CBL202 Sigma-AldrichAnti-Vimentin Antibody, clone VIM 3B4

Effective treatment as well as prognostic biomarker for malignant esophageal squamous cell carcinoma (ESCC) is urgently needed. The present study was aimed at identifying oncogenic genes involving dysregulated intracellular Ca2+ signaling, which is known to function importantly in cellular proliferation and migration. Tumors from patients with ESCC were found to display elevated expression of Orai1, a store-operated Ca2+ entry (SOCE) channel, and the high expression of Orai1 was associated with poor overall and recurrence-free survival. In contrast to the quiescent nature of non-tumorigenic epithelial cells, human ESCC cells exhibited strikingly hyperactive in intracellular Ca2+ oscillations, which were sensitive to treatments with Orai1 channel blockers and to orai1 silencing. Moreover, pharmacologic inhibition of Orai1 activity or reduction of Orai1 expression suppressed proliferation and migration of ESCC in vitro and slowed tumor formation and growth in in vivo xenografted mice. Combined, these findings provide the first evidence to imply Orai1 as a novel biomarker for ESCC prognostic stratification and also highlight Orai1-mediated Ca2+ signaling pathway as a potential target for treatment of this deadly disease.

The conversion of differentiated cells into insulin-producing cells is a promising approach for the autologous replacement of pancreatic cells in patients with type 1 diabetes (T1D). At present, cellular reprogramming strategies encompass ethical problems, epigenetic failure or teratoma formation, which has prompted the development of new approaches. Here, we report a novel technique for the conversion of skin fibroblasts from T1D patients into insulin-expressing clusters using only drug-based induction. Our results demonstrate that skin fibroblasts from diabetic patients have pancreatic differentiation capacities and avoid the necessity of using transgenic strategies, stem cell sources or global demethylation steps. These findings open new possibilities for studying diabetes mechanisms, drug screenings and ultimately autologous transgenic-free regenerative medicine therapies in patients with T1D.

Pancreata from Sprague Dawley rats of different developmental stages were studied to determine the expression and cellular localization of different c-MET isoforms in the developing rat pancreas. Pancreatic mRNA and protein expression levels of c-MET at different developmental stages from embryo to adult were detected by reverse transcription-polymerase chain reaction and by western blotting. To identify the cellular localization of c-MET protein in the developing rat pancreas, double immunofluorescent staining was performed using antibodies for cell type-specific markers and for c-MET. The expression of two isoforms of c-MET (190 kDa and 170 kDa) coincided with the development of the pancreas. The 190 kDa isoform of c-MET is expressed during embryonic stages, and its expression is replaced by the expression of the 170 kDa isoform as the pancreas develops. Only the 170 kDa isoform is expressed in the adult rat pancreas. Throughout all stages of pancreatic development, c-MET is expressed by vimentin-positive cells. In contrast, c-MET staining was stronger in rat pancreata from newborn to adult stages and overlapped with insulin-positive beta-cells. The dynamic expression and localization of different c-MET isoforms in the rat pancreas during different developmental stages indicates that distinct c-MET isoform might be involved in different aspects of pancreatic development.

The reproductive system of chickens undergoes dynamic morphological and functional tissue remodeling during the molting period. The present study identified global gene expression profiles following oviductal tissue regression and regeneration in laying hens in which molting was induced by feeding high levels of zinc in the diet. During the molting and recrudescence processes, progressive morphological and physiological changes included regression and re-growth of reproductive organs and fluctuations in concentrations of testosterone, progesterone, estradiol and corticosterone in blood. The cDNA microarray analysis of oviductal tissues revealed the biological significance of gene expression-based modulation in oviductal tissue during its remodeling. Based on the gene expression profiles, expression patterns of selected genes such as, TF, ANGPTL3, p20K, PTN, AvBD11 and SERPINB3 exhibited similar patterns in expression with gradual decreases during regression of the oviduct and sequential increases during resurrection of the functional oviduct. Also, miR-1689* inhibited expression of Sp1, while miR-17-3p, miR-22* and miR-1764 inhibited expression of STAT1. Similarly, chicken miR-1562 and miR-138 reduced the expression of ANGPTL3 and p20K, respectively. These results suggest that these differentially regulated genes are closely correlated with the molecular mechanism(s) for development and tissue remodeling of the avian female reproductive tract, and that miRNA-mediated regulation of key genes likely contributes to remodeling of the avian reproductive tract by controlling expression of those genes post-transcriptionally. The discovered global gene profiles provide new molecular candidates responsible for regulating morphological and functional recrudescence of the avian reproductive tract, and provide novel insights into understanding the remodeling process at the genomic and epigenomic levels.

Epithelial-mesenchymal (EMT) and mesenchymal-epithelial (MET) transitions occur in the development of human tumorigenesis and are part of the natural history of the process to adapt to the changing microenvironment. In this setting, the miR-200 family is recognized as a master regulator of the epithelial phenotype by targeting ZEB1 and ZEB2, two important transcriptional repressors of the cell adherence (E-cadherin) and polarity (CRB3 and LGL2) genes. Recently, the putative DNA methylation associated inactivation of various miR-200 members has been described in cancer. Herein, we show that the miR-200ba429 and miR-200c141 transcripts undergo a dynamic epigenetic regulation linked to EMT or MET phenotypes in tumor progression. The 5'-CpG islands of both miR-200 loci were found unmethylated and coupled to the expression of the corresponding miRNAs in human cancer cell lines with epithelial features, such as low levels of ZEB1/ZEB2 and high expression of E-cadherin, CRB3 and LGL2, while CpG island hypermethylation-associated silencing was observed in transformed cells with mesenchymal characteristics. The recovery of miR-200ba429 and miR-200c141 expression by stable transfection in the hypermethylated cells restored the epithelial markers and inhibited migration in cell culture and tumoral growth and metastasis formation in nude mice. We also discovered, using both cell culture and animal models, that the miR-200 epigenetic silencing is not an static and fixed process but it can be shifted to hypermethylated or unmethylated 5'-CpG island status corresponding to the EMT and MET phenotypes, respectively. In fact, careful laser microdissection in human primary colorectal tumorigenesis unveiled that in normal colon mucosa crypts (epithelia) and stroma (mesenchyma) already are unmethylated and methylated at these loci, respectively; and that the colorectal tumors undergo selective miR-200 hypermethylation of their epithelial component. These findings indicate that the epigenetic silencing plasticity of the miR-200 family contributes to the evolving and adapting phenotypes of human tumors.

To know the embryogenesis of the core and shell regions of the midbrain auditory nucleus, a single dose of [(3)H]-thymidine was injected into the turtle embryos at peak stages of neurogenesis in the shell and core of the torus semicircularis. Following sequential survival times, labeled neurons and the dynamics of cell proliferation were examined. The expression of vimentin (VM), reelin, calbindin, parvalbumin, and substance P were also studied. The results showed that: 1) progenitor cells for the core and shell regions were generated in different sites of the ventricular zone; 2) the length of the cell cycle or S-phase for the shell region were both longer than those for the core region (4.7 and 3.2 hours longer, respectively), suggesting that mitotic activity in the core region is higher than it is in the shell region; 3) the elongated cell bodies of the labeled core and shell cells had close apposition to VM fibers, suggesting that the migration of these cells is guided by VM fibers; 4) the germinal sites of the core and shell constructed by projecting the orientation of radial VM fibers back to the ventricular zone was consistent with those obtained by short and sequential survival [(3)H]-thymidine radiography; and 5) the beginning of positive staining for parvalbumin in the core region was interposed between those for calbindin and substance P in the shell regions. This study contributes to the understanding of how auditory nuclei are organized and how their components developed and evolved.

AIM: To investigate the expression of alpha-fetoprotein (AFP), a cancer-associated fetal glycoprotein, and its involvement during rat colon development. METHODS: Colons from Sprague-Dawley rat fetuses, young and adult (8 wk old) animals were used in this study. Expression levels of AFP in colons of different development stage were detected by reverse-transcriptase PCR (RT-PCR) and Western blotting. To identify the cell location of AFP in the developing rat colons, double-immunofluorescent staining was performed using antibodies to specific cell markers and AFP, respectively. RESULTS: The highest levels of AFP mRNA were detected in colons of rats at embryonic day 18.5 (e18.5). Compared to e18.5 d, the AFP expression was significantly decreased during rat development [85% for e20.5, P 0.05, 58% for postnatal day 0.5 (P0.5), P 0.05, 37% for P7, P 0.05, 24% for P14, P 0.05, and 11% for P21, P 0.05] and undetected in adult rats. Only the 72-kDa isoform of AFP was detected by Western blotting, the expression pattern was similar to AFP mRNA and conformed to the results of mRNA expression. The AFP positive staining was identical to different distribution patterns in fetuses, young and adult animals and positive staining for both AFP and vimentin was overlapped in mesenchymal cells at each stage tested. CONCLUSION: This study has for the first time demonstrated that AFP is localized in the mesenchyme of rat colon from the embryo to the weaning stage by immunofluorescence and presents 72-kDa isoform in the developing rat colons by Western blotting. The dynamic expression of AFP in the various developmental stages of the colon indicates that AFP might be involved in many aspects of colon development.

To define a genetic network that regulates development of the pancreas, we used high-density microarray (Affymetrix) to generate transcriptional profiles of rat pancreas from five biologically significant stages of development: embryonic day 12.5 (E12.5), E15.5, E18.5, postnatal day 0 (P0) and adult. Many genes were notably highly expressed in the later gestation when islet architecture and function are gradually forming. The expression and localization of mesothelin, one of these genes, was further examined. Reverse transcription-polymerase chain reaction and Western blot analysis revealed that mRNA and protein levels of mesothelin were high from later gestation to 2-3 weeks after birth, and with relatively low but detectable expression levels in adult rat pancreas. Immunolocalization indicated that mesothelin localized not only in islet beta-cells but also in the mesenchyme of developing rat pancreas. Transient mesothelin expression was concomitant with the development of islets architecture formation, remodeling and maturation. These findings indicate that mesothelin is dynamically expressed in the developing rat pancreas and that mesothelin might be involved in some developmental events during development of rat pancreas.

The mesodermally derived normal ovarian surface epithelium (OSE) displays both epithelial and mesenchymal characteristics and exhibits remarkable phenotypic plasticity during post-ovulatory repair. The majority of epithelial ovarian carcinomas (EOC) are derived from the OSE and represent the most lethal of all gynecological malignancies, as most patients (approximately 70%) present at diagnosis with disseminated intra-abdominal metastasis. The predominant pattern of EOC metastasis involves pelvic dissemination rather than lymphatic or hematologic spread, distinguishing EOC from other solid tumors. Acquisition of the metastatic phenotype involves a complex series of interrelated cellular events leading to dissociation (shedding) and dispersal of malignant cells. A key event in this process is disruption of cell-cell contacts via modulation of intercellular junctional components. In contrast to most carcinomas that downregulate E-cadherin expression during tumor progression, a unique feature of primary well-differentiated ovarian cancers is a gain of epithelial features, characterized by an increase in expression of E-cadherin. Subsequent reacquisition of mesenchymal features is observed in more advanced tumors with concomitant loss of E-cadherin expression and/or function during progression to metastasis. The functional consequences of this remarkable phenotypic plasticity are not fully understood, but may play a role in modulation of cell survival in suspension (ascites), chemoresistance, and intraperitoneal anchoring of metastatic lesions.