Endothelin-1 decreases gap junctional intercellular communication by inducing phosphorylation of connexin 43 in human ovarian carcinoma cells

Endothelin-1 (ET-1) is overexpressed in ovarian carcinoma and acts as an autocrine factor selectively through the ETA receptor (ETAR) to promote tumor cell proliferation, survival, neovascularization, and invasiveness. Loss of gap junctional intercellular communication (GJIC) is critical for tumor progression by allowing the cells to escape growth control. Exposure of HEY and OVCA 433 ovarian carcinoma cell lines to ET-1 led to a 50-75% inhibition in intercellular communication and to a decrease in the connexin 43 (Cx43)-based gap junction plaques. To investigate the phosphorylation state of Cx43, ovarian carcinoma cell lysates were immunoprecipitated and transient tyrosine phosphorylation of Cx43 was detected in ET-1-treated cells. BQ 123, a selective ETAR antagonist, blocked the ET-1-induced Cx43 phosphorylation and cellular uncoupling. Gap junction closure was prevented by tyrphostin 25 and by the selective c-Src inhibitor, PP2. Furthermore, the increased Cx43 tyrosine phosphorylation was correlated with ET-1-induced increase of c-Src activity, and PP2 suppressed the ET-1-induced Cx43 tyrosine phosphorylation, indicating that inhibition of Cx43-based GJIC is mainly mediated by the Src tyrosine kinase pathway. In vivo, the inhibition of human ovarian tumor growth in nude mice induced by the potent ETAR antagonist, ABT-627, was associated with a reduction of Cx43 phosphorylation. These findings indicate that the signaling mechanisms involved in GJIC disruption on ovarian carcinoma cells depend on ETAR activation, which leads to the Cx43 tyrosine phosphorylation mediated by c-Src, suggesting that ETAR blockade may contribute to the control of ovarian carcinoma growth and progression also by preventing the loss of GJIC.     

Establishment of murine cell lines by constitutive and conditional immortalization

Mouse cell lines were immortalized by introduction of specific immortalizing genes. Embryonic and adult animals and an embryonal stem cell line were used as a source of primary cells. The immortalizing genes were either introduced by DNA transfection or by ecotropic retrovirus transduction. Fibroblasts were obtained by expression of SV40 virus large T antigen (TAg). The properties of the resulting fibroblast cell lines were reproducible, independent of the donor mouse strains employed and the cells showed no transformed properties in vitro and did not form tumors in vivo. Endothelial cell lines were generated by Polyoma virus middle T antigen expression in primary embryonal cells. These cell lines consistently expressed relevant endothelial cell surface markers. Since the expression of the immortalizing genes was expected to strongly influence the cellular characteristics fibroblastoid cells were reversibly immortalized by using a vector that allows conditional expression of the TAg. Under inducing conditions, these cells exhibited properties that were highly similar to the properties of constitutively immortalized cells. In the absence of TAg expression, cell proliferation stops. Cell growth is resumed when TAg expression is restored. Gene expression profiling indicates that TAg influences the expression levels of more than 1000 genes that are involved in diverse cellular processes. The data show that conditionally immortalized cell lines have several advantageous properties over constitutively immortalized cells.     

Mitochondrial respiratory chain dysfunction, a non-receptor-mediated effect of synthetic PPAR-ligands: biochemical and pharmacological implications

Peroxisome proliferator activated receptors (PPARs) are a class of nuclear receptors involved in lipid and glucidic metabolism, immune regulation, and cell differentiation. Many of their biological activities have been studied by using selective synthetic activators (mainly fibrates and thiazolidinediones) which have been already employed in therapeutic protocols. Both kinds of drugs, however, showed pharmacotoxicological profiles, which cannot be ascribed by any means to receptor activation. To better understand these non-receptorial or extrareceptorial aspects, the effect of different PPAR-ligands on the metabolic status of human HL-60 cell line has been investigated. At this regard, NMR analysis of cell culture supernatants was accomplished in order to monitor modifications at the level of cell metabolism. Cell growth and chemiluminescence assays were employed to verify cell differentiation. Results showed that all the considered PPAR-ligands, although with different potencies and independently from their PPAR binding specificity, induced a significant derangement of the mitochondrial respiratory chain consisting in a strong inhibition of NADH-cytochrome c reductase activity. This derangement has been shown to be strictly correlated to the adaptive metabolic modifications, as evidenced by the increased formation of lactate and acetate, due to the stimulation of anaerobic glycolysis and fatty acid beta-oxidation. It is worthy noting that the mitochondrial dysfunction appeared also linked to the capacity of any given PPAR-ligand to induce cell differentiation. These data could afford an explanation of biochemical and toxicological aspects related to the therapeutic use of synthetic PPAR-ligands and suggest a revision of PPAR pathophysiologic mechanisms.     

Metabolic reduction of chromium,as related to its carcinogenic properties

At variance with Cr(III), Cr(VI) compounds easily cross cell membranes and exert genotoxic effects. No metabolic oxidation of Cr(III) could be detected, whereas Cr(VI) reduction was observed in the presence of body fluids and subcellular fractions of various tissues from several animal species. The differential efficiency of this process may account for the selection of target tissues in Cr(VI) carcinogenesis. For instance, reduction by saliva and gastric juice may explain a lack of carcinogenicity by the oral route; reduction inside erythrocytes may explain a lack of carcinogenicity at a distance from administration sites; reduction by the epithelial-lining fluid of terminal airways and by alveolar macrophages may be consistent with the occurrence of thresholds in lung carcinogenesis. Liver preparations displayed the top efficiency in reducing Cr(VI), whereas skeletal muscle, i.e., a typical target in experimental Cr(VI) carcinogenesis, had no detectable activity. Bronchial tree and peripheral lung parenchyma preparations from almost 100 individuals reduced Cr(VI) to a variable extent. The efficiency of lung parenchyma and of isolated alveolar macrophages was enhanced in cigarette smokers. In rats, Cr(VI) reduction by lung preparations was significantly stimulated by the repeated i.t. instillation of Cr(VI) itself. Among the electron donors (chiefly GSH) and enzymatic mechanisms responsible for the intracellular Cr(VI) reduction, such as cytochrome P-450 reductase, glutathione redactase, and aldehyde oxidase, an important role can be ascribed to cytosolic DT diaphorase activity, usually catalyzing a 2-electron reduction.     

G4 motifs affect origin positioning and efficiency in two vertebrate replicators

DNA replication ensures the accurate duplication of the genome at each cell cycle. It begins at specific sites called replication origins. Genome‐wide studies in vertebrates have recently identified a consensus G‐rich motif potentially able to form G‐quadruplexes (G4) in most replication origins. However, there is no experimental evidence to demonstrate that G4 are actually required for replication initiation. We show here, with two model origins, that G4 motifs are required for replication initiation. Two G4 motifs cooperate in one of our model origins. The other contains only one critical G4, and its orientation determines the precise position of the replication start site. Point mutations affecting the stability of this G4 in vitro also impair origin function. Finally, this G4 is not sufficient for origin activity and must cooperate with a 200‐bp cis‐regulatory element. In conclusion, our study strongly supports the predicted essential role of G4 in replication initiation.     

Serum levels of anti-heat shock protein 27 antibodies in patients with chronic liver disease

Heat shock protein 27 (HSP27), an intracellular molecular chaperone, is involved in the pathogenesis of cancer by promoting both tumor cell proliferation and resistance to therapy. HSP27 is also present in the circulation and circulating HSP27 (sHSP27) can elicit an autoimmune response with production of antibodies. Levels of sHSP27 are enhanced in patients with hepatocellular carcinoma (HCC); it is, however, unknown whether changes in HSP27 antibody levels occur in patients with HCC and can be exploited as a circulating biomarker of HCC. Our aim was to assess the potential association between newly diagnosed HCC and serum anti-HSP27 antibody levels. In this cross-sectional study, anti-HSP27 antibody levels were measured in serum samples from 71 HCC patients, 80 subjects with chronic liver disease, and 38 control subjects by immunoenzymatic assay. Anti-HSP27 antibody levels did not differ significantly among groups. However, in patients with chronic active hepatitis/cirrhosis, anti-HSP27 levels were significantly higher in subjects with a positive history of alcoholism (p = 0.03). Our data do not support the hypothesis that anti-HSP27 antibody levels may help identify patients with HCC among subjects with chronic liver disease. However, our finding that alcohol-related liver disease is associated with higher anti-HSP27 levels is novel and deserves further investigations.     

Modeling human osteosarcoma in mice through 3AB-OS cancer stem cell xenografts

Osteosarcoma is the second leading cause of cancer‐related death for children and young adults. In this study, we have subcutaneously injected—with and without matrigel—athymic mice (Fox1nu/nu) with human osteosarcoma 3AB‐OS pluripotent cancer stem cells (CSCs), which we previously isolated from human osteosarcoma MG63 cells. Engrafted 3AB‐OS cells were highly tumorigenic and matrigel greatly accelerated both tumor engraftment and growth rate. 3AB‐OS CSC xenografts lacked crucial regulators of beta‐catenin levels (E‐cadherin, APC, and GSK‐3beta), and crucial factors to restrain proliferation, resulting therefore in a strong proliferation potential. During the first weeks of engraftment 3AB‐OS‐derived tumors expressed high levels of pAKT, beta1‐integrin and pFAK, nuclear beta‐catenin, c‐Myc, cyclin D2, along with high levels of hyperphosphorylated‐inactive pRb and anti‐apoptotic proteins such as Bcl‐2 and XIAP, and matrigel increased the expression of proliferative markers. Thereafter 3AB‐OS tumor xenografts obtained with matrigel co‐injection showed decreased proliferative potential and AKT levels, and undetectable hyperphosphorylated pRb, whereas beta1‐integrin and pFAK levels still increased. Engrafted tumor cells also showed multilineage commitment with matrigel particularly favoring the mesenchymal lineage. Concomitantly, many blood vessels and muscle fibers appeared in the tumor mass. Our findings suggest that matrigel might regulate 3AB‐OS cell behavior providing adequate cues for transducing proliferation and differentiation signals triggered by pAKT, beta1‐integrin, and pFAK and addressed by pRb protein. Our results provide for the first time a mouse model that recapitulates in vivo crucial features of human osteosarcoma CSCs that could be used to test and predict the efficacy in vivo of novel therapeutic treatments. J. Cell. Biochem. 113: 3380–3392, 2012. © 2012 Wiley Periodicals, Inc.