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Biomedical subjects

F Hirsch

Publications and source records attributed to F Hirsch.

At least 19 recordsLinked to original sources
PubMed2003

Potentiation of tumour apoptosis by human growth hormone via glutathione production and decreased NF-kappaB activity.

In addition to its primary role as growth factor, human growth hormone (hGH) can also participate in cell survival, as already documented by its protective effect on human monocytes or human promyelocytic leukaemia U937 cells exposed to a Fas-mediated cell death signal. However, despite similarities in the molecular events following Fas and TNF-alpha receptor engagement, we report that U937 cells, genetically engineered to constitutively produce hGH, were made more sensitive to TNF-alpha-induced apoptosis than parental cells. This was due to overproduction of the antioxidant glutathione, which decreased the nuclear factor (NF)-kappaB activity known to control the expression of survival genes. These findings were confirmed in vivo, in nude mice bearing U937 tumours coinjected with recombinant hGH and the NF-kappaB -inducing anticancer drug daunorubicin, to avoid the in vivo toxicity of TNF-alpha. This study therefore highlights one of the various properties of hGH that may have potential clinical implications.

Animals↗
PubMed2002

Human growth hormone gene transfer into tumor cells may improve cancer chemotherapy.

Chemotherapy remains the main tool for the treatment of cancers, but is often hampered by tumor cell resistance. In this context, the transfer of genes able to accentuate the effect of anticancer drugs may constitute a useful approach, as exemplified by inactivation of nuclear factor (NF)-kappa B via direct transfer of a gene encoding a negative dominant of its natural inhibitor I kappa B, leading to improved response to cancer chemotherapy. Following our previous report that transfection of human growth hormone (hGH) gene into human monocytic cell lines may also inactivate NF-kappa B in another situation, we decided to test the consequences of hGH gene transfer on cancer treatments. We demonstrated that hGH-transfected human myeloid leukemia U937 cells were sensitized to an apoptotic signal mediated by the anticancer drugs. In parallel, we found that, by inhibiting degradation of I kappa B, hGH gene transfer diminished NF-kappa B entry into the nuclei of U937 cells exposed to daunorubicin. Finally, we report that hGH-transfected tumor cells engrafted in nude mice responded in vivo to chemotherapy with nontoxic doses of daunorubicin whereas, under the same conditions, control tumor cells remained insensitive. Overall, this study therefore suggests that hGH gene transfer may offer new therapeutic prospects in cancer therapy.

Antibiotics, Antineoplastic↗
PubMed2002

Consensus statements from the Second International Lung Cancer Molecular Biomarkers Workshop: a European strategy for developing lung cancer molecular diagnostics in high risk populations.

The Second Molecular Biomarkers Workshop was held at the Roy Castle International Centre for Lung Cancer Research in Liverpool, in June 2001 and it brought together experts in the clinical, epidemiological and molecular-pathology of lung cancer from Europe and the USA, to address issues surrounding the development of a European strategy for early lung cancer detection. The 2001 Workshop Breakout Groups concentrated on the current challenges in the early detection of lung cancer which need to be addressed in the light of the recent surge in interest in many countries for mounting new clinical trials to evaluate the utility of Spiral CT in early lung cancer detection. If population-based trials of CT screening are mounted it will also be a favorable clinical environment in which to evaluate efficiently recent advances in molecular screening and genotyping. The Workshop focused specifically on: a) clinical and molecular biomarkers, b) sputum as an early detection and diagnostic tool, c) validation of molecular markers prior to their use in early detection trials and d) ethical issues that have to be considered in early lung cancer detection trials. A distillation of the Workshop discussions is given in this article.

Biomarkers, Tumor↗
PubMed2001

Multicenter phase II study of gemcitabine in previously untreated patients with advanced epithelial ovarian cancer.

Gemcitabine has activity in advanced ovarian cancer, with responses in platinum-resistant disease. This study assessed the activity of gemcitabine in previously untreated patients with advanced epithelial ovarian cancer. All patients had histologically verified invasive epithelial ovarian cancer, International Federation of Gynecology and Obstetrics (FIGO) stage III/IV disease and no prior chemotherapy. Patients received gemcitabine 1250 mg/m(2) on days 1, 8 and 15 of a 28-day cycle. Radiological response was assessed after two cycles. Between December 1992 and October 1995, 35 patients were enrolled. Of 33 evaluable patients, there was one complete response and five partial responses, for an overall response rate of 18% (95% confidence interval 7-36%). Forty-two percent of patients had a greater than 50% decrease in their CA-125 levels. Of the 25 patients who received platinum-based chemotherapy following treatment with gemcitabine, 12 achieved an overall response rate of 48%. Toxicity was mild, with two episodes of WHO grade 4 neutropenia (not associated with fever) and two episodes of grade 4 thrombocytopenia (not associated with bleeding). Gemcitabine has single-agent activity for poor-prognosis patients with advanced ovarian cancer. Similar results with subsequent platinum-based therapy indicate a lack of cross-resistance. This, combined with gemcitabine's favorable toxicity profile, warrants testing in comparative trials.

Adult↗
PubMed2000

Caspase-independent commitment phase to apoptosis in activated blood T lymphocytes: reversibility at low apoptotic insult.

Little is known about the mechanisms of programmed death triggered in T lymphocytes by stimuli that can bypass caspase activation. Anti-CD2 monoclonal antibody and staurosporine are such apoptosis inducers because they operate in the presence of broad-spectrum caspase inhibitors BOC-D.fmk and Z-VAD.fmk. A system was devised, based on the isolation according to density of activated blood T cells progressively engaged in the apoptotic process. This allowed definition of a sequence of caspase-dependent and caspase-independent apoptogenic events that are triggered by anti-CD2 and staurosporine. Thus, a commitment phase to apoptosis was defined that is entirely caspase independent and that is characterized by cell volume loss, partial chromatin condensation, and release into the cytosol and the nucleus of mitochondrial "apoptosis-inducing factor " (AIF). Committed cells were viable, displayed a high mitochondrial inner transmembrane potential (triangle upPsim), and lacked large-scale and oligonucleosomal DNA fragmentation. Mitochondrial release of AIF was selective because cytochrome c was retained in mitochondria of the very same cells. Mitochondrial release of cytochrome c occurred later, at the onset of the execution phase of apoptosis, concurrently with triangle upPsim collapse, poly (ADP-ribose) polymerase cleavage, and DNA fragmentation. The apoptogenic events of this commitment phase are reversible if the strength of the stimulus is low and of short duration.

Apoptosis↗
PubMed2000

IFN-gamma induces apoptosis in mouse embryonic stem cells, a putative mechanism of its embryotoxicity.

It has been reported that interferon (IFN)-gamma should inhibit in vitro mouse embryo growth by direct cell toxicity. However, the mechanism involved has not been clearly established. In the present study, this question was addressed using the embryonic stem (ES) cell model. It was found that IFN-gamma, induces a dose-dependent apoptosis in ES cells, as assessed by trypan-blue staining, by Annexin-V labeling and DNA analysis, Moreover, IFN-gamma treatment cooperates with Fas-mediated apoptosis, a phenomenon that has been recently reported. As Bcl-2 oncoprotein functions as a death repressor molecule in an evolutionarily conserved cell death pathway, its expression was analyzed by flow cytometry. It was demonstrated that Bcl-2 is expressed in ES cells. When compared to untreated ES cells, IFN-gamma-treated, apoptotic cells expressed a lower Bcl-2 level and a normal level of Fas, whereas surviving cells expressed a normal level of Bcl-2 but a lower Fas expression. Altogether, these data suggest that IFN-gamma may influence early mouse embryo development by promoting apoptosis, which may constitute a novel mechanism of IFN-gamma embryotoxicity.

Animals↗
PubMed2000

Fas-mediated apoptosis of mouse embryo stem cells: its role during embryonic development.

PROBLEM: Fas antigen (APO-1/CD95) can regulate the activity of various cells during adulthood. This study aimed at determining whether Fas may also be involved in the regulation of very early events such as the embryo preimplantation stage. METHOD OF STUDY: We used mouse embryo stem (ES) cell line as a model for testing the effect of Fas crosslinking upon anti-Fas monoclonal antibody (MoAb) treatment. In addition, this treatment was also applied to in-vitro mouse-embryo culture. RESULTS: Flow-cytometry analysis of cultured ES cells demonstrated an increase in Fas expression. unchanged in the presence of mouse interleukin-2, while greatly upregulated in the presence of lipopolysaccharide (LPS). As determined by various means, ES cells may undergo a Fas-mediated apoptosis, slightly but significantly intensified by the addition of LPS to cell cultures. We also report that anti-Fas MoAb directly inhibited two-cell stage mouse-embryo (preimplantation) development in in-vitro culture conditions. CONCLUSION: These data suggest a novel mechanism controlling the regulation of physiological cell turnover as well as blastocyst implantation in early embryo development.

Animals↗
PubMed1999

Lonidamine triggers apoptosis via a direct, Bcl-2-inhibited effect on the mitochondrial permeability transition pore.

The molecular mode of action of lonidamine, a therapeutic agent employed in cancer chemotherapy, has been elusive. Here we provide evidence that lonidamine (LND) acts on mitochondria to induce apoptosis. LND provokes a disruption of the mitochondrial transmembrane potential which precedes signs of nuclear apoptosis and cytolysis. The mitochondrial and cytocidal effects of LND are not prevented by inhibitors of caspases or of mRNA or protein synthesis. However, they are prevented by transfection-enforced overexpression of Bcl-2, an oncoprotein which inhibits apoptosis by stabilizing the mitochondrial membrane barrier function. Accordingly, the cell death-inducing effect of LND is amplified by simultaneous addition of PK11195, an isoquinoline ligand of the peripheral benzodiazepine receptor which antagonizes the cytoprotective effect of Bcl-2. When added to isolated nuclei, LND fails to provoke DNA degradation unless mitochondria are added simultaneously. In isolated mitochondria, LND causes the dissipation of the mitochondrial inner transmembrane potential and the release of apoptogenic factors capable of inducing nuclear apoptosis in vitro. Thus the mitochondrion is the subcellular target of LND. All effects of LND on isolated mitochondria are counteracted by cyclosporin A, an inhibitor of the mitochondrial PT pore. We therefore tested the effect of LND on the purified PT pore reconstituted into liposomes. LND permeabilizes liposomal membranes containing the PT pore. This effect is prevented by addition of recombinant Bcl-2 protein but not by a mutant Bcl-2 protein that has lost its apoptosis-inhibitory function. Altogether these data indicate that LND represents a novel type of anti-cancer agent which induces apoptosis via a direct effect on the mitochondrial PT pore.

Amino Acid Chloromethyl Ketones↗
PubMed1999

Growth hormone prevents human monocytic cells from Fas-mediated apoptosis by up-regulating Bcl-2 expression.

Apoptosis and particularly Fas-mediated apoptosis has been proposed to play a key role in controlling monocyte homeostasis. We and others have documented the regulatory function of human growth hormone (hGH) on monocytic cells, which prompted us to investigate the role of hGH on their response to Fas antigen cross-linking. Using human promonocytic U937 cells constitutively producing hGH upon gene transfer and human primary monocytes cultured in the presence of recombinant hGH, we demonstrated that hGH diminished Fas-mediated cell death by enhancing the expression of the antiapoptotic oncoprotein Bcl-2 as well as the level of bcl-2alpha mRNA. In parallel, we established that overexpression of Bcl-2 through gene transfer into normal U937 cells also diminished Fas-induced apoptosis. Further, as a result of Bcl-2 overexpression, we found that hGH greatly depressed Fas-induced activation of the cysteine protease caspase-3 (CPP32), which in turn affected the cleavage of poly(ADP-ribose) polymerase. Altogether, these data provide evidence that hGH mediates its protective effect through a Bcl-2-dependent pathway, clearly a crucial step in enhanced survival of monocytic cells exposed to Fas-induced death.

Apoptosis↗
PubMed1999

Targeted transfection of the IL-3 gene into primary human hematopoietic progenitor cells through the c-kit receptor.

We recently showed that an antibody-mediated gene transfer procedure termed antifection can be used for targeted gene delivery into lymphoid cells in vitro and in vivo. We here report that antifection also is effective for targeted gene transfer to immature hematopoietic cells. A human IL3-expressing plasmid was chemically linked to an anti-human CD117 antibody. Delivery of the IL3 plasmid into IL-3-dependent myeloid TF-1 cells (bearing the CD117 antigen) was specific and resulted in the transient proliferation of the targeted cells in the absence of exogenous IL-3. Transfection of primary human CD34+ hematopoietic stem/progenitor cells led to transient production of IL-3 and transient proliferation of the target cells. Interestingly, by using a semisolid progenitor cell assay, we found that transfected primary CD34+ cells were able to generate normal numbers of cell colonies in the absence of exogenous IL-3. Polymerase chain reaction analysis confirmed the presence and expression of the IL-3 transgene in the progenitor-derived colonies. In conclusion, our data show that CD117 is a suitable cell surface target to specifically transfer gene by antifection into primary CD34+ cells and that delivery of IL-3 gene in these cells resulted in the expression of a functional IL-3 able to support cell growth in absence of exogenous cytokine. Thus, antifection may provide new therapeutic modality relying on the transient production of appropriate growth factors acting via autocrine and/or paracrine mechanisms.

Antigens, CD34↗
PubMed1999

A signalling accessory molecule revealed by a new anti-fibroblastoid L cell monoclonal antibody.

Fibroblastoid mouse L-cells are widely used in immunological models because when transfected with class II-coding genes they become efficient antigen presenting cells. Little is known, however, about the cell surface markers borne by L-cells and their putative involvement/Interference with the experimental models studied. Rats were immunized against DAP.3 cells (subclone of L-cells) and monoclonal antibodies (mAbs) were prepared. One of them, 4D4, was studied in detail. It recognizes an epitope which is neither cell lineage- nor strain- nor species-restricted since, in addition to DAP.3 cells, it binds, as determined by flow cytometry and immunohistochemistry, to various cells such as CD8+ T cells from thymus, spleen, lymph node or intestinal epithelium, mouse peritoneal B cells and various tissues such as renal, pulmonary or intestinal epithelia. 4D4 mAb immunoprecipitates an undescribed 68 kDa protein. Functionally, this mAb inhibits the IL-2 secretion of a T cell clone in response to its peptide presented by appropriate class II-transfected L-cells and induces a negative selection of double positive CD4+CD8+ thymocytes. Since the 4D4 ligand is found on cells which are submitted to selection (T cells) and on cells which mediate selection (epithelial and antigen presenting cells), we conclude that 4D4 mAb defines a cell surface antigen involved, as an accessory molecule, in a cell selection process.

Animals↗
PubMed1998

Caspase-independent cell death induced by anti-CD2 or staurosporine in activated human peripheral T lymphocytes.

We examined the effects of the cell-permeable, broad spectrum peptide caspase inhibitors, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD.fmk), and BOC-Asp(OMe)-fluoromethyl ketone (BOC-D.fmk), on apoptosis induced by anti-CD2, anti-Fas, and the protein kinase inhibitor staurosporine in activated human peripheral T lymphocytes. We monitored ultrastructural, flow cytometric, and biochemical apoptotic changes, including externalization of phosphatidylserine, cleavage of poly(ADP-ribose) polymerase (PARP) and lamins, activation of caspase-3 and caspase-7, decrease in mitochondrial membrane potential, and DNA fragmentation. Z-VAD.fmk and BOC-D.fmk completely inhibited all the biochemical and ultrastructural changes of apoptosis in anti-Fas-treated cells. In marked contrast, neither Z-VAD.fmk nor BOC-D.fmk inhibited CD2- or staurosporine-mediated cell shrinkage, dilatation of the endoplasmic reticulum (seen in anti-CD2-treated cells), externalization of phosphatidylserine, and loss of mitochondrial membrane potential that accompanied cell death. However, these inhibitors did inhibit the cleavage of PARP and lamins and the formation of hypodiploid cells, and partially inhibited chromatin condensation. These results demonstrate that in activated T cells, anti-CD2 and staurosporine induce a caspase-independent cell death pathway that exhibits prominent cytoplasmic features of apoptosis. However, caspase activation is required for the proteolytic degradation of nuclear substrates such as PARP and lamins together with the DNA fragmentation and extreme chromatin condensation that occur in apoptotic cells.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Potent apoptotic signaling and subsequent unresponsiveness induced by a single CD2 mAb (BTI-322) in activated human peripheral T cells.

Manipulation of CD2 molecules with CD2 mAb pairs has been shown to deliver apoptotic signals to activated mature T cells. We show that BTI-322, a CD2 mAb directed at a peculiar epitope of CD2, can trigger on its own the apoptotic death of IL-2-activated peripheral T cells and of OKT3-stimulated T cells, contrasting in this respect with a series of other mouse or rat CD2 mAb. F(ab')2 fragments were as potent as the whole Ab. BTI-322-induced apoptosis proceeded in a few hours and was independent of the Fas/Fas ligand system. Less than 5 ng/ml of BTI-322, added at the beginning of culture, were able to eliminate within 4 days most CD3+ cells from OKT3- and IL-2-stimulated lymphocytes, the only cells remaining being CD16+CD2- NK cells. T cell proliferative responses induced by a mitogenic CD2 mAb pair or by PHA-P (which mainly binds to CD2) were not inhibited by BTI-322. In this case, the apoptotic effect was successfully counteracted by simultaneous enhancement of T cell divisions. Thus, the killing effect of BTI-322 was most effective when T cells were exclusively stimulated through the CD3/TCR complex. Apoptosis of the responding T cells may explain why T cells recovered from a primary MLC performed in the presence of BTI-322 responded to third party cells but not to the primary stimulatory cells. These data constitute the rational basis for the use of BTI-322 for inducing tolerance in human allotransplantation.

Animals↗
PubMed1998

Pattern of cytokine expression in circulation CD57+ T cells from long-term renal allograft recipients.

We made a quantitative analysis of the lymphokine mRNA and of proteins produced by CD57+ and CD57- circulating T cells isolated from long-term kidney-transplanted patients with expanded CD4+/CD57+ and CD8+/CD57+ T cells, and from normal individuals. We concentrated on IL-2 and IFN-gamma, which define a Th1-like type of lymphokine production, and on IL-4 which defines a Th-2-like type. We also analysed the production of IL-10 which is endowed with inhibitory effects on IL-2 and IFN-gamma synthesis, and of TNF-alpha, a pleiotropic inflammatory cytokine. On ionomycin + PMA stimulation, which reveals the intrinsic potential of lymphokine production by T cells, the CD57+ T cell subsets from all individuals produced high amounts of IFN-gamma and TNF-alpha mRNA and protein. They also produced IL-2, but to a much lesser extend than their CD57- counterparts, and little IL-4 and IL-10. They were no more capable of producing IL-2 when stimulated through the CD3/TCR in the presence of monocytes, yet still synthesized IFN-gamma. Our data suggest that the in vivo expansion of CD57+ T cells in stable allograft renal recipients might correspond to Th1 energized cells which on triggering of cell surface receptors hardly secrete lymphokines involved in cell cycle progression, but can still exert some effector functions, including IFN-gamma secretion.

CD57 Antigens↗
PubMed1997

Inhibitory effect of growth hormone on TNF-alpha secretion and nuclear factor-kappaB translocation in lipopolysaccharide-stimulated human monocytes.

Several studies have pointed to a link between immune and endocrine systems, including a regulatory function of GH on monocyte activation. The present study demonstrates that human THP-1 promonocytic cells, engineered by gene transfer to constitutively produce human growth hormone (hGH), secreted depressed amounts of TNF-alpha in response to challenge by LPS. The effect of GH appears to occur in an autocrine fashion, since the inhibitory effect on TNF-alpha secretion by constitutive GH production could be abolished in the presence of anti-hGH mAb. The GH-induced inhibitory effect was also observed using normal human monocytes and monocyte-derived macrophages. Inhibition of TNF-alpha production by THP-1-hGH-transfected cells cultured in the presence of LPS is dependent on a selective pathway, since no inhibition of TNF-alpha production was observed when cells were cultured in the presence of PMA. Inhibition of TNF-alpha secretion by LPS-stimulated THP-1-hGH cells was associated with a decrease in nuclear translocation of nuclear factor-kappaB. The capacity of GH to inhibit LPS-induced TNF-alpha production by monocytes without altering other pathways leading to TNF-alpha production may be of potential relevance in septic shock, since GH is available for clinical use.

CD4 Antigens↗
PubMed1997

Costimulatory molecules in human atherosclerotic plaques: an indication of antigen specific T lymphocyte activation.

Atherosclerotic plaques contain inflammation, composed largely of macrophages and lymphocytes. A proportion of lymphocytes shows signs of activation, but the question arises whether they are activated in an antigen specific way. The expression of costimulatory molecules-receptors that provide accessory signals during antigen-specific activation is a prerequisite for such a condition. This aspect of inflammation in atherosclerotic lesions has not been investigated. Human arterial segments with diffuse intimal thickening, fatty streaks and atherosclerotic plaques were studied with immuno-single and double staining methods. Macrophages and T lymphocytes were stained with CD68 and CD3, respectively, and pan-B cell markers CD19 and CD22 were also used. Costimulatory molecules B7-1 and B7-2, together with their common ligand CD28, and CD27 with its ligand CD70, were stained with specific monoclonal antibodies. The results show that most T lymphocytes were CD27 positive and that only a subpopulation of these (5-15%) was positive also for B7-1, CD28 and CD70. Macrophages expressed B7-1, B7-2, CD28 and CD70, while macrophages positive for CD28 and CD70 have not been reported yet. The expression of costimulatory molecules was most pronounced in the superficial layers at the fibrous cap, but decreased towards the lipid core. This study shows, therefore, that atherosclerotic plaques provide costimulatory signals generally accepted as a prerequisite for adequate T cell stimulation. In addition, this study reveals that only approximately 5-15% of the lymphocytes appears actively involved in the inflammatory reaction.

Aged↗
PubMed1997

Thiol-mediated inhibition of FAS and CD2 apoptotic signaling in activated human peripheral T cells.

Fas and CD2 receptors can transduce apoptotic signals through two independent biochemical pathways. In this study, we first evaluated the role of intracellular GSH in these signaling pathways by inducing variations in the GSH pool of activated peripheral T lymphocytes. Increasing the concentration of intracellular GSH by means of N-acetyl-L-cysteine (NAC) and GSH ethyl ester (OEt) resulted in total protection against cell death, while inhibiting GSH synthesis with buthionine sulfoximine (BSO) greatly enhanced cell sensitivity to Fas and CD2 apoptotic signaling. The protection exerted by NAC and GSH OEt was essentially based on their capacity to establish an intracellular reducing environment as it still occurred in BSO-treated cells. Thiol-containing compounds (cysteine, captopril, D-penicillamine and 2-mercaptoethanol) inhibited apoptosis while a series of non-thiol antioxidants (including catalase and vitamin E) failed to do so, suggesting that protection was secondary to thiols/disulfides exchange reactions at the level of cysteine residues in proteins and not to detoxification of reactive oxygen intermediates. This conclusion was further supported by the finding that no enhanced generation of O.-2 and H2O2 could be detected in cells experiencing early stages of apoptosis such as a decreased concentration of intracellular GSH and cell shrinkage. Also, protection occurred in the presence of protein synthesis inhibitors, indicating that it was due to post-translational sulfhydryl redox regulation of critical molecules involved in the apoptotic cascade. These data suggest that GSH, the most abundant intracellular thiol antioxidant, may be important in counteracting Fas- and CD2-mediated apoptosis of T lymphocytes.

Acetylcysteine↗