To investigate the expressions of Fas and Fas ligand (FasL) in human placenta, we studied the expressions of Fas and FasL in placenta with RT-PCR, immunoblotting and immunostaining. We observed amplified products of Fas and FasL transcripts, the band of Fas (52 kDa) and multiple bands of FasL (42-52 kDa) in pla-centa. Fas and FasL localized mainly on fetal vessels and on syncytiotrophoblasts respectively. The differential distribution of Fas and FasL in human placenta may reflect intrinsic expressions of them by trophoblasts during differentiation. The increased expression of Fas in trophoblasts may promote apoptosis of placenta in pathologic condition such as preeclampsia.
Antigens, CD95/biosynthesis/*genetics ; Fas Ligand Protein ; *Gene Expression ; Gene Expression Profiling ; Glycosylation ; Humans ; Immunoblotting/methods ; Membrane Glycoproteins/biosynthesis/*genetics ; Placenta/*metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Trophoblasts/cytology/metabolism

Fas transduces apoptotic signals upon cross-linking with the Fas ligand (FasL), which is experimentally replaced by agonistic anti-Fas monoclonal antibodies (mAb). Of eight human malignant hematopoietic cell lines (HL-60, KG-1, THP-1, K562, U937, Jurkat, IM-9, RPMI-8226) examined by flow cytometric analysis, all, except K562, were found to be positive for surface Fas antigen. However, despite surface Fas expression, the agonistic anti-Fas mAb (7C11) induced apoptosis in only three of seven Fas-expressing cell lines (KG-1, Jurkat and IM-9). This Fas-resistance did not correlated with high levels of mRNA either for DcR3, a decoy receptor for FasL, or for FAP-1, a Fas-associated phosphatase that can block the apoptotic function of Fas. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis did not show consistent differences in the expression of Bcl-2 and Bax between Fas-sensitive and Fas-resistant cell lines examined. These findings indicated that the presence or absence of mRNA expression of DcR3, FAP-1, Bcl-2 and Bax did not always correlate with relative sensitivity to Fas-mediated apoptosis. Treatment of cells with cycloheximide converted the phenotype of resistant cell lines from Fas-resistant to Fas-sensitive, and enhanced the sensitivity of Fas-sensitive cell lines. These results suggest that the Fas-resistance is dependent on the presence of labile proteins that determine resistance to Fas-mediated apoptosis and the apoptotic machinery is already in place in Fas-resistant cell lines.
Antigens, CD95/*metabolism ; Apoptosis/drug effects/*genetics ; Carrier Proteins/biosynthesis/genetics ; Comparative Study ; Cycloheximide/pharmacology ; Gene Expression Regulation, Neoplastic ; Hematologic Neoplasms/*genetics/metabolism ; Human ; Membrane Glycoproteins/*metabolism ; Protein Synthesis Inhibitors/pharmacology ; Protein-Tyrosine-Phosphatase/biosynthesis/genetics ; Proto-Oncogene Proteins/biosynthesis/genetics ; Proto-Oncogene Proteins c-bcl-2/biosynthesis/genetics ; Receptors, Cell Surface/biosynthesis/genetics ; Signal Transduction ; Support, Non-U.S. Gov't ; Tumor Cells, Cultured

The pathogenesis of chronic cyclosporine A (CsA) nephrotoxicity has not been elucidated, but apoptosis is thought to play an important role in CsA induced tubular atrophy. Recently Fas-Fas ligand system mediated apoptosis has been frequently reported in many epithelial cells as well as in T lymphocytes. We investigated the ability of CsA to induce apoptosis in cultured human proximal tubular epithelial cells and also the effect of -MSH on them. Fas, Fas ligand, and an intracellular adaptor protein, Fas-associating protein with death domain (FADD) expression, and poly-ADP ribose polymerase (PARP) cleavage were also studied. CsA induced apoptosis in cultured tubular epithelial cells demonstrated by increased number of TUNEL positive cells and it was accompanied by a significant increase in Fas mRNA and Fas ligand protein expressions. FADD and the cleavage product of PARP also increased, indicating the activation of caspase. In -MSH co-treated cells, apoptosis markedly decreased with downregulation of Fas, Fas ligand and FADD expressions and also the cleavage product of PARP. In conclusion, these data suggest that tubular cell apoptosis mediated by Fas system may play a role in tubular atrophy in chronic CsA nephrotoxicity and pretreatment of -MSH may have a some inhibitory effect on CsA induced tubular cell apoptosis.
Antigens, CD95/genetics ; Apoptosis/*drug effects ; Carrier Proteins/biosynthesis ; Caspases/physiology ; Cells, Cultured ; Cyclosporine/*toxicity ; Human ; Immunosuppressive Agents/*toxicity ; Kidney Tubules, Proximal/cytology/*drug effects/metabolism ; Membrane Glycoproteins/biosynthesis ; NAD+ ADP-Ribosyltransferase/metabolism ; RNA, Messenger/analysis ; alpha-MSH/*pharmacology

To characterize the TGF-beta1 response of monocytic leukemia cells, we analyzed the effects of TGF-beta1 on cell proliferation, differentiation, and apoptosis of human monoblastic U937 cells. Treatment of cells with TGF-beta1 in the absence of growth factors significantly enhanced cell viability. Flow cytometric analysis of DNA content and CD14 expression revealed that TGF-beta1 does not affect cell proliferation and differentiation. Consistent with these results was the finding that no transcriptional induction of Cdk inhibitors such as p21Waf1, p15Ink4b, and p27Kip1 was detected following TGF-beta1 treatment. Interestingly, however, pretreatment of TGF-beta1 significantly inhibited Fas-, DNA damage-, and growth factor deprivation-induced apoptosis. This antiapoptotic effect was totally abrogated by anti-TGF-beta1 antibody. Quantitative RT-PCR analysis demonstrated a dose- and time-dependent transcriptional up-regulation of Bcl-X(L), suggesting its implication in the TGF-1-mediated antiapoptotic pathway. We also observed elevated expression of c-Fos and PTEN/MMAC1. But, no detectable change was recognized in expression of c-Jun, Fas, Fadd, Fap-1, Bcl-2, and Bax. Taken together, our study shows that TGF-beta1 enhancement of cellular viability is associated with its antiapoptotic effect, which may result from the transcriptional up-regulation of Bcl-X(L).
Antigens, CD14/metabolism ; Antigens, CD95/metabolism ; Apoptosis/drug effects* ; Cell Cycle/drug effects ; Cell Differentiation/drug effects ; Cell Division/drug effects ; Cell Survival/drug effects ; DNA/analysis ; DNA Damage ; Gene Expression Regulation, Neoplastic/genetics* ; Genes, Suppressor, Tumor/genetics ; Human ; Leukemia, Myeloid/genetics ; Neoplasm Proteins/metabolism ; Phosphoric Monoester Hydrolases/genetics ; Proto-Oncogene Proteins c-bcl-2/genetics ; Proto-Oncogene Proteins c-bcl-2/biosynthesis* ; RNA, Messenger/metabolism ; Receptors, Antigen, T-Cell/genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; Transforming Growth Factor beta/pharmacology* ; U937 Cells ; Up-Regulation (Physiology)