The evolutionary conservation of the core components necessary for the extrinsic apoptotic signaling pathway, in Medaka fish

<p>Abstract</p> <p>Background</p> <p>Death receptors on the cell surface and the interacting cytosolic molecules, adaptors and initiator caspases, are essential as core components of the extrinsic apoptotic signaling pathway. While the apoptotic machinery governing the extrinsic signaling pathway is well characterized in mammals, it is not fully understood in fish.</p> <p>Results</p> <p>We identified and characterized orthologs of mammalian Fas, FADD and caspase-8 that correspond to the death receptor, adaptor and initiator caspase, from the Medaka fish (<it>Oryzias latipes</it>). Medaka Fas, caspase-8 and FADD exhibited protein structures similar to that of their mammalian counterparts, containing a death domain (DD), a death effector domain (DED) or both. Functional analyses indicated that these molecules possess killing activity in mammalian cell lines upon overexpression or following activation by apoptotic stimuli, suggesting similar pro-apoptotic functions in the extrinsic pathway as those in mammals. Genomic sequence analysis revealed that the Medaka <it>fas </it>(<it>tnfrsf6</it>), <it>fadd </it>and <it>caspase-8 </it>(<it>casp8</it>) genes are organized in a similar genomic structure as the mammalian genes. Database search and phylogenetic analysis revealed that the <it>fas </it>gene, but not the <it>fadd </it>and <it>casp8 </it>genes, appear to be present only in vertebrates.</p> <p>Conclusion</p> <p>Our results indicate that the core components necessary for the extrinsic apoptotic pathway are evolutionarily conserved in function and structure across vertebrate species. Based on these results, we presume the mechanism of apoptosis induction via death receptors was evolutionarily established during the appearance of vertebrates.</p

LATS1/2 kinases trigger self-renewal of cancer stem cells in aggressive oral cancer

Cancer stem cells (CSCs), which play important roles in tumor initiation and progression, are resistant to many types of therapies. However, the regulatory mechanisms underlying CSC-specific properties, including self-renewal, are poorly understood. Here, we found that LATS1/2, the core Hippo pathway-kinases, were highly expressed in the oral squamous cell carcinoma line SAS, which exhibits high capacity of CSCs, and that depletion of these kinases prevented SAS cells from forming spheres under serum-free conditions. Detailed examination of the expression and activation of LATS kinases and related proteins over a time course of sphere formation revealed that LATS1/2 were more highly expressed and markedly activated before initiation of self-renewal. Moreover, TAZ, SNAIL, CHK1/2, and Aurora-A were expressed in hierarchical, oscillating patterns during sphere formation, suggesting that the process consists of four sequential steps. Our results indicate that LATS1/2 trigger self-renewal of CSCs by regulating the Hippo pathway, the EMT, and cell division

cAMP-responsive element in TATA-less core promoter is essential for haploid-specific gene expression in mouse testis

Promoters, including neither TATA box nor initiator, have been frequently found in testicular germ cell-specific genes in mice. These investigations imply that unique forms of the polymerase II transcription initiation machinery play a role in selective activation of germ cell-specific gene expression programs during spermatogenesis. However, there is little information about testis-specific core promoters, because useful germ cell culture system is not available. In this study, we characterize the regulatory region of the haploid-specific Oxct2b gene in detail by using in vivo transient transfection assay in combination with a transgenic approach, with electrophoretic mobility shift and chromatin immunoprecipitation assays. Expression studies using mutant constructs demonstrate that a 34 bp region, which extends from −49 to −16, acts as a core promoter in an orientation-dependent manner. This promoter region includes the cAMP-responsive element (CRE)-like sequence TGACGCAG, but contains no other motifs, such as a TATA box or initiator. The CRE-like element is indispensable for the core promoter activity, but not for activator in testicular germ cells, through the binding of a testis-specific CRE modulator transcription factor. These results indicate the presence of alternative transcriptional initiation machinery for cell-type-specific gene expression in testicular germ cells

Pattern formation of reaction-diffusion system having self-determined flow in the amoeboid organism of Physarum plasmodium

The amoeboid organism, the plasmodium of Physarum polycephalum, behaves on the basis of spatio-temporal pattern formation by local contraction-oscillators. This biological system can be regarded as a reaction-diffusion system which has spatial interaction by active flow of protoplasmic sol in the cell. Paying attention to the physiological evidence that the flow is determined by contraction pattern in the plasmodium, a reaction-diffusion system having self-determined flow arises. Such a coupling of reaction-diffusion-advection is a characteristic of the biological system, and is expected to relate with control mechanism of amoeboid behaviours. Hence, we have studied effects of the self-determined flow on pattern formation of simple reaction-diffusion systems. By weakly nonlinear analysis near a trivial solution, the envelope dynamics follows the complex Ginzburg-Landau type equation just after bifurcation occurs at finite wave number. The flow term affects the nonlinear term of the equation through the critical wave number squared. Contrary to this, wave number isn't explicitly effective with lack of flow or constant flow. Thus, spatial size of pattern is especially important for regulating pattern formation in the plasmodium. On the other hand, the flow term is negligible in the vicinity of bifurcation at infinitely small wave number, and therefore the pattern formation by simple reaction-diffusion will also hold. A physiological role of pattern formation as above is discussed.Comment: REVTeX, one column, 7 pages, no figur

Distinct DNA Methylation Dynamics of Spermatogenic Cell-Specific Intronless Genes Is Associated with CpG Content

<div><p>In mammals, DNA methylation is restricted to cytosines of CpG dinucleotides, which are frequently found in short genomic regions including gene promoters. Methylation within CpG-rich regions around promoters tends to repress gene expression; thus, the CpG islands of housekeeping genes are normally unmethylated. We previously described a testis-specific single-exon gene containing a CpG-rich sequence that is methylated and thus repressed in somatic cells, whereas its expression in spermatogenic cells requires that it be hypomethylated. However, the relationship among the specific expression of spermatogenic genes, their methylation dynamics, and their CpG frequencies are poorly understood. Here, we analyzed the methylation patterns of the sphort genomic region around the transcription start site in spermatogenic cell-specific single-exon genes of various CpG contents. By using UniGene and Ensembl database analyses of the mouse genome and reverse transcription-PCR, we identified 39 single-exon genes that are exclusively expressed in spermatogeniccells. Regardless of their specific expression characteristics, genes containing higher (7 to 14 CpGs in 200 bp; mean = 12) and lower (2 to 6 CpGs in 200 bp; mean = 3.1) number ofCpG were hypo- and hyper-methylated, respectively, in all cell types examined, including spermatogeniccells. We found that genes with intermediate number of CpG (2 to 11 CpGs in 200 bp; mean = 6.9) are methylated in somatic cells, but not in male germ cells. These results suggest that DNA methylation dynamics of spermatogenic cell-specific single-exon genes are associated with CpG content, and the methylation status are stably maintained throughout male germ cell development.</p> </div

Ligand-Independent EGFR Activation by Anchorage-Stimulated Src Promotes Cancer Cell Proliferation and Cetuximab Resistance via ErbB3 Phosphorylation

Activation of the epidermal growth factor receptor (EGFR) pathway plays an important role in the progression of cancer and is associated with a poor prognosis in patients. The monoclonal antibody cetuximab, which displays EGFR extracellular domain-specific binding, has proven effective in the treatment of locally advanced disease and relapsed/metastatic disease. However, the effects of cetuximab are weaker than those of EGFR tyrosine kinase inhibitors (TKIs). This study investigates differences in the effects on cell growth of cetuximab and EGFR TKI AG1478 at the molecular level using oral squamous cell carcinoma (OSCC) cell lines. First, we found that there were EGFR-inhibitor-sensitive (EIS) and EGFR-inhibitor-resistant cell lines. The EIS cell lines expressed not only EGFR but also ErbB3, and both were clearly phosphorylated. The levels of phosphorylated ErbB3 were unaffected by cetuximab but were reduced by AG1478. EGFR ligand treatment increased the levels of phosphorylated EGFR but not phosphorylated ErbB3. Moreover, when EIS cell lines that were only capable of anchorage-dependent growth were grown in suspension, cell growth was suppressed and the levels of phosphorylated focal adhesion kinase (FAK), Src, and ErbB3 were significantly reduced. The levels of phosphorylated ErbB3 were unaffected by the FAK inhibitor PF573228, but were reduced by Src inhibition. Finally, combining cetuximab and a Src inhibitor produced an additive effect on the inhibition of EIS cell line growth