S100A6 (calcyclin) enhances the sensitivity to apoptosis via the upregulation of caspase-3 activity in Hep3B cells.

S100A6 (calcyclin) is a small calcium-binding protein which has been implicated in several cellular processes such as cell cycle progression, cytoskeleton rearrangement, and exocytosis. Also the upregulation of S100A6 has been reported in a variety of tumors and linked to metastasis. However, exact intracellular roles of S100A6 related with apoptosis have not been clarified yet. Here we demonstrated that the upregulation of S100A6 enhances the cell death rate compared to the control under the apoptotic conditions. In exogenously S100A6 induced Hep3B cells, cell viability was significantly decreased compared with mock and S100A6-knockdown cells under calcium ionophore A23187 treatment. The exogenously introduced S100A6 significantly affected the caspase-3-like activity in programmed cell death through the enhanced caspase-3 expression, which was verified by promoter assay in wild or mutant S100A6-transfected Hep3B cells. Next, the promoter activity of caspase-3 was increased by 2.5-folds in wild-type S100A6-transfected cells compared to mutant 2 (E67K, mutant of EF-hand motif) or control. Our results suggest that S100A6 might be involved in the processing of apoptosis by modulating the transcriptional regulation of caspase-3.

Enhancement of cell survival by stromal cell-derived factor-1/CXCL12 involves activation of CREB and induction of Mcl-1 and c-Fos in factor-dependent human cell line MO7e.

Stromal cell-derived factor-1 (SDF-1/CXCL12) enhances the survival of hematopoietic stem and progenitor cells in synergy with other cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), steel factor, and thrombopoietin (TPO), and both the PI3K/Akt and MAPK pathways have been linked to this survival. To further evaluate intracellular signaling involved in SDF-1/CXCL12 survival effects, we investigated modulation of downstream signaling molecules. The synergistic survival enhancement of SDF-1/CXCL12 plus other cytokines were directly linked to enhanced phosphorylation of p70/85S6K and cAMP responsive element binding protein (CREB), as well as enhanced induction of the Bcl-2 family member Mcl-1. Most prominently, c-Fos, a component of AP1 transcription factor, was synergistically induced by SDF-1/CXCL12 plus other cytokines. These results suggest that SDF-1/CXCL12 enhanced cell survival in synergy with other cytokines involves activation of CREB and induction of Mcl-1 and c-Fos.

The Bmi-1 oncoprotein is overexpressed in human colorectal cancer and correlates with the reduced p16INK4a/p14ARF proteins.

To clarify the roles of Bmi-1 in colorectal carcinoma, we examined the expression of Bmi-1 in 41 samples out of 46 colorectal carcinomas by reverse transcription-PCR, whereas all 46 were analyzed by immunostaining. In addition, we analyzed the expression patterns of Bmi-1 in association with p16INK4a and p14ARF (in mouse p19ARF) in a series of colorectal carcinomas. The level of Bmi-1 mRNA in the carcinoma tissues was significantly higher than those of the adjacent non-neoplastic colonic mucosal tissues. Immunohistochemistry for Bmi-1 showed moderate or strong expression levels in 65% (30/46) of colorectal carcinomas. Colorectal carcinomas with moderate or strong Bmi-1 expression were more likely to have low levels of the INK4 locus proteins (p16INK4a/p14ARF) (P<0.07). These results suggested that modulation of Bmi-1 protein might be involved in human colorectal carcinogenesis by repressing the INK4a/ARF proteins.

Synergistic activation of p70S6 kinase associated with stem cell factor in MO7e cells.

Stem cell factor (SCF) is an early-acting cytokine inducing proliferative synergy with other cytokines in hematopoietic cells. We earlier showed that p21 was synergistically induced in SCF synergy and the p44/42 MAPK pathway was essential for the transcriptional control of p21. SCF synergy accompanies protein synthesis. p70S6K implicated in translational control in many other systems has not been shown in SCF synergy induced system. GM-CSF dependent human cell line MO7e was stimulated with GM-CSF with SCF, and investigated activation of p70S6K by using phospho-specific antibody. A possible contribution of p70S6K to SCF synergy was examined by measuring p21 induction as a model system. p70S6K was slightly activated by GM-CSF alone and markedly activated by SCF alone. Combined stimulation with these two cytokines synergistically activated p70S6K resulting in persistent activation. Addition of the pathway-specific inhibitors for P13K or FRAP/TOR, two up-stream pathways of p70S6K resulted in abolishment of p70S6K phosphorylation and also significant reduction of p21 protein level. These data suggest that synergistically activated p70S6K by GM-CSF plus SCF involves, at least in part, protein translational control including regulation of p21 protein.

HPV E6 antisense induces apoptosis in CaSki cells via suppression of E6 splicing.

Cervical cancer is known to be highly associated with viral oncogene E6 and E7 of human papilloma virus. Down-regulation of oncogene expression by antisense-based gene therapy has been extensively studied. To investigate the effect of HPV 16 E6 antisense nucleic acid (AS) on cervical cancer cells, human cervical cancer cell lines, CaSki and SiHa cells harboring HPV 16 genome were transfected with plasmid containing E6(AS). The decreased viability and the apoptotic morphology were observed in E6(AS)-transfected cervical cancer cell lines. By 6 h after transfection, inhibition of E6 splicing, rapid upregulations of p53 and a p53-responsive protein, GADD45, were displayed in E6(AS)-transfected CaSki cells. Furthermore, E6(AS) induced loss of mitochondrial transmembrane potential, release of mitochondrial cytochrome c into the cytoplasm, and subsequent activation of caspase-9 and caspase-3. These results indicate that HPV 16 E6(AS) induces apoptosis in CaSki cells via upregulation of p53 and release of cytochrome c into cytoplasm, consequently activating procaspase-9 and procaspase-3.

Overexpression of Bmi-1 oncoprotein correlates with axillary lymph node metastases in invasive ductal breast cancer.

The modulation of Bmi-1 is observed in several tumor tissues, and its heightened protein level is suspected to be involved in tumorigenesis by acting as a transcriptional repressor in the INK4a/ARF locus. To elucidate the modulation of Bmi-1 in invasive ductal breast cancers, we examined its transcript and protein levels. The bmi-1 mRNA level by reverse transcription-polymerase chain reaction (RT-PCR) showed that it was significantly up-regulated in 28 specimens out of 33 breast carcinoma tissues compared with those of non-neoplastic tissues just adjusted to tested specimens. Immunohistochemical staining for Bmi-1 also showed that 44 specimens out of 71 breast carcinoma tissues (62%) had strong positive signals with a more intense staining pattern in the invading fronts than in the central portions of primary invasive breast cancers. Univariate and multivariate analyses showed that a high level of Bmi-1 expression was significantly correlated with axillary lymph node metastases and positive estrogen receptor status. These findings suggested that Bmi-1 might be involved in the tumor progression and metastasis of invasive ductal breast cancer.

Overexpression and clinical significance of carcinoembryonic antigen-related cell adhesion molecule 6 in colorectal cancer.

BACKGROUND: Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) inhibits anoikis and affects the malignant phenotype of cancer cells. In this study, we analyzed CEACAM6 as a gene that is highly upregulated in colon cancer tissues, and examined the assertion that CEACAM6 might be a suitable candidate tumor marker for the diagnosis of colon cancer. METHODS: CEACAM6 gene expression in human colon tissues was performed by tissue microarray and analyzed using RT-PCR (each of normal and tumor tissue, n=40) and immunohistochemical and clinicopathological (colon cancer patients, n=143) analyses. RESULTS: CEACAM6 transcriptional and translational levels were significantly upregulated in human tumor tissues compared to non-tumor regions, and clinicopathological analysis revealed a significant correlation between CEACAM6 protein expression and Dukes' stage (p<0.001). High expression levels of CEACAM6 were significantly associated with lower overall survival (p<0.001) and shorter recurrence-free survival (p<0.001). We demonstrated that knockdown of CEACAM6 with CEACAM6-specific small interfering RNA in colorectal cancer cells attenuated invasivity (35%); conversely, the overexpression of CEACAM6 increased invasiveness. CONCLUSIONS: CEACAM6 is significantly upregulated in colon cancer tissues and is closely associated with poor prognosis, indicating that CEACAM6 might be used as a tumor biomarker and a potential therapeutic target for colon cancer.

Over-expression of human UREB1 in colorectal cancer: HECT domain of human UREB1 inhibits the activity of tumor suppressor p53 protein.

Many fundamental processes, including oncogenesis, have implicated HECT domain proteins with ubiquitin ligase activity. The protein human upstream regulatory element binding protein 1 (hUREB1) is a HECT domain protein whose function is not defined yet. Here, we investigate the function of hUREB1 as a ubiquitin-protein ligase in human colorectal cells. Ectopic expression of the HECT domain of hUREB1 reduces the protein level and transcriptional activity of the p53 tumor suppressor, which is abrogated by the deletion in the HECT domain or point mutations in the essential residues of the HECT domain. The ubiquitination and destabilization of p53 is observed in cells treated with the protease inhibitor MG132, implying that the HECT domain of hUREB1 suppresses the transcriptional activity of p53 through a ubiquitin-dependent degradation pathway. Based on the results of Northern blot analysis, RT-PCR, and immunohistochemical analyses, the over-expression of hUREB1 is associated with colorectal carcinoma. Moreover, protein levels of hUREB1 and p53 were inversely correlated. These findings suggest that hUREB1 can function, at least in part, as a negative regulator of p53 during the colorectal carcinoma progression through the ubiquitination pathway mediated by the HECT domain.

Involvement of NF-kappaB in the regulation of S100A6 gene expression in human hepatoblastoma cell line HepG2.

S100A6 (calcyclin) is an acidic calcium binding protein with two EF-hand motifs and overexpressed in several tumors including intrahepatic carcinoma. TNFalpha, a strong NF-kappaB activator required for hepatocyte proliferation during liver regeneration, triggered the expression of S100A6 mRNA in human hepatoblastoma cell line HepG2. Transient expression of NF-kappaB (p65) increased S100A6 promoter activity and expression of inhibitor of NF-kappaB (IkappaBalpha) decreased TNFalpha-induced S100A6 promoter activity. To confirm the involvement of NF-kappaB in S100A6 promoter activation, we analyzed serially deleted promoter constructs of the S100A6 gene by luciferase reporter assay and found a NF-kappaB-responsive DNA fragment at the position between -584 and -361. Electrophoretic mobility shift assays showed that TNFalpha induced p65 binding to a potential NF-kappaB binding site at -460/-451. Furthermore, treatment of cells with CAPE (caffeic acid phenethyl ester), a specific NF-kappaB (p65) inhibitor, decreased NF-kappaB binding and promoter activity. These results suggest that NF-kappaB transcription factor contributes to the activation of S100A6 gene expression in response to TNFalpha in HepG2 cells.