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Objective: LNK is an adapter protein negatively regulating the JAK/STAT cell signaling pathway. In this study, we observed the correlation between variation in LNK gene and the clinical type of myeloproliferative neoplasms (MPN). Methods: A total of 285 MPN cases were recruited, including essential thrombocythemia (ET) 154 cases, polycythemia vera (PV) 76 cases, primary myelofibrosis (PMF) 19 cases, and chronic myeloid leukemia (CML) 36 cases. Ninety-three healthy individuals were used as normal controls. V617F mutation in JAK2 was identified by allele-specific PCR method, RT-PCR was used for the detection of BCR/ABL1 fusion gene, and mutations and variations in coding exons and their flanking sequences of LNK gene were examined by PCR-sequencing. Results: Missense mutations of A300V, V402M, and R415H in LNK were found in 8 patients including ET (4 cases, all combined with JAK2-V617F mutation), PV (2 cases, one combined with JAK2-V617F mutation), PMF (one case, combined with JAK2-V617F mutation) and CML (one case, combined with BCR/ABL1 fusion gene). The genotype and allele frequencies of the three SNPs (rs3184504, rs111340708 and rs78894077) in LNK were significantly different between MPN patients and controls. For rs3184504 (T/C, in exon2), the T allele (p.262W) and TT genotype were frequently seen in ET, PV and PMF (P<0.01), and C allele (p.262R) and CC genotype were frequently seen in CML (P<0.01). For rs78894077 (T/C, in exon1), the T allele (p.242S) was frequently found in ET (P<0.05). For rs111340708 (TGGGGx5/TGGGGx4, in intron 5), the TGGGG x4 allele was infrequently found in ET, PMF and CML(P<0.01). Conclusion: Mutations in LNK could be found in some of MPN patients in the presence or absence of JAK2-V617F mutation. Several polymorphisms in LNK gene may affect the clinical type or the genetic predisposition of MPN. [ABSTRACT FROM AUTHOR]

The retinoblastoma protein (pRb) is required for cell-cycle exit of embryonic mammalian hair cells but is not required for hair cell fate determination and early differentiation, and this provides a strategy for hair cell regeneration by manipulating the pRb pathway. To reveal the mechanism of pRb functional modification in the inner ear, we compared the effects of attenuated pRb phosphorylation by an inhibitor of the Mitogen-Activated Protein (MAP) kinase pathway and an inhibitor of the Rb–Raf-1 interaction on cultured chicken otocysts. We demonstrated that the activity of pRb is correlated with its phosphorylation state, which is regulated by a newly established cell cycle-independent pathway mediated by the physical interaction between Raf-1 and pRb. The phosphorylation of pRb plays an important role during the early stage of inner ear development, and attenuated phosphorylation in progenitor cells leads to cell cycle arrest and increased apoptosis along with a global down-regulation of the genes involved in cell cycle progression. Our study provides novel routes to modulate pRb function for hair cell regeneration. [ABSTRACT FROM AUTHOR]

Glycosaminoglycans are important regulators of multiple signaling pathways. As a major constituent of the heart extracellular matrix, glycosaminoglycans are implicated in cardiac morphogenesis through interactions with different signaling morphogens. Ext1 is a glycosyltransferase responsible for heparan sulfate synthesis. Here, we evaluate the function of Ext1 in heart development by analyzing Ext1 hypomorphic mutant and conditional knockout mice. Outflow tract alignment is sensitive to the dosage of Ext1. Deletion of Ext1 in the mesoderm induces a cardiac phenotype similar to that of a mutant with conditional deletion of UDP-glucose dehydrogenase, a key enzyme responsible for synthesis of all glycosaminoglycans. The outflow tract defect in conditional Ext1 knockout(Ext1f/f:Mesp1Cre) mice is attributable to the reduced contribution of second heart field and neural crest cells. Ext1 deletion leads to downregulation of FGF signaling in the pharyngeal mesoderm. Exogenous FGF8 ameliorates the defects in the outflow tract and pharyngeal explants. In addition, Ext1 expression in second heart field and neural crest cells is required for outflow tract remodeling. Our results collectively indicate that Ext1 is crucial for outflow tract formation in distinct progenitor cells, and heparan sulfate modulates FGF signaling during early heart development. [ABSTRACT FROM AUTHOR]

The brightness of fluorescently labeled proteins provides an excellent marker for identifying protein interactions in living cells. Quantitative interpretation of brightness, however, hinges on a detailed understanding of the processes that affect the signal fluctuation of the fluorescent label. Here, we focus on the cumulative influence of photobleaching on brightness measurements in cells. Photobleaching within the finite volume of the cell leads to a depletion of the population of fluorescently labeled proteins with time. The process of photodepletion reduces the fluorescence signal which biases the analysis of brightness data. Our data show that even small reductions in the signal can introduce significant bias into the analysis of the data. We develop a model that quantifies the bias and introduce an analysis method that accurately determines brightness in the presence of photodepletion as verified by experiments with mammalian and yeast cells. In addition, photodepletion experiments with the fluorescent protein EGFP reveal the presence of a photoconversion process, which leads to a marked decrease in the brightness of the EGFP protein. We also identify conditions where the effect of EGFP's photoconversion on brightness experiments can be safely ignored. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is one of the most malignant tumors and the biggest obstacle in curing HCC is its high metastasis potential. Alteration of p53 is the most frequent genetic change found in HCC. Although the biological function of p53 in tumor initiation and progression has been well characterized, whether or not p53 is implicated in metastasis of HCC is largely unknown. In this study, we analyzed the potential functions of p53 in epithelial-mesenchymal transition (EMT) and metastasis of HCC cells. Both insulin- and TGF-β1-induced changes of critical EMT markers were greatly enhanced by p53 knockdown in HCC cells. The insulin- and TGF-β1-stimulated migration of HCC cells were enhanced by p53 knockdown. Furthermore, in vivo metastasis of HCC cells using different mouse models was robustly enhanced by p53 knockdown. In addition, we found that p53 regulation on EMT and metastasis involves β-catenin signaling. The nuclear accumulation and transcriptional activity of β-catenin was modulated by p53. The enhanced EMT phenotype, cell migration and tumor metastasis of HCC cells by p53 knockdown were abrogated by inhibiting β-catenin signal pathway. In conclusion, this study reveals that p53 plays a pivotal role in EMT and metastasis of HCC cells via its regulation on β-catenin signaling. [ABSTRACT FROM AUTHOR]

Background and Aims: EGFR activation and PKM2 expression are instrumental in tumorigenesis. EGFR activation regulates PKM2 functions in a subcellular compartment-dependent manner and promotes gene transcription and tumor growth. In addition, PKM2 is upregulated in EGFR-induced pathways in glioma malignancies. However, we found that PKM2 could also regulate the activity of the EGF/EGFR signaling pathway in gastric cancer cells. We aimed to define the biological mechanisms for PKM2 in regulating the cell motility and invasion. Methods: We employed stable transfection with short hairpin RNA to stably silence the expression of PKM2 in the BGC823, SGC7901 and AGS gastric cancer cell lines. The effects of PKM2 in vitro were determined by assessing cell migration and invasion. Immunohistochemical analysis was used to explore the relationship among PKM2 and other proteins. Results: Our results indicate that the knockdown of PKM2 decreased the activity of E-cadherin and enhanced the EGF/EGFR signaling pathway in the gastric cell lines BGC823 and SGC7901 that were positive for E-cadherin expression. However, in the undifferentiated gastric carcinoma cell line AGS, which lacks E-cadherin expression, PKM2 promoted cell migration and invasion. Immunohistochemical analyses showed that the levels of E-cadherin expression, ERK1/2 phosphorylation, and cytoplasmic PKM2 expression were correlated with each other. Conclusion:: PKM2 may play different roles in differently differentiated gastric cancer cell types, and this finding would be consistent with the previous clinical research. The results of our study reveal an important link between PKM2 and E-cadherin during EGFR-stimulated gastric cancer cell motility and invasion. [ABSTRACT FROM AUTHOR]