Fas-induced apoptosis of human B cells can be inhibited by over-expression of Bcl-2

14

Regulation of apoptosis

P.1.08.25

Fas-induced apoptosis of human B ceils can be inhibited by over-expression of Bci-2

M.K. Alam ‘, S. Davison ‘, J.D. Norton *, J.J. Murphy I. ’ hfectionand immunity Research Group, Division of Life S&nces, King’s College London, UK, *Department of Gene Regulation, Paterson InsUrufefor Cancer Research, Manchester, UK Introduction: We have previously reported that over-expression of Bcl-2 can inhibit apoptosis of Ramos B calls induced by calcium ionophore or macromolecular synthesis inhibitors. There are conflicting reports in the literature as to whether Bcl-2 can protect from Fas-induced apoptosis. The object of the study was to test the effects of Bcl-2 over-expression on Fas-induced apoptosis of the human Ramos Burt&t lymphoma B cell line. Materlalsand Methods: Ramos cells were electroporated with either the &I-2 expression vector, pZen bc-2 SV neo, or with the same vector lacking a bcl-2 coding sequence and selected in G418. Levels of expression of Bcl-2 in the transfected pool or individual clones were tested by Western Blot analysis. Cells were stimulated with either CD40 antibody (028-5, 1 &ml) or CD40 ligand (soluble trtmetic (Immunex), 1 &ml) to induce Fas expression and different concentrations of APO-l antibody (a gift from Prof. P.H. Krammer) were used to induce apoptosis. Cell apoptosis was monitored by measuring annexin V and propidium iodide staining on a flwv cytometer or by assessing nuclear condensation following acrtdine orange staining using a fluorescence microscope. Results: CD40 ligation induces Fas expression on human Ramos B cells within 24 h, following which the cells become susceptible to Fas-induced apoptosis. A dose-response curve revealed that 0.1 @/ml of APO-l antibody was the maximal dose required to induce apoptosis of roughly 80% of wild type Ramos cells or vector alone transfected cells over a three day time course following addition of the antibody. In contrast, in a clone of Ramos cells over-expressing Bci-2 (B3) or the Ramos Bcl-2 transfected pool, Fas-induced apoptosis was significantly inhibited over the same time course with only 40% of cells undergoing apoptosis. Levels of induced Fas expression on Ramos cells 24 h following CD40 ligation were identical on wild type, vector alone or Bcl-2 over-expressing (83) cells ruling out the possibility that Bcl-2 over-expression inhibited Fas-induced apoptosls by blocking CD40-mediated upregulation of Fas. Conclusion: The results of the present study suggest that over-expression of B&2 can inhibit Fas-induced apopto-sisin human B cells and this along with our previous results which showed Bcl-2 protected Ramos cells from apoptosis induced by calcium ionophore or macromolecular synthesis inhibitors is consistent with this protein functioning to block a signalling event(s) common to diverse pathways of apoptosis induction in Ramos cells. However, preliminary data on Ramos cells transfected with the related B&XL protein, which is itself induced by CD40, have shown that Bcl-XLconfers much reduced protection in Fas-induced apoptosis compared to Bd-2.

23 June 1997 - Poster presentations

addition of 0.01 mdml of AFP. AFP was shown to enhance the cytotoxicity induced by low doses of TNF and practically completely abrogated the cytotoxic signal, induced by high TNF. Conclusions: Our data suggests that AFP could directly participate in the regulation of cell growth and death mechanisms and could serve as a dual regulator of cell proliferation and apoptosis. AFP was shown to interfere with the death signals generated by TNF or antibiotics, inducing the enhance the low dose induced cytotoxicity and preventing cytotoxicity of high doses.

P.1.08.27 n

The ceil cycle control factors in renal and urotheiiai cancer

S. Roman ‘, D.N. Demir ‘, I. Moldovan ‘, M. Paraoan *, D. Damian *, A. Sulica ‘. ’ Center of Immunology; Bucharest, Romania, *Clinic of Urology, “Prof. Dr. Th. Butghele” Hospital, Bucharest, Romania Since a question of considerable practical interest is whether in a newly detected tumor a relatively stable state of development has been achieved or whether further development towards higher aggressiveness can be expected, apoptosis and other cell cycle control factors (~53, bcl-2) have been studied in patients with renal and urothetium cancer. The study was canted out on 40 patients with renal and bladder cancer. None of the patients was treated before removal of the tumor specimen. Single cell suspension was obtained by the tumor processing and enzymatic digestion. The flow-cytometryc analysis were used for the cell cycle and apoptosis evaluations. The apoptosis was also estimated by DNA electrophoresis and TUNEL technique. The nuclear oncoproteins were estimated by immunofluorescence. The values of apoptosis, as estimated by the three techniques used, were quite comparable, thus demonstrating the accuracy of our results. The most important prognostic factor in renal and urothelium cancer was the value of the cell cycle S phase fraction directly related to the functional integrity of the Gl-S check-point. The intensity of the apoptotic process and ~53 expression were low in transitional cell carcinoma of the bladder, observation which could explain the frequency of recurrences characteristic to this type of tumor. On the contrary, in renal carcinoma the intensity of the apoptotkz process was high and very high, independently by the DNA ploidy. The high values of the apoptosis were associated with the p53 expression and with the absence or the low expression of bcl-2 oncoprotein. The evaluation of the nuclear oncoproteins allows us to estimate whether a tumor cell will enter the apoptotic pathway or opt for Gl arrest (tumor dormancy?) as state of activity of p53 and growth regulatory genes. Therefore, cell cycle profile in correlation with histopathological features could represent a valuaMe tool in renal and bladder cancer monitortzation. Unexpectedly, morphologically normal renal tissue, with normal DNAcontent (diploidy), harvested from the affected kidney showed high percentages of cells undergoing apoptosis, independently by the tumor cells ploidy.

1P.1.08.26 1 Alpha-fetoprotein as a regulator of apoptotic signals induced by different stimuli

1P.1.08.28 1 Expression and function of Fas (COSS)on human renal tubular epitheiiai ceils

E.I. Dudich, L.N. Semenkova, I.V. Dudich, EA. Gorbatova. lnstifute of Engineering lmmunolqy Lyubuchany, Moscow Region, Russia

J.G. Boonstra’, F.J. van der Woude*, J.C. Laterveer’, P.C. Wever3, L.A. van Es’, MR. Daha', C. Van Kooten ‘. ’ Depr of Nephro/w, Leiden Uniwsify Hospital, Leiden, The Netherlands, 2 V Medizinische Klinik, Klinikum Mannheim, Germany 3Dept. of Nephroiogy, Academic Medical Center, Amsterdam, The Netherlands

Introduction: Alpha-fetoprotein (AFP) is known as a 89 kDa oncodevelopmental and immunosuppressive protein with numerous immunoregulative and cell growth regulative activities. AFP is known to bind and to transfer to the developing cells different biologically active ligands, such as fatty acids, steroids, growth factors, drugs etc. We have studied here the effect of purified human AFP on the growth of the different types of tumor and normal developing cells in vitro and its interference with the cytotoxic signals generated by other stimuli. Materials and Methods: It was determined the dose-dependent effect of AFP on the proliferation of tumor cell lines (hepatoma HepG2, fibroblastoma L929, lymphoma Jurkat and MT4) and mouse ConA activated splenocytes, as was assessed by PHI-thymidine incorporation. Also it was measured the effect of AFP on the cytotoxicity and cytostasis induced in these cells by the different doses of tumor necrosis factor (TNF), and antibiotics doxorubycin and mitomycin. Cytotoxicity and cytostasis were measured by MlT calorimetric and PHI-thymidine incorporation assays. Apoptosis was evaluated microscopically by detecting of the morphological changes characteristic of epoptosis and by assessment of the DNA fragmentation. Rewfts: It was demonstrated that high doses of AFP could induce the cytostatic and cytotoxic effect on the proliferation of HepGP, MT4, Jurkat and L929 cells. Moreover, it was shown that high AFP doses (more than 0.1 ms/ml) could induce apoptosis of sensitive tumor cells (HepG2, MT4) with the significant DNA fragmentation and cellular morphological changes, characteristic of apoptosis. AFP was shown to interfere with the apoptotic signals, generated by other stimuli. Physiological doses of AFP (0.01-0.1 mg/ml) were found to enhance the cytotoxicity of low doses of doxorubycin and mitomycin and to decrease the cytotoxicity induced by the high doses in the tumor cells. Doxorubycin-induced cytotoxic response of ConA-activated splenocytes was significantly reduced by

Introduction: During renal allograft rejection, apoptosis can be found specifically in tubular epfthelial cells, which are a main target of the inflammatory reponse in the kidney. Cell mediated cytotoxicity by the infiltrating lymphocytes is theoretically mediated via either the perforinlgranzyme route or the interaction between Fas and Fas ligand. In this study we investigated whether Fas is expressed on tubular epithelial cells of the kidney, and whether these calls are sensitive to Fas-mediated apoptosis. MaterlaIrand Methods:Fas expression in situ was studied by staining normal renal tissue and biopsies from rejecting renal allografts with UB2 (ms lgG1 anti-Fas). Fas expression on cultured primary proximal tubular epithelial cells (PTEC) was studied by FACS, using DX2 (ms IgGl anti-Fas) and by RT-PCR. For induction of apoptosis, PTEC were incubated for 18 or 42 hrs in the presence 1 &ml anti-Fas Ah’s in the presence or absence of CD4OLtransfeced mouse L cells or 10 &ml cycloheximide (CHX). Apoptosis was detected by studying morphological changes (nuclear condensation, appearance of apoptotic bodies) upon staining with Hoechst, and by DNA fragmentation as detemined by the TUNEL method. Results: Fas expression was obsenred on epithelial cells of 50-70% of tubuli in normal and rejecting renal tissue. Cultured PTEC were positive for Fas as determined by FACS and RT-PCR. However, incubation of PTEC with anti-Fas Ab’s did not lead to the induction of apoptosis, while the Ah’s were able to induce >80% apoptosis in Jurkat cells. Pre-treatment with IFN-y, or w-ligation of CD40 by CD40L transfected L cells, did not render the F’TEC susceptible to apoptosis. However, simultaneous treatment of PTEC with anti-Fas and CHX