Interleukin-4 induces human hepatocyte apoptosis through a Fas-independent pathway.

IL-4 is overexpressed in liver grafts during severe recurrent hepatitis C and rejection. Hepatocyte apoptosis is involved in both these phenomena. We therefore examined the proapoptotic effect of IL-4 on HepG2 cells and human hepatocytes in vitro, together with the underlying mechanisms. We first measured IL-4 receptor expression, STAT6 activation by IL-4, and STAT6 inhibition by an anti-IL-4 antibody or by STAT6 siRNA transfection. We then focused on the pathways involved in IL-4-mediated apoptosis and the role of STAT6 activation in apoptosis initiation. The IL-4 receptor was expressed on both cell types, and STAT6 was activated by IL-4. Both anti-IL-4 and STAT-6 siRNA inhibited this activation. IL-4 induced apoptosis of both HepG2 cells (P=0.008 vs. untreated control) and human hepatocytes (P<0.001 vs. untreated control). IL-4 reduced the mitochondrial membrane potential, activated Bid and Bax, and augmented caspase 3, 8, and 9 activity. STAT6 blockade inhibited IL-4-induced apoptosis. Expression of Fas and Fas ligand was unaffected when HepG2 cells and hepatocytes were cultured with IL-4, and Fas/FasL pathway blockade failed to inhibit IL-4-induced apoptosis. These results show that IL-4 induces apoptosis of human hepatocytes through IL-4 receptor binding, STAT6 activation, decreased mitochondrial membrane potential, and increased caspase activation, independently of the Fas pathway. IL-4 might thus contribute to the progression of severe liver graft damage.

Protein kinase PKC delta and c-Abl are required for mitochondrial apoptosis induction by genotoxic stress in the absence of p53, p73 and Fas receptor.

Doxorubicin, cis-diamminedichloroplatinum (II) and 5-fluorouracil used in chemotherapy induce apoptosis in Hep3B cells in the absence of p53, p73, and functional Fas. Since mediators remain unknown, the requirement of PKC delta (PKCdelta) and c-Abl was investigated. Suppression of c-Abl or PKCdelta expression using SiRNAs impaired PARP cleavage, Gleevec and/or rottlerin inhibited the induction of the subG1 phase and the increase of reactive oxygen species level. Co-precipitations and phosphorylations to mitochondria of c-Abl, PKCdelta and Bcl-X(L/s) were induced. A depolarization of the mitochondrial membrane and activations of caspase-2 and -9 were observed. We propose that, in the absence of p53, p73 and Fas, genotoxic drugs could require both PKCdelta and c-Abl to induce apoptosis through the mitochondrial pathway.

Effects of progesterone and anti-progestin (mifepristone) treatment on proliferation and apoptosis of the human ovarian cancer cell line, OVCAR-3.

The study examined the effects of various progesterone and mifepristone concentrations on the proliferation and apoptosis of the human ovarian cancer cell line, OVCAR-3. OVCAR-3 cells were incubated with progesterone and mifepristone at concentrations ranging from 10(-3) to 10(-9) M. Proliferation and apoptosis were studied by means of inverted optical microscopy, DAPI staining, and crystal violet assay. Immunoblotting was used to study the regulation of the apoptosis-related proteins, bcl-2, caspase-3 and PARP, after incubation with various reagents. OVCAR-3 cell density was increased by progesterone concentrations of 10(-5) M or less, and decreased by 10(-3) M progesterone. DAPI staining showed no apoptotic bodies. Mifepristone concentrations of 10(-3) and 10(-4) M reduced the OVCAR-3 cell density. Immunoblotting showed PARP cleavage in the presence of mifepristone 10(-4) M. Caspase-3 and bcl-2 expression was reduced by mifepristone 10(-4) and 10(-7) M. These results suggest that progesterone has a paradoxical effect on OVCAR-3 cell proliferation, stimulating it at low concentrations and inhibiting it at high concentrations, potentially through a caspase-independent non-apoptotic death pathway. Mifepristone seems to inhibit OVCAR-3 cell proliferation by down-regulating bcl-2 and up-regulating caspase-3 activity. These preliminary results suggest that progesterone and mifepristone have beneficial effects in ovarian cancer.

Inducible nitric oxide synthase (iNOS) activity could be responsible for resistance or sensitivity to IFN-gamma-induced apoptosis in several human hepatoma cell lines.

Response to interferon-gamma (IFN-gamma)-induced apoptosis of human hepatoma cell lines (HHCLs) is variable. We analyzed this different behavior in Hep3B, Chang-liver, HepG2, and HuH7 cells. We studied (1) IFN-gamma-induced apoptosis, (2) protein expression of Stat1, (3) binding of nuclear proteins to IFN-gamma activated sequence (GAS), (4) mRNA and expression of proteins acting in apoptosis, and (5) HuH7 sensitivity after inducible nitric oxide synthase (iNOS) siRNA transfection. IFN-gamma induced apoptosis in Hep3B and Chang-liver cells only. In all HHCLs, Stat1 protein increased. Binding of proteins and transactivation activity of GAS increased much more in HuH7. In all HHCLs, caspase activity and apoptotic proteins were not implicated in resistance or sensitivity. iNOS mRNA and protein expression increased in HuH7, disappeared in Hep3B, and remained unchanged in Chang-liver and HepG2. We compared the role of iNOS in Hep3B and HuH7. The iNOS inhibitor, L-NAME, sensitized HuH7 to IFN-gamma, Hep3B/HuH7 coculture partially inhibited Hep3B apoptosis, and HuH7 transfection with iNOS siRNA induced a 50% inhibition of iNOS protein and cell apoptosis. GAS activity and overexpression of iNOS in HuH7, but not in the other HHCLs, suggest that this enzyme could play an important role in the resistance of HuH7 to IFN-gamma-induced apoptosis, perhaps by the antiapoptotic action of NO.

Differential responses of proliferative and non-proliferative leukemia cells to oxidative stress.

The response of three human leukemia cell lines, the proliferative promonocyte THP-1 and the promyeloid HL60 cells and the non-proliferative phorbol ester-treated HL60 cells (HL60/PMA), to oxidative stress induced by tert-butylhydroperoxide (t-BHP) treatment was analyzed by fluorescence microplate assay, anti-oxidant enzyme activity measurements, high performance liquid chromatography, yopro-1/PI incorporation, poly (ADP-ribose) polymerase and caspase 3 cleavages. After t-BHP treatment, the non-proliferative HL60/PMA cells exhibited a weak increase in reactive oxygen species (ROS) production, a better preservation of thiol content, a decrease of glutathione peroxidase activity and a high ability to undergo necrosis rather than apoptosis. Submitted to the same treatment, the proliferative HL60 and THP-1 cells exhibited a high increase of ROS production, a moderate thiol depletion and a high percentage of apoptosis. Under thiol depleting conditions, the oxidative treatment of the HL60/PMA cells resulted in a high ROS production that reached levels similar to those of the two other cell lines and in cell death mainly by necrosis. In conclusion, these results that show proliferative phenotype is essential for cell response towards oxidative stress, are of particular interest in chemotherapy involving an oxidative mechanism.

Progesterone induces BRCA1 mRNA decrease, cell cycle alterations and apoptosis in the MCF7 breast cancer cell line.

BACKGROUND: Inherited mutations of the BRCA1 gene are responsible for hereditary breast and ovarian cancer syndrome. However, little is known of how disruption of BRCA1 functions preferentially increases cancer risk in hormone-dependent organs. We aimed to study whether BRCA1 was regulated by progesterone in the MCF7 breast cancer cell line. MATERIALS AND METHODS: MCF7 breast cancer cells were incubated with 10(-4) or 10(-10) M progesterone for 24 or 48 hours. BRCA1 expression, proliferation and apoptosis were analysed. RESULTS: 10(-4) M progesterone decreased cell proliferation, cell cycle progression and induced apoptosis. In addition, BRCA1 and cyclin A mRNA decreased. In contrast, none of these effects were observed in MCF7 cells incubated with 10(-10) M progesterone. CONCLUSION: The down-regulation of BRCA1 in MCF7 cells incubated with 10(-4) M progesterone seems to be a consequence of cell cycle alterations rather than a direct effect of the hormone on BRCA1.

Disruption of MKK4 signaling reveals its tumor-suppressor role in embryonic stem cells.

The dual Ser/Thr kinase MKK4 and its downstream targets JNK and p38 regulate critical cellular functions during embryogenesis and development. MKK4 has been identified as a putative tumor-suppressor gene in human solid tumors of breast, prostate and pancreas. To clarify the mechanisms underlying the transforming potential of molecular defects targeting MKK4, we have generated totipotent embryonic stem (ES) cells expressing the dominant-negative mutant DN-MKK4(Ala), S257A/T261A. Stably transfected DN-MKK4-ES cells exhibit a transformed fibroblast-like morphology, reduced proliferation rate, were no more submitted to cell contact inhibition, were growing in soft agar, and were much more tumorigenic than parental ES cells in athymic nude mice. These phenotypic changes: (i) are consistent with the protection of DN-MKK4-transfected ES cells from spontaneous, cell density-dependent, and stress-induced apoptosis (DAPI staining and poly (ADP-ribose) polymerase (PARP) cleavage) and (ii) correlated with alterations in JNK, p38, and Erk-1/-2 MAPK/SAPK signaling. Taken together, our data provide a new mechanism linking the MKK4 signaling pathways to cancer progression and identify MKK4 as a tumor-suppressor gene implicated in several transforming functions.

Effects of a Gonadotropin-Releasing Hormone agonist and Follicle Stimulating Hormone on the incidence of apoptosis in human luteinized granulosa cells.

OBJECTIVE: Previous studies have shown the importance of apoptosis in follicular atresia occurring especially in granulosa cells (GC) and its relation to the outcome of in vitro fertilization (IVF). The aim of this study was to evaluate the effects of a Gonadotropin-Releasing Hormone agonist (GnRHa) and of Follicle-Stimulating Hormone (FSH) on the apoptosis rate of human luteinized GC. STUDY DESIGN: GC were isolated from follicular fluids of 15 women undergoing IVF cycles, cultured for 1 day and then treated for 1 day in serum-free medium with triptorelin at 100 or 1000pg/ml or with FSH at 100 or 500ng/ml. GC cultured without any hormone addition were used as controls. Treatment of cultured GC with triptorelin 100pg/ml and FSH 100ng/ml was performed five times each. GC were analysed by flow cytometry after propidium iodide staining to measure the percentage of apoptotic GC. Some triptorelin-treated GC were also examined by electron microscopy. RESULTS: Percentages of GC apoptosis were after hormone treatment respectively: FSH: 100ng/ml, 2.9+/-0.6%; 500ng/ml, 2.9%; triptorelin: 100pg/ml, 18.6+/-2.8%; 1000pg/ml, 86.5% versus 9.8+/-1.8% in GC controls (FSH 100ng/ml versus control; triptorelin 100pg/ml versus control: P<10(-6)). Electron microscopy confirmed apoptosis of GC incubated with triptorelin. CONCLUSIONS: This study demonstrated that FSH decreased apoptosis in human luteinized GC. In contrast, triptorelin was possibly implicated in a dose-dependent increase in the incidence of apoptotic GC. This last result suggests that clinical use of GnRHa should perhaps be reconsidered in the context of its apoptosis-inducing effect.

Could induced apoptosis of human granulosa cells predict in vitro fertilization-embryo transfer outcome? A preliminary study of 25 women.

OBJECTIVE: The aim of this study was to investigate the relationship between induced apoptosis of human luteinized granulosa cells (GCs) and in vitro fertilization (IVF) outcome. STUDY DESIGN: We induced apoptosis with interferon gamma and an anti-human Fas antibody in cultured GCs isolated from follicular fluids coming from 25 different women undergoing 25 consecutive IVF cycles. After examination of 1000 GCs stained by DAPI with a fluorescent microscope, we determined the percentages of apoptotic GCs. Ovarian, endometrial and IVF parameters were recorded for every woman. RESULTS: We classified the women according to their induced GCs apoptosis percentages in two groups. Group 1 with a low percentage of apoptotic GCs (11.6+/-4.8%) had a significantly higher pregnancy rate (P<0.05) than group 2 with a high percentage of apoptotic GCs (59.5+/-14.8%). No other statistically significant differences were observed. CONCLUSION: Resistance of human GCs to apoptosis might be implicated in IVF outcome.

Soluble intercellular adhesion molecule 1 in umbilical cord serum: potential for the diagnosis of neonatal infections.

OBJECTIVE: To evaluate the diagnostic relevance to neonatal infections of the soluble intercellular adhesion molecule 1 (sICAM-1) cord serum level. METHODS: The case-control study included 66 term newborn infants with and without risk factors for neonatal infections. Cord blood serum determinations of white blood cell count, C-reactive protein, fibrinogen, and sICAM-1 were systematically performed associated with bacterial cultures from placenta, ears, and gastric fluids. RESULTS: 6 of 33 infants (18.2%) with risk factors were infected, and 13 (39.4%) were colonized. Two infants included in the group without infection risk factors (n = 33) were colonized. No difference in sICAM-1 cord serum levels was found according to the presence of premature rupture of membrane, fetal tachycardia >160 bpm, meconial amniotic fluid, and duration of labour >10 h. No difference in sICAM-1 was noted between infected and non-infected infants. The cord serum levels of sICAM-1 were significantly higher in infants after forceps extraction (p = 0.01). A correlation was observed between sICAM-1 and C-reactive protein cord serum levels (p = 0.004, r = 0.371) and between sICAM-1 level and neutrophil count (p = 0.01, r = 0.489). CONCLUSIONS: Our results suggest that cord serum sICAM-1 determinations have no diagnostic relevance to neonatal infection. The increase of sICAM-1 cord serum levels in infants after forceps extraction suggests its potential to evaluate cerebral trauma or hypoxia.

Overexpression of the mouse Fas gene in human Hep3B hepatoma cells overcomes their resistance to Fas-mediated apoptosis.

BACKGROUND/AIMS: Fas-induced apoptosis is one of the main forms of apoptosis occurring in hepatocytes. We have previously demonstrated that the human hepatoma cell line Hep3B is resistant to Fas-mediated apoptosis. In this study, we investigated whether the human Fas receptor itself, or the Fas transduction pathway was responsible for the resistant phenotype. METHODS: Clones of Hep3B cells overexpressing the mouse Fas gene (Hep3B(mfas)) were generated by transfection, and apoptosis was studied by (i) chromatin condensation and nuclear fragmentation, (ii) flow cytometry, (iii) DNA fragmentation and (iv) poly (ADP-ribose) polymerase cleavage. RESULTS: Use of the species-specific and agonistic anti-mFas monoclonal antibody (JO2), showed that the mFas receptor was correctly routed to the plasma membrane of Hep3B(mfas) cells. Using the four above-mentioned criteria, we demonstrated that JO2 triggered mFas-mediated apoptosis of Hep3B(mfas), but not of Hep3B(pCi) cells (transfected with an empty vector). CONCLUSIONS: Our data show (i) that the Fas signaling pathway can be completed when a functional mFas receptor is expressed in Hep3B cells, and thus, (ii) that the death-inducing signaling complex components and the effector caspases are functional in Hep3B cells. Moreover, they suggest that the Fas subunits are not pre-assembled at the cell membrane before receptor-ligand interaction.