Spindlin1, a novel nuclear protein with a role in the transformation of NIH3T3 cells

spindlin1, a novel human gene recently isolated by our laboratory, is highly homologous to mouse spindlin gene. In this study, we cloned cDNA full-length of this novel gene and send it to GenBank database as spindlin1 (Homo sapiens spindlin1) with Accession No. AF317228. In order to investigate the function of spindlin1, we studied further the subcellular localization of Spindlin1 protein and the effects of spindlin1 overexpression in NIH3T3 cells. The results showed that the fusion protein pEGFP-N1-spindlin1 was located in the nucleus and the C-terminal is correlated with nuclear localization of Spindlin1 protein. NIH3T3 cells which could stably express spindlin1 as a result of RT-PCR analysis compared with the control cells displayed a complete morphological change; made cell growth faster; and increased the percentage of cells in G2/M and S phase. Furthermore, overexpressed spindlin1 cells formed colonies in soft agar in vitro and formed tumors in nude mice. Our findings provide direct evidence that spindlin1 gene may contribute to tumorigenesis.     

Introduction of G1 phase arrest in Human Hepatocellular carcinoma cells (HHCC) by APMCF1 gene transfection through the down-regulation of TIMP3 and up-regulation of the CDK inhibitors p21

We previously found that there was up-regulation of APMCF1 expression in apoptotic MCF-7 cells. Moreover, bioinformatics analysis has found that APMCF1 molecules had similar size and structure with molecules which belong to small G-protein superfamily. We presume that APMCF1 plays certain biological role in the regulation of cell proliferation and apoptosis. In this study, we first detected the expression pattern of APMCF1 in human hepatocellular carcinoma cell line and find no expression in Human Hepatocellular carcinoma cells (HHCC) and enhanced expression in HepG2 cells. Expression of liposome-mediated ectogenic APMCF1 induced inhibition of HHCC growth and cell cycle, and RNAi inhibited APMCF1 expression and promoted HepG2 cell growth. Results of cell cycle gene chips analysis showed up-regulation of p21 expression and down-regulation of TIMP3 in HHCC cells expressing ectogenic APMCF1, indicating that APMCF1 participates at least partially in cell cycle regulation through regulating genes such as p21 and TIMP3.     

The protein-tyrosine phosphatase SHP-2 is required during angiotensin II-mediated activation of cyclin D1 promoter in CHO-AT1A cells

Angiotensin II (Ang II) binds to specific G protein-coupled receptors and is mitogenic in Chinese hamster ovary (CHO) cells stably expressing a rat vascular angiotensin II type 1A receptor (CHO-AT(1A)). Cyclin D1 protein expression is regulated by mitogens, and its assembly with the cyclin-dependent kinases induces phosphorylation of the retinoblastoma protein pRb, a critical step in G(1) to S phase cell cycle progression contributing to the proliferative responses. In the present study, we found that in CHO-AT(1A) cells, Ang II induced a rapid and reversible tyrosine phosphorylation of various intracellular proteins including the protein-tyrosine phosphatase SHP-2. Ang II also induced cyclin D1 protein expression in a phosphatidylinositol 3-kinase and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)-dependent manner. Using a pharmacological and a co-transfection approach, we found that p21(ras), Raf-1, phosphatidylinositol 3-kinase and also the catalytic activity of SHP-2 and its Src homology 2 domains are required for cyclin D1 promoter/reporter gene activation by Ang II through the regulation of MAPK/ERK activity. Our findings suggest for the first time that SHP-2 could play an important role in the regulation of a gene involved in the control of cell cycle progression resulting from stimulation of a G protein-coupled receptor independently of epidermal growth factor receptor transactivation.     

Possible dual regulatory circuits involving AtS6K1 in the regulation of plant cell cycle and growth

The role of Arabidopsis S6 Kinase 1 (AtS6K1), a downstream target of TOR kinase, in controlling plant growth and ribosome biogenesis was characterized after generating transgenic plants expressing AtS6K1 under auxin-inducible promoter. Down regulation of selected cell cycle regulatory genes upon auxin treatment was observed in the transgenic plants, confirming the negative regulatory role of AtS6K1 in the plant cell cycle progression reported earlier. Callus tissues established from these transgenic plants grew to larger cell masses with more number of enlarged cells than untransformed control, demonstrating functional implication of AtS6K1 in the control of plant cell size. The observed negative correlation between the expression of AtS6K1 and the cell cycle regulatory genes, however, was completely reversed in protoplasts generated from the transgenic plants expressing AtS6K1, suggesting a possible existence of dual regulatory mechanism of the plant cell cycle regulation mediated by AtS6K1. An alternative method of kinase assay, termed "substrate-mediated kinase pull down", was employed to examine the additional phosphorylation on other domains of AtS6K1 and verified the phosphorylation of both amino- and carboxy-terminal domains, which is a novel finding regarding the phosphorylation target sites on plant S6Ks by upstream regulatory kinases. In addition, this kinase assay under the stress conditions revealed the salt- and sugar-dependencies of AtS6K1 phosphorylations.     

Evolution of the Spindlin Gene in Birds: Independent Cessation of the Recombination of Sex Chromosomes at the Spindlin Locus in Neognathous Birds and Tinamous, a Palaeognathous Avian Family

Tinamous (Aves, Palaeognathae, Tinamiformes) are primitive birds, generally considered to be the sister group to the ratites. Tinamous possess a W sex-chromosome, intermediate in heterochromatization between the largely euchromatic W chromosome of the ratites and the highly condensed W chromosome of the neognathous birds. Of the four genes which are known to have diverged copies on the neognathous avian W and Z chromosome (ATP5A1, CHD1, PKC and SPIN) only the spindlin gene has W- and Z-chromosomal forms in the tinamiformes. This paper describes experiments which show that the sequences of these forms are more similar to each other and to the homologous undifferentiated spindlin gene sequences in the ratite genome than to the W or Z forms of the spindlin gene in other, neognathous species. This suggests that cessation of recombination at the spindlin locus of the ancestral W and Z chromosomes of the paleognathous tinamiformes and the neognathous avian species were independent events.     

Structure of an XRCC1 BRCT domain: a new protein-protein interaction module.

The BRCT domain (BRCA1 C-terminus), first identified in the breast cancer suppressor protein BRCA1, is an evolutionarily conserved protein-protein interaction region of approximately 95 amino acids found in a large number of proteins involved in DNA repair, recombination and cell cycle control. Here we describe the first three-dimensional structure and fold of a BRCT domain determined by X-ray crystallography at 3.2 A resolution. The structure has been obtained from the C-terminal region of the human DNA repair protein XRCC1, and comprises a four-stranded parallel beta-sheet surrounded by three alpha-helices, which form an autonomously folded domain. The compact XRCC1 structure explains the observed sequence homology between different BRCT motifs and provides a framework for modelling other BRCT domains. Furthermore, the established structure of an XRCC1 BRCT homodimer suggests potential protein-protein interaction sites for the complementary BRCT domain in DNA ligase III, since these two domains form a stable heterodimeric complex. Based on the XRCC1 BRCT structure, we have constructed a model for the C-terminal BRCT domain of BRCA1, which frequently is mutated in familial breast and ovarian cancer. The model allows insights into the effects of such mutations on the fold of the BRCT domain.     

The major human rhinovirus receptor is ICAM-1

The major human rhinovirus receptor has been identified with monoclonal antibodies that inhibit rhinovirus infection. These monoclonal antibodies recognize a 95 kd cell surface glycoprotein on human cells and on mouse transfectants expressing a rhinovirus binding phenotype. Purified 95 kd protein binds to rhinovirus in vitro. Protein sequence from the 95 kd protein showed an identity with that of intercellular adhesion molecule-1 (ICAM-1); a cDNA clone obtained from mouse transfectants expressing the rhinovirus receptor had essentially the same sequence as ICAM-1. Thus, the major human rhinovirus receptor is ICAM-1. The gene for this receptor maps to human chromosome 19, which also contains the genes for a number of other picornavirus receptors.     

The p53 target protein Wig-1 binds hnRNP A2/B1 and RNA Helicase A via RNA

The p53-induced Wig-1 gene encodes a double stranded RNA-binding zinc finger protein. We generated Saos-2 osteosarcoma cells expressing tetracycline-inducible Flag-tagged human Wig-1. Induction of Wig-1 expression by doxycycline inhibited cell growth in a long-term assay but did not cause any changes in cell cycle distribution nor increased fraction of apoptotic cells. Using co-immunoprecipitation and mass spectrometry, we identified two Wig-1-binding proteins, hnRNP A2/B1 and RNA Helicase A, both of which are involved in RNA processing. The binding was dependent on the presence of RNA. Our results establish a link between the p53 tumor suppressor and RNA processing via hnRNPA2/B1 and RNA Helicase A.