L. Testolin

Acidosis Inhibits Endothelial Cell Apoptosis and Function and Induces Basic Fibroblast Growth Factor and Vascular Endothelial Growth Factor Expression

Endothelial cells are exposed to an acidotic environment in a variety of pathological and physiological conditions. However, the effect of acidosis on endothelial cell function is still largely unknown, and it was evaluated in the present study. Bovine aortic endothelial cells (BAECs) were grown in bicarbonate buffer equilibrated either with 20% CO(2) (pH 7.0, acidosis) or 5% CO(2) (pH 7.4, control). Acidosis inhibited BAEC proliferation in 10% FCS, whereas by day 7 in serum-free medium, cell number was 3-fold higher in acidotic cells than in control cells. Serum deprivation enhanced BAEC apoptosis, and apoptotic cell death was markedly inhibited by acidosis. Additionally, acidosis inhibited FCS-stimulated migration in a modified Boyden chamber assay and FCS-stimulated differentiation into capillary-like structures on reconstituted basement membrane proteins. Conditioned media from BAECs cultured for 48 hours either at pH 7.0 or pH 7.4 enhanced BAEC proliferation and migration at pH 7.4, and both effects were more marked with conditioned medium from BAECs grown in acidotic than in control conditions. Acidosis enhanced vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) mRNA expression as well as bFGF secretion, and a blocking bFGF antibody inhibited enhanced BAEC migration in response to conditioned medium from acidotic cells. These results show that acidosis protects endothelial cells from apoptosis and inhibits their proangiogenic behavior despite enhanced VEGF and bFGF mRNA expression and bFGF secretion.

Positive Selection of Apoptosis-resistant Cells Correlates with Activation of Dominant-Negative STAT5

The STAT5 activation has important roles in cell differentiation, cell cycle control, and development. However, the potential implications of STAT5 in the control of apoptosis remain unexplored. To evaluate any possible link between the erythropoietin receptor (EpoR) JAK2/STAT5 transduction pathway and apoptosis, we have investigated apoptosis-resistant cells (ApoR) that arose from positive selection of the erythroid-committed Ba/F3EpoR cells triggered to apoptosis by ectopic expression of the HOX-B8 homeotic gene. We show that JAK2 is normally activated by Epo in both Ba/F3EpoR and ApoR cells. In contrast, both STAT5a and STAT5b isoforms are uniquely activated in a C-truncated form (86 kDa) only in ApoR cells. Analysis of ApoR and Ba/F3EpoR subclones confirmed that the switch to the truncated STAT5 isoform coincides with apoptosis survival and that ApoR do not derive from preexisting cells with a shortened STAT5. In addition, ApoR cells die in the absence of Epo. This indicates that resistance to apoptosis is not because of a general defect in the apoptotic pathway of ApoR cells. Furthermore, we show that the 86-kDa STAT5 protein presents a dominant-negative (DN) character. We hypothesize that the switch to a DN STAT5 may be part of a mechanism that allows ApoR cells to be selectively advantaged during apoptosis. In conclusion, we provide evidence for a functional correlation between a naturally occurring DN STAT5 and a biological response.

Cross-linked trimers of bovine ribonuclease A: activity on double-stranded RNA and antitumor action

Trimers of bovine pancreatic RNase A were obtained by cross‐linking native RNase A with dimethyl suberimidate. They degrade double‐stranded RNA more efficiently than dimers and monomers of RNase A, and display significant cytotoxic and/or cytostatic actions against C4‐I cells (a human cell line derived from squamous carcinoma of the uterus cervix). On the same cell line cross‐linked dimers of RNase A appear to be ineffective.

The exposure of carcinogen-initiated primary neonatal rat hepatocytes to tumor promoters modulates both the transcripts and the enzymatic activity of nuclear poly(ADP-ribose) polymerase

Four tumor promoters, i.e. PB, TPA, NAF, and DDT, added singly to a calcium-deprived synthetic medium, elicited early and late mitogenic effects and concurrent surges of nuclear poly(ADP-ribose) polymerase (pADPRP) activity in primary neonatal rat hepatocytes mutagenized with an intra-uterine dose of DMN. These actions were fully abated by the pADPRP inhibitor 3-MBA. Conversely, EGF only acted as a full mitogen when mediums calcium was at physiological levels, and its effects could not be blocked by 3-MBA. The same tumor promoters, but not EGF, also evoked a swift and lingering amplification of pADPRP transcripts in DMN-initiated hepatocytes kept in low-calcium medium. Hence, a coordinated modulation of both pADPRP transcripts and activity by xenobiotics is likely to be involved in the clonal expansion of early preneoplastic hepatocytes.

Effects of neoplastic transformation and teniposide (VM26) on protein kinase C isoform expression in rodent fibroblasts

This study examined changes in protein kinase C (PKC) isoforms in rodent fibroblasts (rat F111 and mouse NIH3T3), transformed by the polyoma virus middle T antigen (mT) and undergoing apoptosis in response to teniposide (VM26). The mT-transformed cells up-regulated PKC delta and down-regulated both PKC epsilon and PKC lambda expression, and were more sensitive to the drug than their non-transformed counterparts. The drug treatment further lowered the expression of PKC epsilon, triggered nuclear translocation of PKC delta and its site-specific proteolysis, consistent with the notion that changes in specific PKC isoforms play a role not only in the neoplastic transformation of fibroblasts, but also in their apoptotic response.

Morphology, growth, and gene expression in five newly isolated murine hepatocellular tumor cell lines

Five murine hepatocellular tumor cell lines (HepM-1-5) were isolated and grown in a synthetic medium added with hormones, growth factors and/or serum. The morphology of these lines ranged from a nearly homogeneous epithelial-like shape (HepM-2) to a stromal appearance (HepM-1). The remaining lines displayed a mixed morphology. For their proliferation all of the cell lines retained a clear dependence on the extracellular calcium level and hormonal and/or serum growth factors and, rather homogeneously, they did not express the albumin, alpha-fetoprotein (with the exception of HepM-2 cells), tyrosine aminotransferase, and ornithine transcarbamylase genes, whereas they all exhibited discrete levels of the ornithine aminotransferase mRNA. Only HepM-3 and HepM-5 lines expressed the procollagen type I gene.

Tumor Promoters, But Not EGF, Increase Nuclear Poly(ADP-Ribose) Polymerase Gene Expression in Rat Hepatocytes Initiated in Utero with DMN and Cultured After Birth in Low-Calcium Synthetic Medium

Mixed, in vivo-in vitro,models of liver chemical carcinogenesis have been used only rarely [1,2]. Recently, we devised to utilize a new one of such mixed systems to clarify the role(s) played in the unfolding of liver malignancies by nuclear poly(ADP-ribosyl)ating reactions. Such metabolic events posttranslationally modify manifold nuclear protein species, including the enzyme itself, i.e. poly(ADP-ribose) polymerase (pADPRP), by which they are catalyzed [3]. Nuclear poly(ADP-ribosyl)ations are involved not only in DNA repair, but even in the modulation of gene expression related to normal and abnormal cell proliferation, as the occurrence of malignancy is prevented by established inhibitors of pADPRP [4,5]. An increased activity of nuclear pADPRP is indispensable for the multiplication of fully transformed hepatocytes [6,7]. Likewise, both partial hepatectomy and the administration of lead nitrate increase the activity and genetic expression of nuclear pADPRP in rat hepatocytes in vivo [8]. Previously, we showed that the activation of nuclear pADPRP played a pivotal role in the enactment of the mitogenic effects elicited by several tumor promoters administered to bona fide normal, primary neonatal rat hepatocytes [9,10]. In this communication we relate that the in utero initiation with a transplacental genotoxic carcinogen like dimethylnitrosamine (DMN) [11,12] and the in vitro postnatal promotion of the offspring’s initiated hepatocytes with divers xenobiotics [9,10] do increase the expression of the nuclear pADPRP gene.