 80 min. More than 85% of control cells remained uncapacitated. Addition of 0.3% SVA to the cell incubation did not affect the cell status. Approximately 65% of cells were capacitated in the incubation medium containing 0.3% BSA. Only 30% of the cells became capacitated after incubation with 0.3% BSA and 0.3% SVA together. The decapacitation effect by 0.3% SVA could be subdued by more than 3% BSA in the cell incubation. Whereas BSA did, SVA did not cause removal of Zn2+ from sperm, but SVA could suppress the BSA effect. The tyrosine phosphorylated proteins in sperm were detected after incubation in a modified HEPES medium containing 0.3% BSA and/or 0.3% SVA at 37°C for 90 min. Whereas BSA enhanced greatly, SVA did not cause phosphorylation of proteins in the range of Mr 40 000–120 000. The BSA-stimulated protein tyrosine phosphorylation could be suppressed by SVA in the cell incubation.

Magnetic cryopreservation for dental pulp stem cells

Magnetic cryopreservation has been successfully used for tooth banking with satisfactory implantation outcomes, suggesting that the method preserves human periodontal ligament cells and dental pulp stem cells (DPSCs). Therefore, magnetic cryopreservation may be applied for the preservation of DPSCs; however, this method has not been evaluated yet. A reliable cryopreservation method for live-cell preservation is important for the clinical applications of regenerative medicine. The conventional slow-freezing procedure with 10% dimethylsulfoxide (DMSO) may not be appropriate for stem cell-based therapies because DMSO is cytotoxic. The objective of this study was to investigate whether magnetic cryopreservation can be applied for DPSC cryopreservation. Cells isolated from human dental pulp were subjected to magnetic cryopreservation. Postthawing cell viability, adhesion, proliferation, expression of markers for mesenchymal stem cells (MSCs), differentiation ability of magnetically cryopreserved DPSCs and DNA stability were compared to those of cells subjected to the conventional slow-freezing method. The results indicated that a serum-free cryopreservation medium (SFM) containing 3% DMSO is optimal for magnetic cryopreservation. Post-thaw magnetically cryopreserved DPSCs express MSC markers, and perform osteogenesis and adipogenesis after induction similarly to fresh MSCs. No significant DNA damage was found in magnetically cryopreserved DPSCs. Magnetic cryopreservation is thus a reliable and effective method for storage of DPSCs. The smaller amount of DMSO required in SFM for cryopreservation is beneficial for the clinical applications of post-thaw cells in regenerative medicine.

Alpha-Fetoprotein Measurement Benefits Hepatocellular Carcinoma Surveillance in Patients with Cirrhosis

OBJECTIVES: Liver cirrhosis is a major risk factor for hepatocellular carcinoma (HCC), and all liver study societies recommend HCC surveillance in patients with cirrhosis. However, no ideal modality for HCC surveillance has been determined. The aim of this study is to assess the effectiveness of α‐fetoprotein (AFP) measurement in HCC surveillance. METHODS: In this retrospective analysis, all patients with cirrhosis, who received HCC surveillance through ultrasound (US) and AFP measurement between January 2002 and July 2010, were followed up until June 2013. The performance effectiveness of surveillance using AFP, US, or both in HCC detection was compared. RESULTS: Overall, 1,597 patients were followed for a median duration of 4.75 (range 1.42–12) years. Over the 8563.25‐person‐year follow‐up period, 363 patients (22.7%) developed HCCs. For HCC detection, the area under the receiver operator characteristic curve of surveillance AFP was 0.844 (95% confidence interval: 0.820−0.868, P<0.001). When the traditional cutoff value of 20 ng/ml was used, the sensitivity and specificity of AFP were 52.9% and 93.3%, respectively. US exhibited a sensitivity and specificity of 92.0% and 74.2%, respectively. A combination of US and AFP exhibited a sensitivity and specificity of 99.2% and 68.3%, respectively. By using cut‐off at 20 ng/ml and AFP level increase ≥2 × from its nadir during the previous 1 year, the combination of US and AFP yielded a sensitivity of 99.2% and an improved specificity of 71.5%. CONCLUSIONS: The complementary use of AFP and US improved the effectiveness of HCC surveillance in patients with cirrhosis.

Meiotic Competent Human Germ Cell-like Cells Derived from Human Embryonic Stem Cells Induced by BMP4/WNT3A Signaling and OCT4/EpCAM (Epithelial Cell Adhesion Molecule) Selection

Background: Pluripotent human embryonic stem cells (hESCs) are crucial for studying the molecular processes governing human germ cell specification. Results: Human germ cells highly expressed epithelial cell adhesion molecule (EpCAM) and the synergistic effects of BMP4/WNT3A promote hESCs toward germline differentiation. Conclusion: BMP4/WNT3A stimulation and OCT4/EpCAM selection allow enrichment of germ cell-like cells from differentiating hESCs. Significance: This study provides a robust system to elucidate the molecular mechanisms of human germ cell development. The establishment of an effective germ cell selection/enrichment platform from in vitro differentiating human embryonic stem cells (hESCs) is crucial for studying the molecular and signaling processes governing human germ cell specification and development. In this study, we developed a germ cell-enriching system that enables us to identify signaling factors involved in germ cell-fate induction from differentiating hESCs in vitro. First, we demonstrated that selection through an OCT4-EGFP reporter system can successfully increase the percentage of meiotic-competent, germ cell-like cells from spontaneously differentiating hESCs. Furthermore, we showed that the pluripotency associated surface marker, epithelial cell adhesion molecule (EpCAM), is also expressed in human fetal gonads and can be used as an effective selection marker for germ cell enrichment from differentiating hESCs. Combining OCT4 and EpCAM selection can further enrich the meiotic-competent germ cell-like cell population. Also, with the percentage of OCT4+/EpCAM+ cells as readout, we demonstrated the synergistic effect of BMP4/pSMAD1/5/8 and WNT3A/β-CATENIN in promoting hESCs toward the germline fate. Combining BMP4/WNT3A induction and OCT4/EpCAM selection can significantly increase the putative germ cell population with meiotic competency. Co-transplantation of these cells with dissociated mouse neonatal ovary cells into SCID mice resulted in a homogenous germ cell cluster formation in vivo. The stepwise platform established in this study provides a useful tool to elucidate the molecular mechanisms of human germ cell development, which has implications not only for human fertility research but regenerative medicine in general.

Activation of IL6/IGFIR Confers Poor Prognosis of HBV-Related Hepatocellular Carcinoma through Induction of OCT4/NANOG Expression

Purpose: To unravel the role of interleukin (IL)-6 and insulin-like growth factor (IGF)-I receptor (IGFIR) in expressing stemness-related properties and to evaluate the prognostic values of pluripotent transcription factor OCT4/NANOG, and IGFIR in hepatocellular carcinoma (HCC). Experimental Design: Serum levels of IL6 were detected using ELISA assays (n = 120). The effects of IL6/IGFI on stemness expression in HCC were examined using OCT4/NANOG promoter luciferase reporter, RNA interference, secondary sphere formation, side population, and xenograft animal models. The OCT4/NANOG protein and phospho-IGFI receptor (p-IGFIR) in tissues were detected by Western blotting (n = 8) and immunohistochemical staining (n = 85). OCT4, NANOG, and IGFIR expression levels in tissues (n = 191) were analyzed by real-time qRT-PCR and was correlated with early tumor recurrence using the Kaplan–Meier survival analysis. Results: A high positive correlation between the expression levels of OCT4/NANOG and IGFIR/p-IGFIR in human HCC tissues was observed. The concurrent expression of OCT4/NANOG/IGFIR was mostly confined to hepatitis B virus (HBV)–related HCC (HBV-HCC) and was significantly correlated with early tumor recurrence. High serum levels of IL6 were significantly correlated with high OCT4/NANOG expression. IL6 stimulated an autocrine IGFI/IGFIR expression STAT3 dependently, which stimulated stemness-related properties in both the cell lines and the xenografted mouse tumors. The inhibition of IGFIR activation by either RNA interference or by treatment with the inhibitor picropodophyllin (PPP) significantly suppressed the IL6-induced stemness-related properties both in vitro and in vivo. Conclusions: The expression of pluripotency-related genes is associated with early tumor recurrence and is regulated by IL6-induced IGF/IGFIR activation, particularly in HBV-HCC. Clin Cancer Res; 21(1); 201–10. ©2014 AACR.

Expression of the pluripotent transcription factor OCT4 promotes cell migration in endometriosis.

OBJECTIVE To identify the impact of the pluripotent transcription factor OCT4 in endometrial cell migration and endometriosis. DESIGN The OCT4 expression and cell migration study. SETTING Research institution and reproductive medical clinic. PATIENT(S) Nine subjects with normal endometrium, 3 subjects with normal myometrium, 36 patients with hyperplastic endometrium, and 58 patients with endometriosis. INTERVENTION(S) The expression of OCT4 messenger RNA in normal endometrium, normal myometrium, hyperplastic endometrium, and ectopic endometriotic tissues was analyzed using reverse transcription and quantitative real-time polymerase chain reaction (PCR). The effect of OCT4 expression on the migration activity of the endometrial cells was examined. MAIN OUTCOME MEASURE(S) Reverse transcription and quantitative real-time PCR, Western blotting, and wound closure and transwell assays. RESULT(S) The expression of OCT4 and NANOG messenger RNA was significantly higher in ectopic endometriotic tissues, compared with that of the normal endometrium, the normal myometrium, and the hyperplastic endometrium. The level of OCT4 messenger RNA in endometriotic tissues was positively correlated with the expression of genes associated with cell migration. Overexpression of the OCT4 protein in primary human endometriotic stromal cells and human RL95-2 and HEC1A endometrial carcinoma cell lines resulted in decreased levels of E-CADHERIN, the increased expression of the VIMENTIN, TWIST, and SLUG proteins, and an increase in the migration activity of endometrial cells in transwell and wound closure assays. CONCLUSION(S) The transcription of the OCT4 gene is significantly up-regulated in human ectopic endometriotic tissues. The expression of OCT4 may contribute to the pathology of ectopic endometrial growth by stimulating the migration activity of endometrial cells.

Chemotherapeutic sensitivity of testicular germ cell tumors under hypoxic conditions is negatively regulated by SENP1-controlled sumoylation of OCT4.

Testicular germ cell tumors (TGCT) generally respond well to chemotherapy, but tumors that express low levels of the transcription factor OCT4 are associated with chemoresistance and poor prognosis. Hypoxia is known to induce drug resistance in TGCTs; however, the mechanistic basis for reduced expression of OCT4 and drug resistance is unclear. Here we show that hypoxia reduces OCT4 levels and increases the resistance of embryonal carcinoma (EC) cells to cisplatin and bleomycin. Furthermore, we show that the loss of OCT4 expression under hypoxia can be triggered by sumoylation, which was regulated by SUMO1 and the SUMO1 peptidase SENP1. Under hypoxic conditions, overexpression of SUMO1gg (the active form of SUMO1) not only increased the level of sumoylated OCT4 (Su-OCT4), but also decreased the stability of OCT4 protein. In addition, overexpression of SENP1 reduced the Su-OCT4 level induced by SUMO1gg overexpression, thereby maintaining OCT4 levels and enhancing chemosensitivity. Mechanistic investigations revealed that OCT4 sumoylation occurred at K123, as overexpression of an OCT4-K123R mutant effectively reduced the level of Su-OCT4 under hypoxic conditions. Taken together, our results showed that hypoxia reduces OCT4 expression levels in ECs to increase drug resistance and that these effects could be countered to ablate the suppressive effects of hypoxia on chemosensitivity. Our findings also highlight SENP1 as a potential therapeutic target for drug resistant TGCTs.

Cloning, Expression, Characterization, and Role in Autocrine Cell Growth of Cell Surface Retention Sequence Binding Protein-1

Cell surface retention sequence binding protein-1 (CRSBP-1) is a cell surface binding protein for the cell surface retention sequence (CRS) motif of the v-sis gene product (platelet-derived growth factor-BB). It has been shown to be responsible for cell surface retention of the v-sis gene product in v-sis-transformed cells (fibroblasts) and has been hypothesized to play a role in autocrine growth and transformation of these cells. Here we demonstrate that the CRSBP-1 cDNA cloned from bovine liver libraries encodes a 322-residue type I membrane protein containing a 23-residue signal peptide, a 215-residue cell surface domain, a 21-residue transmembrane domain, and a 63-residue cytoplasmic domain. CRSBP-1 expressed in transfected cells is an ∼120-kDa disulfide-linked homodimeric glycoprotein and exhibits dual ligand (CRS-containing growth regulators (v-sis gene product and insulin-like growth factor binding protein-3, IGFBP-3) and hyaluronic acid) binding activity. CRSBP-1 overexpression (by stable transfection of cells with CRSBP-1 cDNA) enhances autocrine loop signaling, cell growth, and tumorigenicity (in mice) of v-sis-transformed cells. CRSBP-1 expression also enhances autocrine cell growth mediated by IGFBP-3 in human lung carcinoma cells (H1299 cells), which express very little, if any, endogenous CRSBP-1 and exhibits a mitogenic response to exogenous IGFBP-3, stably transfected with IGFBP-3 cDNA. However, CRSBP-1 overexpression does not affect growth of normal and transformed cells that do not produce these CRS-containing growth regulators. These results suggest that CRSBP-1 plays a role in autocrine regulation of cell growth mediated by growth regulators containing CRS.

Dental Stem Cells and Tooth Banking for Regenerative Medicine

Abstract Stem cell (SC) therapy has a promising future for tissue regenerative medicine. However, because SC technology is still in its infancy, interdisciplinary cooperation is needed to achieve successful clinical applications. Dental SCs have drawn attention in recent years because of their accessibility, plasticity, and high proliferative ability. Several types of dental SCs have been identified, including dental pulp SCs from adult human dental pulp, SCs from human primary exfoliated deciduous teeth, periodontal ligament SCs, and dental follicle SCs from human third molars. Similar to mesenchymal SCs, these dental SCs can undergo self-renewal and have multipotent differentiation ability, but do not have the ethical issues associated with other sources of SCs. Therefore, appropriate preservation procedures for dental SCs and teeth are now needed. Here, we discuss the opportunities for tooth-banking (as it is now clinically feasible and commercially available), the advantages and limitations of current cryopreservation techniques for dental SCs/teeth or tissues, and the current status of tooth banks.