ASAP,a novel protein complex involved in RNA processing and apoptosis

Different isoforms of a protein complex termed the apoptosis- and splicing-associated protein (ASAP) were isolated from HeLa cell extract. ASAP complexes are composed of the polypeptides SAP18 and RNPS1 and different isoforms of the Acinus protein. While Acinus had previously been implicated in apoptosis and was recently identified as a component of the spliceosome, RNPS1 has been described as a general activator of RNA processing. Addition of ASAP isoforms to in vitro splicing reactions inhibits RNA processing mediated by ASF/SF2, by SC35, or by RNPS1. Additionally, microinjection of ASAP complexes into mammalian cells resulted in acceleration of cell death. Importantly, after induction of apoptosis the ASAP complex disassembles. Taken together, our results suggest an important role for the ASAP complexes in linking RNA processing and apoptosis.     

Identification of a novel centrosomal protein CrpF46 involved in cell cycle progression and mitosis

A novel centrosome-related protein CrpF46 was detected using a serum F46 from a patient suffering from progressive systemic sclerosis. We identified the protein by immunoprecipitation and Western blotting followed by tandem mass spectrometry sequencing. The protein CrpF46 has an apparent molecular mass of ~60 kDa, is highly homologous to a 527 amino acid sequence of the C-terminal portion of the protein Golgin-245, and appears to be a splice variant of Golgin-245. Immunofluorescence microscopy of synchronized HeLa cells labeled with an anti-CrpF46 monoclonal antibody revealed that CrpF46 localized exclusively to the centrosome during interphase, although it dispersed throughout the cytoplasm at the onset of mitosis. Domain analysis using CrpF46 fragments in GFP-expression vectors transformed into HeLa cells revealed that centrosomal targeting is conferred by a C-terminal coiled-coil domain. Antisense CrpF46 knockdown inhibited cell growth and proliferation and the cell cycle typically stalled at S phase. The knockdown also resulted in the formation of poly-centrosomal and multinucleate cells, which finally became apoptotic. These results suggest that CrpF46 is a novel centrosome-related protein that associates with the centrosome in a cell cycle-dependent manner and is involved in the progression of the cell cycle and M phase mechanism.     

Altered cell cycle gene expression and apoptosis in post-implantation dog parthenotes

Mature oocytes can be parthenogenetically activated by a variety of methods and the resulting embryos are valuable for studies of the respective roles of paternal and maternal genomes in early mammalian development. In the present study, we report the first successful development of parthenogenetic canine embryos to the post-implantation stage. Nine out of ten embryo transfer recipients became pregnant and successful in utero development of canine parthenotes was confirmed. For further evaluation of these parthenotes, their fetal development was compared with artificially inseminated controls and differentially expressed genes (DEGs) were compared using ACP RT-PCR, histological analysis and immunohistochemistry. We found formation of the limb-bud and no obvious differences in histological appearance of the canine parthenote recovered before degeneration occurred; however canine parthenotes were developmentally delayed with different cell cycle regulating-, mitochondria-related and apoptosis-related gene expression patterns compared with controls. In conclusion, our protocols were suitable for activating canine oocytes artificially and supported early fetal development. We demonstrated that the developmental abnormalities in canine parthenotes may result from defective regulation of apoptosis and aberrant gene expression patterns, and provided evidence that canine parthenotes can be a useful tool for screening and for comparative studies of imprinted genes.     

ASK3, a novel member of the apoptosis signal-regulating kinase family, is essential for stress-induced cell death in HeLa cells

Apoptosis signal-regulating kinase 1 (ASK1) and ASK2 are both members of mitogen-activated protein kinase kinase kinase (MAP3K) family that are implicated in apoptotic cell death, stress responses, and various diseases. We have determined that NT2RI3007443, TESTI4031745, SGK341, and human MAP3K15 are all transcribed from the same genomic locus, which we designate “ASK3 gene” based on sequence homology to ASK1 and ASK2. NT2RI3007443, TESTI4031745, and SGK341 displayed distinct expression profiles among human tissues. TESTI4031745 was expressed in relatively high levels. The expression of TESTI4031745 was increased in rectum tumor and Alzheimer’s disease hippocampus and decreased in kidney tumor and Alzheimer’s disease frontal lobe. NT2RI3007443 showed moderate levels of ubiquitous expression in normal adult tissues. They did not drastically change in diseases except for increase in cirrhosis liver. Expression of SGK341 was restricted. It was highly expressed in fetal brain, and moderately expressed in normal hippocampus, pancreas, spleen, lung, and kidney. Further, its expression was dramatically increased in hepatic cirrhosis and decreased in lung tumor. Target proteins encoded by NT2RI3007443 and TESTI4031745 were translated in cell-free protein synthesis system. They exhibited protein kinase activity indicated by ATP consumption and phosphorylation of Syntide 2 as a substrate. We demonstrated that knockdown of ASK3 protected HeLa cells against cytotoxicity induced by anti-Fas monoclonal antibody, TNF-alpha, or oxidative stress. These findings suggest that “ASK3 gene” is a novel member of apoptosis signal-regulating kinases and that it plays a pivotal role in the signal transduction pathway implicated in apoptotic cell death triggered by cellular stresses. It can be a putative therapeutic drug target for multiple human diseases.     

Scrib, a tumor suppressor gene involved in cell polarity

Polarity is a fundamental feature of all organisms both during development and in the adult. This reflects the key role of cell polarity during basic fundamental processes such as cell division, cell differentiation and cell migration. The control of cell polarity relies on functionally conserved proteins. Among these, Scribble, initially identified as a tumor suppressor gene in Drosophila, has been first involved in epithelial polarity. More recently Scribble function has been implicated in neuronal polarity and polarized cell migration. Scribble joins the growing family of tumor suppressors that play a key and conserved function in cell polarity. Scribble illustrates the more and more obvious link between regulation of cell polarity, cell transformation and tumor formation.     

ei24, a p53 response gene involved in growth suppression and apoptosis

DNA damage and/or hyperproliferative signals activate the wild-type p53 tumor suppressor protein, which induces a G(1) cell cycle arrest or apoptosis. Although the mechanism of p53-mediated cell cycle arrest is fairly well defined, the p53-dependent pathway regulating apoptosis is poorly understood. Here we report the functional characterization of murine ei24 (also known as PIG8), a gene directly regulated by p53, whose overexpression negatively controls cell growth and induces apoptotic cell death. Ectopic ei24 expression markedly inhibits cell colony formation, induces the morphological features of apoptosis, and reduces the number of beta-galactosidase-marked cells, which is efficiently blocked by coexpression of Bcl-X(L). The ei24/PIG8 gene is localized on human chromosome 11q23, a region frequently altered in human cancers. These results suggest that ei24 may play an important role in negative cell growth control by functioning as an apoptotic effector of p53 tumor suppressor activities.     

RACH2, a novel human gene that complements a fission yeast cell cycle checkpoint mutation.

We have identified a novel human gene by virtue of its ability to complement the rad1-1 checkpoint mutant of Schizosaccharomyces pombe. This gene, called RACH2, rescues the temperature-sensitive lethality of a rad1-1 wee1-50 double mutant of S. pombe. Expression of RACH2 in S. pombe rad1-1 strains partially restores UV resistance to the rad1-1 mutant strain. Expression of RACH2 in a rad1-1 cdc25-22 double mutant partially restores the dose-dependent delay in mitotic entry after irradiation that is lost in rad1-1 checkpoint-deficient mutants. Overexpression of RACH2 in human tissue culture cells induces apoptosis.     

Simulated colon fiber metabolome regulates genes involved in cell cycle, apoptosis, and energy metabolism in human colon cancer cells

High level of dietary fiber has been epidemiologically linked to protection against the risk for developing colon cancer. The mechanisms of this protection are not clear. Fermentation of dietary fiber in the colon results in production of for example butyrate that has drawn attention as a chemopreventive agent. Polydextrose, a soluble fiber that is only partially fermented in colon, was fermented in an in vitro colon simulator, in which the conditions mimic the human proximal, ascending, transverse, and distal colon in sequence. The subsequent fermentation metabolomes were applied on colon cancer cells, and the gene expression changes studied. Polydextrose fermentation down-regulated gene ontology classes linked with cell cycle, and affected number of metabolically active cells. Furthermore, up-regulated effects on classes linked with apoptosis, with increased caspase 2 and 3 activity, implicate that polydextrose fermentation plays a role in induction of apoptosis in colon cancer cells. The up-regulated genes involved also key regulators of lipid metabolism, such as PPARα and PGC-1α. These results offer hypotheses for the mechanisms of two health benefits linked with consumption of dietary fiber, reducing risk of development of colon cancer, and dyslipidemia.     

CT2-3, a novel magnolol analogue suppresses NSCLC cells through triggering cell cycle arrest and apoptosis

Magnolol, a major bioactive component found in Magnolia officinalis with anti-inflammation and anti-oxidation activities as well as minimized cytotoxic effects. Although magnolol has a wide range of clinical applications, the anti-tumor activity of magnolol is not efficient. Herein, we reported the synthesis and anti-cancer activities of three novel magnolol analogues CT2-1, CT2-2, CT2-3, among which CT2-3 revealed more efficient anti-non-small cell lung cancer (NSCLC) activity than magnolol. Our data showed that CT2-3 could significantly inhibit the proliferation of human NSCLC cells in a dose-dependent manner. In addition, we revealed CT2-3 could induce cell cycle arrest through down-regulating mRNA expression of CDK4, CDK6 and cyclin D1. Moreover, we verified that CT2-3 could cause ROS generation, leading to apoptosis of human NSCLC cells. Further more, we also provided strong evidences that CT2-3 down-regulates the expression of c-Myc and topoisomerases, and contributes to the apoptosis of human NSCLC cells. Taken together, the current study is the first to report a promising new chemotherapeutic drug candidate CT2-3 that can efficiently eliminate human NSCLC cells through triggering cell cycle arrest as well as ROS-mediated and c-Myc/topoisomerases-mediated apoptosis.