Analysis of the anti-apoptotic v-Bcl2 and v-Flip genes and effect on in vitro programmed cell death of Argentinean isolates of bovine gammaherpesvirus 4 (BoHV-4).

Some viruses encode inhibitory factors of apoptosis during infection to prolong cell viability and then to achieve a higher production of viral progeny or facilitate persistent infections. There is evidence that some gammaherpesviruses, including BoHV-4, carry genes that can both inhibit or induce apoptosis. BoHV-4 possesses two genes (ORF16 and ORF71) that code for proteins with anti-apoptotic functions, such as v-Bcl2 and v-Flip, respectively. Thus, it is relevant to study BoHV-4 in relation to the modulation of apoptosis in infected cells as a strategy for persistence in the host. The objective of this work was to analyze whether variations in v-Flip and v- Bcl2 of six phylogenetically divergent Argentinean isolates of BoHV-4 can influence the capacity of these strains to induce apoptosis in cell cultures. In this study, variations were mainly detected in the v-Flip gene and protein of the BoHV-4 strains belonging to genotype 3. Thus, it is possible to infer that sequence variations could be associated with some BoHV-4 genotype. Induction of apoptosis was not a significant event for any of the genetically distinct local isolates of BoHV-4 and there was not an evident relationship between the variability of both genes with the apoptotic effect of the phylogenetically distinct strains.

Estradiol Upregulates c-FLIPlong Expression in Anterior Pituitary Cells.

Anterior pituitary cell turnover depends on a tight balance between proliferation and apoptosis. We have previously shown that estrogens sensitize anterior pituitary cells to pro-apoptotic stimuli. c-FLIP (cellular-FLICE-inhibitory-protein) isoforms are regulatory proteins of apoptosis triggered by death receptors. c-FLIPshort isoform competes with procaspase-8 inhibiting its activation. However, c-FLIPlong isoform may have a pro- or anti-apoptotic function depending on its expression level. In the present study, we explored whether estrogens modulate c-FLIP expression in anterior pituitary cells from ovariectomized (OVX) rats and in GH3 cells, a somatolactotrope cell line. Acute administration of 17ß-estradiol to OVX rats increased c-FLIPlong expression in the anterior pituitary gland without changing c-FLIPshort expression as assessed by Western blot. Estradiol in vitro also increased c-FLIPlong expression in anterior pituitary cells but not in GH3 cells. As determined by flow cytometry, the percentage of anterior pituitary cells expressing c-FLIP was higher than in GH3 cells. However, c-FLIP fluorescence intensity in GH3 cells was higher than in anterior pituitary cells. FasL increased the percentage of TUNEL-positive GH3 cells incubated either with or without estradiol suggesting that the pro-apoptotic action of Fas activation is estrogen-independent. Our results show that unlike what happens in nontumoral pituitary cells, estrogens do not modulate either c-FLIPlong expression or FasL-induced apoptosis in GH3 cells. The stimulatory effect of estradiol on c-FLIPlong expression could be involved in the sensitizing effect of this steroid to apoptosis in anterior pituitary cells. The absence of this estrogenic action in tumor pituitary cells could be involved in their tumor-like behavior.

Changes in cFLIP expression in the corpus luteum throughout the estrous cycle of Shibagoats.

Changes in the expression of the anti-apoptotic protein cFLIP (cellular FLICE inhibitory protein) were examined in the caprine corpus luteum (CL), during development and subsequent maintenance. Corpora lutea at four different stages were collected from Shiba goats, to measure the expression of cFLIP mRNA, protein and immunolocalization. Expression of short form cFLIP (cFLIPS) mRNA was highest at the early CL stage, and decreased during late and regressed stages (P < 0.05). In contrast, long form cFLIP (cFLIPL) mRNA expression was high during early, mid and late stages, and only decreased at the regressed stage (P< 0.01). Protein expression of cFLIPS was highest at the late CL stage, and decreased at the regressed stage (P < 0.01). Protein expression of cFLIPL was highest at the early and mid CL stages, and decreased by the late and regressed stages (P < 0.01). Further expression of cFLIPL was higher at the early CL stage than at the mid stage (P < 0.01). cFLIP protein expression was detectable by immunostaining in the early, mid and late CL stages, but not at the regressed CL stage. These results are consistent with the hypothesis that cFLIP acts as a survival factor in the maintenance of CL function in goats.

Expression and localization of cFLIP anti-apoptotic protein in the porcine corpus luteum and corpora albicans during the estrous cycle and pregnancy.

We determined expression and localization of the anti-apoptotic cellular FLICE inhibitory protein (cFLIP) in the porcine corpora lutea (CL) and corpus albicans (CA) during estrous and pregnancy. The CL and CA were collected at different stages of estrous to determine cFLIP immunolocalization, and mRNA and protein expression. The mRNA expression of the short cFLIP isoform (cFLIPS) was higher at the early and mid CL stages, and lower by the late CL stage (P < 0.01); mRNA expression of the long cFLIP isoform (cFLIPL) was higher at the mid CL stage, and lower at the early and late CL stages (P < 0.01). Levels of cFLIPS and cFLIPL were steady and high during the early and mid CL stages, and had significantly decreased (P < 0.01) by the late stage. The cFLIP protein was highly expressed in the early and mid CL stages of estrous, but weakly ex-pressed in the late stage. Expression of cFLIPS showed no significant difference between preovulatory corpus albicans (CA1) and corpus albicans (CA2) coexistent with the CL from the previous estrus, but cFLIPL mRNA expression was higher during CA1 than CA2. The expression of cFLIPS showed no significant difference between CA1 and CA2, but cFLIPL was not detected. The cFLIP protein was weak-ly expressed in the CA. Expression of cFLIPS and cFLIPL mRNA and proteins was observed in the CL, and the cFLIP protein was highly expressed during pregnancy. We propose that cFLIPS/L acts as a survival factor, and performs an anti-apoptotic function in the porcine CL.

c-FLIP is involved in erythropoietin-mediated protection of erythroid-differentiated cells from TNF-alpha-induced apoptosis.

The TNF-alpha (tumour necrosis factor) affects a wide range of biological activities, such as cell proliferation and apoptosis. Cell life or death responses to this cytokine might depend on cell conditions. This study focused on the modulation of factors that would affect the sensitivity of erythroid-differentiated cells to TNF-alpha. Hemin-differentiated K562 cells showed higher sensitivity to TNF-induced apoptosis than undifferentiated cells. At the same time, hemin-induced erythroid differentiation reduced c-FLIP (cellular FLICE-inhibitory protein) expression. However, this negative effect was prevented by prior treatment with Epo (erythropoietin), which allowed the cell line to maintain c-FLIP levels. On the other hand, erythroid-differentiated UT-7 cells - dependent on Epo for survival - showed resistance to TNF-alpha pro-apoptotic action. Only after the inhibition of PI3K (phosphatidylinositol-3 kinase)-mediated pathways, which was accompanied by negative c-FLIP modulation and increased erythroid differentiation, were UT-7 cells sensitive to TNF-alpha-triggered apoptosis. In summary, erythroid differentiation might deregulate the balance between growth promotion and death signals induced by TNF-alpha, depending on cell type and environmental conditions. The role of c-FLIP seemed to be critical in the protection of erythroid-differentiated cells from apoptosis or in the determination of their sensitivity to TNF-mediated programmed cell death. Epo, which for the first time was found to be involved in the prevention of c-FLIP down-regulation, proved to have an anti-apoptotic effect against the pro-inflammatory factor. The identification of signals related to cell life/death switching would have significant implications in the control of proliferative diseases and would contribute to the understanding of mechanisms underlying the anaemia associated with inflammatory processes.