Spindlin 1, A Novel Nuclear Protein With A Role In The Transformation Of NIH3T3 Cells

Ovarian cancer is the leading cause of death from gynecological malignancy and the fourth leading cause of cancer death among women all over the world. The overall 5 years survival rate of ovarian cancer is about 46% and has remained essentially unchanged for 25 years. 75% of epithelial ovarian cancers are diagnosed at advanced stages. This is in part due to the lack of symptoms early in the disease course and the absence of a sensitive and specific antioncogene genes and oncogenes involved in the ovarian cancer genesis. Thus isolation of new candidate genes and definition of their role in the ovary cancer genesis and development will help understand the molecular mechanisms and develop protocols for early clinical diagnosis and therapy of ovarian carcinomas.In our previous study, we used differential displayed gene isolation methods to screen ovarian cancer related genes and got a novel gene named Homo sapiens spindlinl gene (Genbank accession no. AF317228).Here we reported the characterization of human spindlinl gene. The cDNA full length of spindlinl was 4375bp which contains an open reading frame of 714 bp and predicts a protein of 237 amino acids. Data base search showed spindlinl was very high homology to mouse spindlin. A same-frame termination codons was found in the upstreamsite to the initiator condons and AATAAA was also found in the 3 ena of the cDNA. The splicing of five exons and four introns included in the genomic DNA was according to GT-AT role.Further bioinformatic analysis revealed that the human spindlin 1 gene is localized at chromosome 9q22.1-22.3, and spans a region of 52.3 kb with five exons separated by four introns.spindlinl gene was expressed in some cancer cell lines, for example OVCAr3, HepG-2, MCF-7, Hela, K562. U937, no expression in HFCL, HLF, A549. spindlinl gene was also expressed in stem cells ( CD34 and AMSC), little expression in HEK293. Morever, spindlinl was expressed in the ovary and adrenal glands of 4-month embryo tissues, but notin 8-month embryo tissues.Based on the above research, we speculated that spindlinl might belong to a specific gene family expressed during the early period of embryo development and act as a tumor embryo antigen during tumorgenesis.All functional information about the human Spindlinl gene and its homologous genes has been lacking. Identification of Spindlinl subcellular localization in mammalian cells may play an important role in the functional study of this gene. To determine the subcellular localization of Spindlinl, the eukaryon expression vectors pEGFP-N I-spindlinl was constructed and transfected into COS-7 cells. The results showed that Spindlinl was localized in the nuclear, while COS-7 cells transfected with the empty vector display cytoplasmic localization. Meanwhile, we found karyokinesis or multi-nuclear formation in transfected COS-7 cells.To further investigation the function of spindlinl gene, the eukaryon expression vector pEGFP-Nl-spindlinl and pEGFP-Nl were transfected into mouse NIH3T3 cells. After three weeks of G418 selection, the resistant clones were obtained and identified by RT- PCR. We named them 3T3-spin. The 3T3-spin cells were morphologically distinguishable from control, showing a thinner and slimmer morphology contrast to the control.The clones named 3T3-spin was examined for cells proliferation by MTT assay. The result indicated that the numbers of 3T3-spin cells was obviously increased when compared with the cells transfected with empty vector (P <0.05). And overexpression of spindlinl increased the cells percentage in S and G2/ M phase of NIH3T3 cells compared with the control transfectants (P<0.05).Since the ability to grow without anchorage to a substratum was thought to be typical of the transformed cells, we assessed the effect of spindlinl overexpression on transformation using a soft agar colony formation assay. The result showed that the cells expressing spindlinl gene had acquired the ability to grow in soft agar, and colony formation rate was (5.4 ± 2.4)%. In contrast, transfected empty vectors cells produced few anchorage independent colonies. These data demonstrated that spindlinl overexpression is sufficient to induce NIH3T3 cell transformation in vitro.To determine whether the overexpression of spindlinl could stimulate the growth of the non-tumorigenic NIH3T3 cells in nude mice, the transfected spindlinl cells were inoculated subcutaneously and tumorigenicity of the cells overexpressing spindlinl was compared with...