Cytoplasmic TSC-22 (transforming growth factor-beta-stimulated clone-22) markedly enhances the radiation sensitivity of salivary gland cancer cells

We transfected a salivary gland cancer cell line, TYS, with three different forms of TSC-22 (transforming growth factor-beta-stimulated clone-22) gene: full-length TSC-22 (TSC-22FL) containing nuclear export signal, TSC-box and leucine zipper, truncated TSC-22 (TSC-22LZ) containing only TSC-box and leucine zipper, and truncated TSC-22 with nuclear localization signal (NLS-TSC-22LZ). High expression of TSC-22FL in the cytoplasm markedly enhanced the radiation-sensitivity of TYS cells, while, moderate expression of TSC-22FL marginally affected the radiation-sensitivity. TSC-22LZ, which was expressed in the cytoplasm and the nucleus, enhanced the radiation-sensitivity of TYS cells irrespective to its expression level. NLS-TSC-22LZ, which was expressed only in the nucleus, marginally affected the radiation-sensitivity of the cells even at high expression level. Interestingly, cytoplasmic TSC-22 translocates to nucleus concomitant with radiation-induced apoptosis. These results suggest that cytoplasmic localization of TSC-22 and translocation of TSC-22 from cytoplasm to nucleus is important for regulating the cell death signal after irradiation-induced DNA damage.     

Role of TSC-22 during early embryogenesis in Xenopus laevis

Transforming growth factor-beta1-stimulated clone 22 (TSC-22) encodes a leucine zipper-containing protein that is highly conserved. During mouse embryogenesis, TSC-22 is expressed at the site of epithelial-mesenchymal interaction. Here, we isolated Xenopus laevis TSC-22 (XTSC-22) and analyzed its function in early development. XTSC-22 mRNA was first detected in the ectoderm of late blastulae. Translational knockdown using XTSC-22 antisense morpholino oligonucleotides (XTSC-22-MO) caused a severe delay in blastopore closure in gastrulating embryos. This was not due to mesoderm induction or convergent-extension, as confirmed by whole-mount in situ hybridization and animal cap assay. Cell lineage tracing revealed that migration of ectoderm cells toward blastopore was disrupted in XTSC-22-depleted embryos, and these embryos had a marked increase in the number of dividing cells. In contrast, cell division was suppressed in XTSC-22 mRNA-injected embryos. Co-injection of XTSC-22-MO and mRNA encoding p27Xic1, which inhibits cell cycle promotion by binding cyclin/Cdk complexes, reversed aberrant cell division. This was accompanied by rescue of the delay in blastopore closure and cell migration. These results indicate that XTSC-22 is required for cell movement during gastrulation though cell cycle regulation.     

Crucial Role of TSC-22 in Preventing the Proteasomal Degradation of p53 in Cervical Cancer

The p53 tumor suppressor function can be compromised in many tumors by the cellular antagonist HDM2 and human papillomavirus oncogene E6 that induce p53 degradation. Restoration of p53 activity has strong therapeutic potential. Here, we identified TSC-22 as a novel p53-interacting protein and show its novel function as a positive regulator of p53. We found that TSC-22 level was significantly down-regulated in cervical cancer tissues. Moreover, over-expression of TSC-22 was sufficient to inhibit cell proliferation, promote cellular apoptosis in cervical cancer cells and suppress growth of xenograft tumors in mice. Expression of also TSC-22 enhanced the protein level of p53 by protecting it from poly-ubiquitination. When bound to the motif between amino acids 100 and 200 of p53, TSC-22 inhibited the HDM2- and E6-mediated p53 poly-ubiquitination and degradation. Consequently, ectopic over-expression of TSC-22 activated the function of p53, followed by increased expression of p21(Waf1/Cip1) and PUMA in human cervical cancer cell lines. Interestingly, TSC-22 did not affect the interaction between p53 and HDM2. Knock-down of TSC-22 by small interfering RNA clearly enhanced the poly-ubiquitination of p53, leading to the degradation of p53. These results suggest that TSC-22 acts as a tumor suppressor by safeguarding p53 from poly-ubiquitination mediated-degradation.     

Babesia gibsoni: Identification, expression, localization, and serological characterization of a Babesia gibsoni 22-kDa protein

Babesia gibsoni causes canine babesiosis. Here, we describe the identification and characterization of a novel gene, BgP22, containing an open reading frame of 621 bp and encoding a 22-kDa protein from B. gibsoni, as a serodiagnostic candidate. The recombinant BgP22 (rBgP22) was expressed and used as an antigen to produce anti-rBgP22 sera in mice. Using these anti-rBgP22 sera, a native 22-kDa protein was recognized by Western blot analysis and observed in the membrane of the parasites by immunofluorescent antibody tests (IFAT). The enzyme-linked immunosorbent assay (ELISA) using the rBgP22 detected specific antibodies to this protein in the sera of dogs experimentally and naturally infected with B. gibsoni in chronic stage. Furthermore, it did not show a cross reaction with the closely related apicomplexan parasites, indicating that the rBgP22 could be used as a diagnostic antigen for a detection of the chronic carrier stages of B. gibsoni infection.     

Characterization of fortilin, a novel antiapoptotic protein

Apoptosis is meticulously controlled in living organisms. Its dysregulation has been shown to play a key role in a number of human diseases, including neoplastic, cardiovascular, and degenerative disorders. Bcl-2 family member proteins and inhibitors of apoptosis proteins are two major negative regulators of apoptosis. We report here the characterization of novel antiapoptotic protein, fortilin, which we identified through yeast two-hybrid library screening. Sequence analysis of fortilin revealed it to be a 172-amino acid polypeptide highly conserved from mammals to plants. Fortilin is structurally unrelated to either Bcl-2 family member proteins or inhibitors of apoptosis proteins. Northern blot analysis showed the fortilin message to be ubiquitous in normal tissue but especially abundant in the liver, kidney, and small intestine. Western blot analysis using anti-fortilin antibody showed more extensive expression in cancerous cell lines (H1299, MCF-7, and A549) than in cell lines derived from normal tissue (HEK293). Immunocytochemistry using HeLa cells transiently expressing FLAG-tagged fortilin and immunohistochemistry using human breast ductal carcinoma tissue and anti-fortilin antibody both showed that fortilin is predominantly localized in the nucleus. Functionally, the transient overexpression of fortilin in HeLa cells prevented them, in a dose-dependent fashion, from undergoing etoposide-induced apoptosis. Consistently, U2OS cells stably expressing fortilin protected the cells from cell death induced by etoposide over various concentrations and durations of exposure. In addition, fortilin overexpression inhibited caspase-3-like activity as assessed by the cleavage of fluorogenic substrate benzyloxycarbonyl-DEVD-7-amido-4-(trifluoromethyl)coumarin. Furthermore, the antisense depletion of fortilin from breast cancer cell line MCF-7 was associated with massive cell death. These data suggest that fortilin represents a novel antiapoptotic protein involved in cell survival and apoptosis regulation.     

EBNA1 may prolong G(2)/M phase and sensitize HER2/neu-overexpressing ovarian cancer cells to both topoisomerase II-targeting and paclitaxel drugs

We have shown previously that the Epstein-Barr virus nuclear antigen-1 (EBNA1) can act as a transforming suppressor in the HER2/neu-overexpressing ovarian cancer cells. In the present study, by using flow cytometric analysis, we demonstrate that EBNA1 could prolong G(2)/M phase and sensitize to Taxol-induced apoptosis in the EBNA1-expressing ovarian cancer cell stable transfectants. In addition, EBNA1 could also significantly increase topoisomerase IIalpha protein expression, indicating that the up-regulation of topoisomerase IIalpha may be one of the mechanisms by which EBNA1 enhances the sensitivity of ovarian cancer cells to topoisomerase II-targeting anticancer drugs, such as VP-16 and Adriamycin. These data suggest that EBNA1 not only prolongs cell cycle at G(2)/M phase and up-regulates topoisomerase IIalpha expression in HER2/neu-overexpressing ovarian cancer cells, but also increases cellular apoptosis through sensitization of cancer cells to topoisomerase II-directing anticancer drugs.     

Akt2 kinase suppresses glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-mediated apoptosis in ovarian cancer cells via phosphorylating GAPDH at threonine 237 and decreasing its nuclear translocation

Protein kinase B (Akt) plays important roles in regulation of cell growth and survival, but while many aspects of its mechanism of action are known, there are potentially additional regulatory events that remain to be discovered. Here we detected a 36-kDa protein that was co-immunoprecipitated with protein kinase Bβ (Akt2) in OVCAR-3 ovarian cancer cells. The protein was identified to be glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by MALDI-TOF/TOF MS, and the interaction of Akt2 and GAPDH was verified by reverse immunoprecipitation. Our further study showed that Akt2 may suppress GAPDH-mediated apoptosis in ovarian cancer cells. Overexpression of GAPDH increased ovarian cancer cell apoptosis induced by H(2)O(2), which was inhibited by Akt2 overexpression and restored by the PI3K/Akt inhibitor wortmannin or Akt2 siRNA. Akt2 phosphorylated Thr-237 of GAPDH and decreased its nuclear translocation, an essential step for GAPDH-mediated apoptosis. The interaction between Akt2 and GAPDH may be important in ovarian cancer as immunohistochemical analysis of 10 normal and 30 cancerous ovarian tissues revealed that decreased nuclear expression of GAPDH correlated with activation (phosphorylation) of Akt2. In conclusion, our study suggests that activated Akt2 may increase ovarian cancer cell survival via inhibition of GAPDH-induced apoptosis. This effect of Akt2 is partly mediated by its phosphorylation of GAPDH at Thr-237, which results in the inhibition of GAPDH nuclear translocation.     

Arsenic trioxide induces gallbladder carcinoma cell apoptosis via downregulation of Bcl-2

Gallbladder carcinoma (GBC), an aggressive and mostly lethal malignancy, is known to be resistant to a number of apoptotic stimuli. Here, we report for the first time the pro-apoptosis role of arsenic trioxide (As2O3) in gallbladder carcinoma and identify the contribution of Bcl-2 in the As2O3-induced apoptosis. The treatment of As2O3 in gallbladder carcinoma cells could induce apoptosis in a dose-dependent manner and downregulate the expression of anti-apoptotic protein Bcl-2 at mRNA level. Moreover, Bcl-2 overexpression could protect gallbladder carcinoma cells from As2O3-induced apoptosis, indicating the contribution of Bcl-2 in As2O3-induced apoptosis. Taken together, these results suggest that arsenic trioxide induces gallbladder carcinoma cell apoptosis via downregulation of Bcl-2, which may have important therapeutic implications in gallbladder carcinoma patients.