14-3-3σ induces heat shock protein 70 expression in hepatocellular carcinoma.

BACKGROUND: 14-3-3σ is implicated in promoting tumor development of various malignancies. However, the clinical relevance of 14-3-3σ in hepatocellular carcinoma (HCC) tumor progression and modulation and pathway elucidation remain unclear. METHODS: We investigated 14-3-3σ expression in 109 HCC tissues by immunohistochemistry. Overexpression and knockdown experiments were performed by transfection with cDNA or siRNA. Protein expression and cell migration were determined by Western blot and Boyden chamber assay. RESULTS: In this study, we found that 14-3-3σ is abundantly expressed in HCC tumors. Stable or transient overexpression of 14-3-3σ induces the expression of heat shock factor-1α (HSF-1α) and heat shock protein 70 (HSP70) in HCC cells. Moreover, expression of 14-3-3σ significantly correlates with HSF-1α/HSP70 in HCC tumors and both 14-3-3σ and HSP70 overexpression are associated with micro-vascular thrombi in HCC patients, suggesting that 14-3-3σ/HSP70 expression is potentially involved in cell migration/invasion. Results of an in vitro migration assay indicate that 14-3-3σ promotes cell migration and that 14-3-3σ-induced cell migration is impaired by siRNA knockdown of HSP70. Finally, 14-3-3σ-induced HSF-1α/HSP70 expression is abolished by the knockdown of ß-catenin or activation of GSK-3ß. CONCLUSIONS: Our findings indicate that 14-3-3σ participates in promoting HCC cell migration and tumor development via ß-catenin/HSF-1α/HSP70 pathway regulation. Thus, 14-3-3σ alone or combined with HSP70 are potential prognostic biomarkers for HCC.

High FOXA1 levels induce ER transcriptional reprogramming, a pro-metastatic secretome, and metastasis in endocrine-resistant breast cancer.

Aberrant activation of the forkhead protein FOXA1 is observed in advanced hormone-related cancers. However, the key mediators of high FOXA1 signaling remain elusive. We demonstrate that ectopic high FOXA1 (H-FOXA1) expression promotes estrogen receptor-positive (ER+) breast cancer (BC) metastasis in a xenograft mouse model. Mechanistically, H-FOXA1 reprograms ER-chromatin binding to elicit a core gene signature (CGS) enriched in ER+ endocrine-resistant (EndoR) cells. We identify Secretome14, a CGS subset encoding ER-dependent cancer secretory proteins, as a strong predictor for poor outcomes of ER+ BC. It is elevated in ER+ metastases vs. primary tumors, irrespective of ESR1 mutations. Genomic ER binding near Secretome14 genes is also increased in mutant ER-expressing or mitogen-treated ER+ BC cells and in ER+ metastatic vs. primary tumors, suggesting a convergent pathway including high growth factor receptor signaling in activating pro-metastatic secretome genes. Our findings uncover H-FOXA1-induced ER reprogramming that drives EndoR and metastasis partly via an H-FOXA1/ER-dependent secretome.

Dynamic profiles of Oct-4, Cdx-2 and acetylated H4K5 in in-vivo-derived rabbit embryos.

This study documents the spatial and temporal distribution of Oct-4, Cdx-2 and acetylated H4K5 (H4K5ac) by immunocytochemistry staining using in-vivo-derived rabbit embryos at different stages: day-3 compact morulae, day-4 early blastocysts, day-4 expanded blastocysts, day-5 blastocysts, day-6 blastocysts and day-7 blastocysts. The Oct-4 signal was stronger in the inner cell mass (ICM)/epiblast cells than in the trophectoderm (TE) cells in all blastocyst stages except day-4 expanded blastocysts, where the signal was similarly weak in both the ICM and TE cells. The Cdx-2 signal was first detected in a small number of TE cells of day-4 early blastocysts, and became evident in the TE cells exclusively afterwards. A consistently strong H4K5ac signal was observed in the TE cells in all blastocyst stages examined. In particular, this signal was stronger in the TE than in the ICM cells in day-4 early blastocysts, day-4 expanded blastocysts and day-5 blastocysts. Double staining of H4K5ac with either Oct-4 or Cdx-2 on embryos at different blastocyst stages confirmed these findings. This work suggests that day 4 is a critical timing for lineage formation in rabbit embryos. A combination of Oct-4, Cdx-2 and H4K5ac can be used as biomarkers to identify different lineage cells in rabbit blastocysts.

Spatial and temporal distribution of Oct-4 and acetylated H4K5 in rabbit embryos.

Rabbit is a unique species to study human embryology; however, there are limited reports on the key transcription factors and epigenetic events of rabbit embryos. This study examined the Oct-4 and acetylated H4K5 (H4K5ac) patterns in rabbit embryos using immunochemistry staining. The average intensity of the Oct-4 signal in the nuclei of the whole embryo spiked upon fertilization, then decreased until the 8-cell stage and increased afterwards until the compact morula (CM) stage. It decreased thereafter from the CM stage to the early blastocyst (EB) stage, with a minimum at the expanded blastocyst (EXPB) stage and came back to a level similar to that of the CM-stage embryos in the hatching blastocysts (HB). The Oct-4 signal was observed in both the inner cell mass (ICM) and the trophectoderm (TE) cells of blastocysts. The average H4K5ac signal intensity of the whole embryo increased upon fertilization, started to decrease at the 4-cell stage, reached a minimum at the 8-cell stage, increased again at the EXPB stage and peaked at the HB stage. While TE cells maintained similar levels of H4K5ac throughout the blastocyst stages, ICM cells of HB showed higher levels of H4K5ac than those of EB and EXPB. Understanding key genetic and epigenetic events during early embryo development will help to identify factors contributing to embryo losses and consequently improve embryo survival rates. As a preferred laboratory species for many human disease studies such as atherosclerosis, rabbit is also a pioneer species in the development of several embryo biotechnologies, such as IVF, transgenesis, animal cloning, embryo cryopreservation and embryonic stem cells. However, there are limited reports on key transcription factors and epigenetic events of rabbit embryos. In the present study, we documented the temporal and spatial distribution of Oct-4 protein and H4K5 acetylation during early embryo development using the immunostaining approach. We also compared the patterns of these two important biomarkers between the inner cell mass (ICM) and the trophectoderm (TE) cells in blastocyst-stage embryos. Our findings suggest that a combination of Oct-4, H4K5ac and possibly other biomarkers such as Cdx-2 is needed to accurately identify different lineages of cells in morula and blastocyst stage rabbit embryos. Importantly, we revealed a novel wave of Oct-4 intensity change in the ICM cells of rabbit blastocysts. The signal was high at the early blastocyst stage, reached a minimum at the expanded blastocyst stage and returned to a high level at the hatching blastocyst stage. We hypothesize that the signal may have reflected the regulation of Oct-4 through enhancer switching and therefore may be related to cell lineage formation in rabbit embryos. These findings enrich our understanding on key genetic and epigenetic programming events during early embryo development in rabbits.

A Novel Neoplastic Fusion Transcript, RAD51AP1-DYRK4, Confers Sensitivity to the MEK Inhibitor Trametinib in Aggressive Breast Cancers.

PURPOSE: Luminal B breast tumors are more aggressive estrogen receptor-positive (ER+) breast cancers characterized by aggressive clinical behavior and a high risk of metastatic dissemination. The underlying pathologic molecular events remain poorly understood with a paucity of actionable genetic drivers, which hinders the development of new treatment strategies. EXPERIMENTAL DESIGN: We performed large-scale RNA sequencing analysis to identify chimerical transcripts preferentially expressed in luminal B breast cancer. The lead candidate was validated by reverse transcription PCR in breast cancer tissues. The effects of inducible ectopic expression or genetic silencing were assessed by phenotypic assays such as MTS, transwell, and transendothelial migration assays, and by clonogenic assays to assess MEK inhibitor sensitivity. Subcellular fractionation, Western blots, and immunoprecipitation were performed to characterize the protein products and elucidate the engaged mechanisms. RESULTS: Here we report a novel tumor-specific chimeric transcript RAD51AP1-DYRK4 preferentially expressed in luminal B tumors. Analysis of 200 ER+ breast tumors detected RAD51AP1-DYRK4 overexpression in 19 tumors (9.5%), which is markedly enriched in the luminal B tumors (17.5%). Ectopic expression of RAD51AP1-DYRK4, but not wild-type RAD51AP1, leads to marked activation of MEK/ERK signaling, and endows increased cell motility and transendothelial migration. More importantly, RAD51AP1-DYRK4 appears to endow increased sensitivity to the MEK inhibitor trametinib through attenuating compensatory activation of HER2/PI3K/AKT under MEK inhibition. CONCLUSIONS: This discovery sheds light on a new area of molecular pathobiology of luminal B tumors and implies potential new therapeutic opportunities for more aggressive breast tumors overexpressing this fusion.

Therapeutic Targeting of Nemo-like Kinase in Primary and Acquired Endocrine-resistant Breast Cancer.

PURPOSE: Endocrine resistance remains a major clinical challenge in estrogen receptor (ER)-positive breast cancer. Despite the encouraging results from clinical trials for the drugs targeting known survival signaling, relapse is still inevitable. There is an unmet need to discover new drug targets in the unknown escape pathways. Here, we report Nemo-like kinase (NLK) as a new actionable kinase target that endows previously uncharacterized survival signaling in endocrine-resistant breast cancer. EXPERIMENTAL DESIGN: The effects of NLK inhibition on the viability of endocrine-resistant breast cancer cell lines were examined by MTS assay. The effect of VX-702 on NLK activity was verified by kinase assay. The modulation of ER and its coactivator, SRC-3, by NLK was examined by immunoprecipitation, kinase assay, luciferase assay, and RNA sequencing. The therapeutic effects of VX-702 and everolimus were tested on cell line- and patient-derived xenograft (PDX) tumor models. RESULTS: NLK overexpression endows reduced endocrine responsiveness and is associated with worse outcome of patients treated with tamoxifen. Mechanistically, NLK may function, at least in part, via enhancing the phosphorylation of ERα and its key coactivator, SRC-3, to modulate ERα transcriptional activity. Through interrogation of a kinase profiling database, we uncovered and verified a highly selective dual p38/NLK inhibitor, VX-702. Coadministration of VX-702 with the mTOR inhibitor, everolimus, demonstrated a significant therapeutic effect in cell line-derived xenograft and PDX tumor models of acquired or de novo endocrine resistance. CONCLUSIONS: Together, this study reveals the potential of therapeutic modulation of NLK for the management of the endocrine-resistant breast cancers with active NLK signaling.

Ambient exposure to criteria air pollutants and risk of death from bladder cancer in Taiwan.

To investigate the relationship between air pollution and risk of death from bladder cancer, the authors conducted a matched case-control study using deaths that occurred in Taiwan from 1995 through 2005. Data on all eligible bladder cancer deaths were obtained from the Bureau of Vital Statistics of the Taiwan Provincial Department of Health. The control group consisted of people who died from causes other than cancer or diseases associated with genitourinary problems. The controls were pair matched to the cases by sex, year of birth, and year of death. Each matched control was selected randomly from the set of possible controls for each case. Classification of exposure to municipality air pollution was based on the measured levels of nitrogen dioxide and sulfur dioxide. The results of the present study show that there is a significant positive association between the levels of air pollution and bladder cancer mortality. The adjusted odds ratios (95% confidence interval) were 1.37 (1.03-1.82) for the group with medium air pollution level and 1.98 (1.36-2.88) for the group with high air pollution level when compared to the group with the low air pollution level. Trend analyses showed statistically significant trend in risk of death from bladder cancer with increasing air pollution level. The findings of this study warrant further investigation of the role of air pollutants in the etiology of bladder cancer.

Laparoscopic loop duodenaljejunal bypass with sleeve gastrectomy in type 2 diabetic patients.

BACKGROUND: Laparoscopic loop duodenojejunal bypass with sleeve gastrectomy (LDJB-SG) is a new metabolic procedure. Our initial data on type 2 diabetes (T2D) remission after LDJB-SG were promising. OBJECTIVES: The aim of this study was to look at our intermediate outcomes after LDJB-SG. SETTING: An academic medical center. METHODS: A prospective analysis of T2D patients who underwent LDJB-SG between October 2011 and October 2014 was performed. Data collected included baseline demographic, body mass index, fasting blood glucose, glycosylated hemoglobin, C-peptide, resolution of co-morbidities, and postoperative complications. RESULTS: A total of 163 patients with minimum of follow-up >1 year were enrolled in this study (57 men and 106 women). The mean age and body mass index were 47.7 (±10.7) years and a 30.2 (±5.1) kg/m2, respectively. There were 119 patients on oral hypoglycemic agents only, 29 patients were on oral hypoglycemic agents and insulin, 3 patients were on insulin only, and the other 12 patients were not on diabetic medication. Mean operation time and length of hospital stay were 144.7 (± 45.1) minutes and 2.4 (± 1.0) days, respectively. Seven patients (3.6%) needed reoperation due to bleeding (n = 1), anastomotic leak (n = 2), sleeve strictures (n = 2), and incisional hernia (n = 2). At 2 years of follow-up, there were 56 patients. None of the patients were on insulin and only 20% of patients were on oral hypoglycemic agents. Mean body mass index significantly dropped to 22.9 (±5.6) kg/m2 at 2 years. The mean preoperative fasting blood glucose, glycosylated hemoglobin, and C-peptide levels were 174.7 mg/dL (± 61.0), 8.8% (±1.8), and 2.6 (±1.7) ng/mL, respectively. The mean fasting blood glucose, glycosylated hemoglobin, and C-peptide at 2 years were 112.5 (±60.7) mg/dL, 6.4% (±2.0), and 1.5 (±0.6) ng/mL, respectively. No patient needed revisional surgery because of dumping syndrome, marginal ulcer, or gastroesophageal reflux disease at the last follow up period. CONCLUSION: At 2 years, LDJB-SG is a relatively safe and effective metabolic surgery with significant weight loss and resolution of co-morbidities.

Association of brain cancer with residential exposure to petrochemical air pollution in Taiwan.

To investigate the relationship between petrochemical air pollution and brain cancer (29 yr of age or younger), the authors conducted a matched case-control study using deaths that occurred in Taiwan from 1995 through 2005. Data on all eligible brain cancer deaths were obtained from the Bureau of Vital Statistics of the Taiwan Provincial Department of Health. The control group consisted of subjects who died from causes other than neoplasms or diseases that were not associated with respiratory problems. The controls were pair matched to the cases by sex, year of birth, and year of death. Each matched control was selected randomly from the set of possible controls for each case. The proportion of a municipality's total population employed in the petrochemical industry in a municipality was used as an indicator of a resident's exposure to air emissions from the petrochemical industry. The subjects were divided into tertiles according to the levels of the index just described. Subjects who lived in the group of municipalities characterized by the highest levels of petrochemical air pollution had a statistically significant higher risk of developing brain cancer than the group that lived in municipalities with the lowest petrochemical air pollution levels after controlling for possible confounders (OR = 1.65, 95% CI = 1.00-2.73). The findings of this study warrant further investigation of the role of petrochemical air pollution in the etiology of brain cancer.

Ambient exposure to criteria air pollutants and female lung cancer in Taiwan.

To investigate the relationship between air pollution and female lung cancer, the authors conducted a matched case-control study using female deaths that occurred in Taiwan from 1995 through 2005. Data on all eligible female lung cancer deaths were obtained from the Bureau of Vital Statistics of the Taiwan Provincial Department of Health. The control group consisted of women who died from causes other than cancer or diseases associated with respiratory problems. The controls were pair-matched to the cases by sex, year of birth, and year of death. Each matched control was selected randomly from the set of possible controls for each case. Classification of exposure to municipality air pollution was based on the measured levels of nitrogen dioxide and carbon monoxide. The results of the present study show that there is a significant positive association between the levels of air pollution and female lung cancer mortality. The adjusted odds ratios (95% confidence interval) were 1.24 (1.03-1.50) for the group with medium air pollution level and 1.46 (1.18-1.81) for the group with high air pollution level when compared to the group with the low air pollution level. Trend analyses showed statistically significant trend in risk of female lung cancer with increasing air pollution level. The findings of this study warrant further investigation of the role of air pollutants in the etiology of lung cancer.

Correction: Regulation of Aldo-keto-reductase family 1 B10 by 14-3-3ε and their prognostic impact of hepatocellular carcinoma.

[This corrects the article DOI: 10.18632/oncotarget.5734.].

Fifteen-year experience of children with Henoch-Schönlein purpura in southern Taiwan, 1991-2005.

BACKGROUND AND PURPOSE: Although Henoch-Schönlein purpura (HSP) is the most common cause of systemic vasculitis in children, long-term and large-scale Taiwanese studies on HSP are rare. We reviewed the records of 107 Taiwanese pediatric patients diagnosed with HSP at our institution between 1991 and 2005. METHODS: The first clinical manifestations, laboratory findings, and outcome evaluations of the patients were analyzed. Data were grouped according to the presence of fever and upper respiratory tact infection (URI) as a presenting symptom and also by gender. Chi-squared test was used for statistical analysis. RESULTS: The children had a mean age of 6.2 +/- 2.5 years (range, 2 to 13 years), with a male-to-female ratio of 1.0:0.7. Main clinical symptoms included skin rashes (95.3%), gastrointestinal (GI) symptoms (72.0%), joint involvement (46.7%), and kidney involvement (28.0%). The most common first manifestations were skin rashes (56.1%), GI symptoms (35.5%), and joint involvement (12.1%). There was no significant association between first manifestations and fever presence or gender. However, the non-URI patients had a significantly higher incidence of GI problems than the URI group (p=0.01). Fever as a symptom was not associated with elevation of C-reactive protein (p=0.45). Immunoglobulin A levels were within the normal range. No chronic renal failure or end-stage renal disease was detected, and overall the prognosis of patients was good. CONCLUSIONS: The categories used did not predict the expression of HSP, with the exception of an association between absence of URI and GI manifestations. Overall, HSP showed a good prognosis.

Paracrine regulation of matrix metalloproteinases contributes to cancer cell invasion by hepatocellular carcinoma-secreted 14-3-3σ.

14-3-3σ overexpression results in enhanced hepatocellular carcinoma (HCC) cell migration and HCC tumor vascular-invasion is significantly associated with 14-3-3σ expression. However, increased expression of 14-3-3σ paradoxically suppresses in vitro cell invasion of HCC. We hypothesize that surrounding tumor-associated stromal cells play a crucial role in 14-3-3σ-regulated HCC cell invasion. In this study, H68 fibroblasts, THP-1 and phorbol-12-myristate-13-acetate (PMA)-treated THP-1 (PMA-THP-1) cells were incubated with conditioned media of control (control-CM) and 14-3-3σ-overepxressing cells (14-3-3σ-CM), followed by co-culture with HCC cells. Invasiveness of HCC cells was examined by a Boyden chamber assay. HCC cells co-cultured with 14-3-3σ-CM treated cells significantly enhanced their invasive ability compared with control-CM treated cells. Moreover, incubation with 14-3-3σ-CM induced differential expression profiles of matrix metalloproteinases (MMPs) in fibroblasts (MMP-1, MMP-2, MMP-9, MMP-12 and MMP-14), THP-1 (MMP-1 and MMP-12) and PMA-THP-1 cells (MMP-2, MMP-12 and MMP-14). In contrast, silencing of 14-3-3σ by siRNA significantly abolished 14-3-3σ-CM induced MMPs. In addition, treatment with recombinant 14-3-3σ (r14-3-3σ) protein exhibits a similar expression profile of MMPs induced by 14-3-3σ-CM in fibroblasts, THP-1 and PMA-THP-1 cells. Finally, knockdown of aminopeptidase N (APN) significantly abrogated r14-3-3σ induced expression of MMPs in HS68 fibroblasts. These results suggest that HCC-secreted 14-3-3σ promotes expression of MMPs in cancerous surrounding cells via an APN dependent mechanism. 14-3-3σ has a paracrine effect in educating stromal cells in tumor-associated microenvironment.

Regulation of aldo-keto-reductase family 1 B10 by 14-3-3ε and their prognostic impact of hepatocellular carcinoma.

14-3-3ε is overexpressed in hepatocellular carcinoma (HCC) and its expression significantly associates with a poor prognostic outcome. To uncover how 14-3-3ε contributes to the tumor progression of HCC, we investigated the potential downstream targets regulated by 14-3-3ε. We found that 14-3-3ε increases expression and nuclear translocation of ß-catenin and that 14-3-3ε-induced cell proliferation is attenuated by ß-catenin silencing in HCC cells. Moreover, 14-3-3ε induces aldo-keto reductase family 1 member B10 (AKR1B10) expression through the activation of ß-catenin signaling. Knockdown of AKR1B10 by siRNAs abolished 14-3-3ε-induced in vitro cell proliferation, anchorage-independent growth as well as in vivo tumor growth. Furthermore, AKR1B10 silencing increased retinoic acid (RA) levels in the serum of tumor-bearing mice and RA treatment attenuated 14-3-3ε-induced HCC cell proliferation. We further examined 14-3-3ε and AKR1B10 expression and clinicopathological characteristics of HCC tumors. Although the expression of AKR1B10 was significantly correlated with 14-3-3ε, an increase of AKR1B10 expression in 14-3-3ε positive patients paradoxically had better overall survival and disease-free survival rates as well as lower metastatic incidence than those without an AKR1B10 increase. Finally, we found a loss of AKR1B10 expression in cells exhibiting a high capacity of invasiveness. Silencing of AKR1B10 resulted in inducing snail and vimentin expression in HCC cells. These results indicate that AKR1B10 may play a dual role during HCC tumor progression. Our results also indicate that 14-3-3ε regulates AKR1B10 expression by activating ß-catenin signaling. A combination of 14-3-3ε with AKR1B10 is a potential therapeutic target and novel prognostic biomarker of HCC.

Increased incidence of preterm delivery in mothers residing in an industrialized area in Taiwan.

The objective of this study was to determine whether women living in the vicinity of industrial districts where emission levels of air contaminants from multiple sources including petrochemical, petroleum, steel, and shipbuilding industries are highly correlated with increased incidence of preterm births. The prevalence of delivery of preterm-birth infants was significantly higher in mothers living in the industrial study area compared to control regions of Taiwan. After controlling for several possible confounders (including maternal age, season, marital status, maternal education, infant gender, and birthplace), the adjusted odds ratio was 1.11 (95% CI = 1.02-1.21) for delivery of preterm infants in the industrial area. Data provide further support for the hypothesis that air pollution can affect the outcome of pregnancy, although a semiecological study cannot confirm a direct causal relationship.

Cordycepin suppresses integrin/FAK signaling and epithelial-mesenchymal transition in hepatocellular carcinoma.

Cordycepin, also known as 3-deoxyadenosine, is an analogue of adenosine extracted from the traditional Chinese medicine "Dong Chong Xia Cao". Cordycepin is an active small molecular weight compound and is implicated in modulating multiple physiological functions including immune activation, anti-aging and anti-tumor effects. Several studies have indicated that cordycepin suppresses tumor progression. However, the signaling pathways involved in cordycepin regulating cancer cell motility, invasiveness and epithelial-mesenchymal transition (EMT) remain unclear. In this study, we found that cordycepin inhibits hepatocellular carcinoma (HCC) cell proliferation and migration/invasion. Treatment of cordycepin results in the increasing expression of epithelial marker, Ecadherin while no significant effect was found on N-cadherin α-catenin and ß-catenin. Furthermore, although the expression of focal adhesion kinase (FAK) was slightly reduced, the level of phosphorylated FAK was significantly reduced by the treatment of cordycepin. In addition, cordycepin significantly suppresses the expression of integrin α3, integrin α6 and integrin ß1 which are crucial interacting partners of FAK in regulating the focal adhesion complex. These results suggest cordycepin may contribute to EMT, antimigration/ invasion and growth inhibitory effects of HCC by suppressing E-cadherin and integrin/FAK signaling. Thus, cordycepin is a potential therapeutic or supplementary agent for preventing HCC tumor progression.

Telomere elongation and naive pluripotent stem cells achieved from telomerase haplo-insufficient cells by somatic cell nuclear transfer.

Haplo-insufficiency of telomerase genes in humans leads to telomere syndromes such as dyskeratosis congenital and idiopathic pulmonary fibrosis. Generation of pluripotent stem cells from telomerase haplo-insufficient donor cells would provide unique opportunities toward the realization of patient-specific stem cell therapies. Recently, pluripotent human embryonic stem cells (ntESCs) have been efficiently achieved by somatic cell nuclear transfer (SCNT). We tested the hypothesis that SCNT could effectively elongate shortening telomeres of telomerase haplo-insufficient cells in the ntESCs with relevant mouse models. Indeed, telomeres of telomerase haplo-insufficient (Terc(+/-)) mouse cells are elongated in ntESCs. Moreover, ntESCs derived from Terc(+/-) cells exhibit naive pluripotency as evidenced by generation of Terc(+/-) ntESC clone pups by tetraploid embryo complementation, the most stringent test of naive pluripotency. These data suggest that SCNT could offer a powerful tool to reprogram telomeres and to discover the factors for robust restoration of telomeres and pluripotency of telomerase haplo-insufficient somatic cells.

14-3-3σ regulates ß-catenin-mediated mouse embryonic stem cell proliferation by sequestering GSK-3ß.

BACKGROUND: Pluripotent embryonic stem cells are considered to be an unlimited cell source for tissue regeneration and cell-based therapy. Investigating the molecular mechanism underlying the regulation of embryonic stem cell expansion is thus important. 14-3-3 proteins are implicated in controlling cell division, signaling transduction and survival by interacting with various regulatory proteins. However, the function of 14-3-3 in embryonic stem cell proliferation remains unclear. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, we show that all seven 14-3-3 isoforms were detected in mouse embryonic stem cells. Retinoid acid suppressed selectively the expression of 14-3-3σ isoform. Knockdown of 14-3-3σ with siRNA reduced embryonic stem cell proliferation, while only 14-3-3σ transfection increased cell growth and partially rescued retinoid acid-induced growth arrest. Since the growth-enhancing action of 14-3-3σ was abrogated by ß-catenin knockdown, we investigated the influence of 14-3-3σ overexpression on ß-catenin/GSK-3ß. 14-3-3σ bound GSK-3ß and increased GSK-3ß phosphorylation in a PI-3K/Akt-dependent manner. It disrupted ß-catenin binding by the multiprotein destruction complex. 14-3-3σ overexpression attenuated ß-catenin phosphorylation and rescued the decline of ß-catenin induced by retinoid acid. Furthermore, 14-3-3σ enhanced Wnt3a-induced ß-catenin level and GSK-3ß phosphorylation. DKK, an inhibitor of Wnt signaling, abolished Wnt3a-induced effect but did not interfere GSK-3ß/14-3-3σ binding. SIGNIFICANCE: Our findings show for the first time that 14-3-3σ plays an important role in regulating mouse embryonic stem cell proliferation by binding and sequestering phosphorylated GSK-3ß and enhancing Wnt-signaled GSK-3ß inactivation. 14-3-3σ is a novel target for embryonic stem cell expansion.

Follicular oocytes better support development in rabbit cloning than oviductal oocytes.

This study was conducted to determine the effect of rabbit oocytes collected from ovaries or oviducts on the developmental potential of nuclear transplant embryos. Donor nuclei were obtained from adult skin fibroblasts, cumulus cells, and embryonic blastomeres. Rabbit oocytes were flushed from the oviducts (oviductal oocytes) or aspirated from the ovaries (follicular oocytes) of superovulated does at 10, 11, or 12 h post-hCG injection. The majority of collected oocytes were still attached to the sites of ovulation on the ovaries. We found that follicular oocytes had a significantly higher rate of fusion with nuclear donor cells than oviductal oocytes. There was no difference in the cleavage rate between follicular and oviductal groups, but morula and blastocyst development was significantly higher in the follicular group than in the oviductal group. Two live clones were produced in follicular group using blastomere and cumulus nuclear donors, whereas one live clone was produced in the oviductal group using a cumulus nuclear donor. These results demonstrate that cloned rabbit embryos derived from follicular oocytes have better developmental competence than those derived from oviductal oocytes.