RESUMEN

The HIV-1 encoded apoptogenic protein Vpr induces mitochondrial membrane permeabilization (MMP) via interactions with the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocator (ANT). We have designed a peptide, TEAM-VP, composed of two functional domains, one a tumor blood vessel RGD-like 'homing' motif and the other an MMP-inducing sequence derived from Vpr. When added to isolated mitochondria, TEAM-VP interacts with ANT and VDAC, reduces oxygen consumption and overcomes Bcl-2 protection to cause inner and outer MMP. TEAM-VP specifically recognizes cell-surface expressed alpha(V)beta(3) integrins, internalizes, temporarily localizes to lysosomes and progressively co-distributes with the mitochondrial compartment with no sign of lysosomal membrane permeabilization. Finally TEAM-VP reaches mitochondria of angiogenic endothelial cells to induce mitochondrial fission, dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), cytochrome c release and apoptosis hallmarks. Hence, this chimeric peptide constitutes the first example of a virus-derived mitochondriotoxic compound as a candidate to kill selectively tumor neo-endothelia.

Asunto(s)

Células Endoteliales/fisiología , Productos del Gen vpr/farmacocinética , Integrina alfaVbeta3/metabolismo , Mitocondrias/metabolismo , Péptidos/farmacocinética , Secuencia de Aminoácidos , Animales , Apoptosis , Supervivencia Celular , Relación Dosis-Respuesta a Droga , Células Endoteliales/metabolismo , Productos del Gen vpr/farmacología , Humanos , Lisosomas/metabolismo , Ratones , Ratones Endogámicos BALB C , Membranas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Péptidos/farmacología , Permeabilidad

RESUMEN

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.

Asunto(s)

Apoptosis/efectos de los fármacos , Glutatión/biosíntesis , Hormona de Crecimiento Humana/farmacología , FN-kappa B/metabolismo , Neoplasias Experimentales/patología , Receptor fas/metabolismo , Animales , Antibióticos Antineoplásicos/farmacología , Daunorrubicina/farmacología , Regulación hacia Abajo , Ensayo de Cambio de Movilidad Electroforética , Humanos , Quinasa I-kappa B , Masculino , Ratones , Ratones Desnudos , Neoplasias Experimentales/prevención & control , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Tasa de Supervivencia , Transfección , Factor de Necrosis Tumoral alfa/farmacología , Células U937/trasplante

RESUMEN

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.

Asunto(s)

Técnicas de Transferencia de Gen , Hormona de Crecimiento Humana/genética , Neoplasias/tratamiento farmacológico , Neoplasias/terapia , Antibióticos Antineoplásicos/farmacología , Peso Corporal/efectos de los fármacos , Muerte Celular , Núcleo Celular/metabolismo , Terapia Combinada , Daunorrubicina/farmacología , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos , Citometría de Flujo , Humanos , FN-kappa B/metabolismo , Factores de Tiempo , Transfección , Células Tumorales Cultivadas , Células U937

Asunto(s)

Anticuerpos Monoclonales/farmacología , Antígenos CD2/inmunología , Inmunosupresores/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Animales , Anticuerpos Monoclonales/inmunología , Antígenos CD2/fisiología , Antígenos CD58/fisiología , Rechazo de Injerto/tratamiento farmacológico , Trasplante de Corazón/inmunología , Humanos , Inmunidad Celular , Ratones , Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Tolerancia al Trasplante

RESUMEN

T-cell reconstitution after bone marrow transplant (BMT) is characterized, for at least 1 year, by the expansion of populations of T cells with a primed/memory phenotype and by reverse CD4/CD8 proportions. T lymphocytes from 26 BMT patients (mostly adults) were obtained at various times after transplantation (from 45 to >/=730 days) and were tested for susceptibility to spontaneous apoptosis and anti-Fas triggered apoptosis in vitro. Substantial proportions of CD4(+) and CD8(+) cells generated during the first year after transplantation, but not by day 730, exhibited in these assays decreased mitochondrial membrane potential (triangle upPsim) and apoptotic DNA fragmentation. The apoptotic phenotype tended to disappear late in the follow-up period, when substantial absolute numbers of naive (CD45RA(+)/CD62-L(+)) T cells had repopulated the peripheral blood compartment of the BMT patients. The rate of spontaneous cell death in vitro was significantly correlated with lower levels of ex vivo Bcl-2 protein, as assessed by cytofluorometry and Western blot analysis. In contrast, the levels of Bax protein remained unchanged, resulting in dysregulated Bcl-2/Bax ratios. Cell death primarily concerned the expanded CD8(+)/CD45R0(+) subpopulation, although CD45R0(-) subpopulations were also involved, albeit to a lesser extent. These results show that the T-cell regeneration/expansion occurring after BMT is accompanied by decreased levels of Bcl-2 and susceptibility to apoptosis.

Asunto(s)

Apoptosis , Trasplante de Médula Ósea , Neoplasias Hematológicas/inmunología , Neoplasias Hematológicas/terapia , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/patología , Adulto , Neoplasias Hematológicas/metabolismo , Humanos , Inmunofenotipificación , Subgrupos de Linfocitos T/inmunología , Inmunología del Trasplante , Trasplante Homólogo , Receptor fas

Asunto(s)

Anticuerpos Monoclonales/farmacología , Apoptosis , Antígenos CD2/fisiología , Linfocitos T/fisiología , Receptor fas/fisiología , Antígenos CD2/inmunología , Células Cultivadas , Humanos , Activación de Linfocitos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/fisiología , Linfocitos T/citología , Linfocitos T/inmunología

RESUMEN

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.

Asunto(s)

Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Genes bcl-2/efectos de los fármacos , Hormona de Crecimiento Humana/farmacología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Receptor fas/metabolismo , Secuencia de Bases , Caspasa 3 , Caspasas/metabolismo , Línea Celular , Cartilla de ADN/genética , Activación Enzimática/efectos de los fármacos , Humanos , Monocitos/citología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Transfección , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

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.

Asunto(s)

Anticuerpos Monoclonales/farmacología , Apoptosis/inmunología , Antígenos CD2/inmunología , Caspasas , Cisteína Endopeptidasas/fisiología , Activación de Linfocitos , Estaurosporina/farmacología , Subgrupos de Linfocitos T/inmunología , Clorometilcetonas de Aminoácidos/farmacología , Apoptosis/efectos de los fármacos , Antígenos CD2/efectos de los fármacos , Caspasa 2 , Caspasa 3 , Caspasa 7 , Muerte Celular/efectos de los fármacos , Muerte Celular/inmunología , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Diploidia , Activación Enzimática/efectos de los fármacos , Activación Enzimática/inmunología , Humanos , Hidrólisis/efectos de los fármacos , Laminas , Activación de Linfocitos/efectos de los fármacos , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas/metabolismo , Estaurosporina/antagonistas & inhibidores , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/enzimología , Subgrupos de Linfocitos T/ultraestructura , Receptor fas/efectos de los fármacos , Receptor fas/inmunología , Receptor fas/fisiología

RESUMEN

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.

Asunto(s)

Apoptosis/inmunología , Antígenos CD2 , Tolerancia Inmunológica , Transducción de Señal/inmunología , Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Femenino , Humanos , Técnicas In Vitro , Activación de Linfocitos , Prueba de Cultivo Mixto de Linfocitos , Masculino , Ratones , Ratas , Complejo Receptor-CD3 del Antígeno de Linfocito T

RESUMEN

We examined the relationship between proliferation and susceptibility to Fas- and CD2-mediated apoptosis of human peripheral T lymphocytes that had been exposed in primary culture to CD3- or CD2-derived mitogenic stimuli in the presence of monocytes and exogenous IL-2. After 5 days, activated T cells were fractionated into large (F2) and small (F6) cells on Percoll density gradients and analyzed for their susceptibility to apoptosis and for their position in the cell cycle. Most F6 cells displayed a CD45RA+, CD25-, CD2R- phenotype and were unable to incorporate bromodeoxyuridine (BrdUrd) during the entire culture period. However, they were activated to express Fas Ag and some cell cycle regulatory proteins specific to late G1 phase. T cells with proliferative unresponsiveness were sensitive to Fas-mediated apoptosis whether it was triggered by anti-Fas mAb or by Fas ligand, but were almost completely resistant to CD2 apoptotic signaling. In contrast, F2 cells exhibited classical activation markers (CD45RO, CD25, and CD2R), had crossed S phase at least once, and were sensitive to both Fas and CD2 apoptotic signals. In large cells harvested earlier (on day 3), the signals were operative in both BrdUrd+ and BrdUrd- cells. Thus, S phase entry is not required for Fas- and CD2-mediated apoptosis. The profound proliferative unresponsiveness of F6 cells to CD3 and CD2 stimuli (bypassed by ionomycin plus PMA) and the CD2R- conformation of their CD2 molecules suggest that they may be in vivo anergized cells whose elimination, upon restimulation, is highly dependent on the Fas death pathway.

Asunto(s)

Apoptosis/inmunología , Antígenos CD2/inmunología , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Linfocitos T/patología , Receptor fas/inmunología , División Celular/inmunología , Células Cultivadas , Citometría de Flujo , Humanos , Inmunofenotipificación

RESUMEN

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.

Asunto(s)

Hormona de Crecimiento Humana/fisiología , Monocitos/metabolismo , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Antígenos CD4/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Hormona de Crecimiento Humana/farmacología , Humanos , Lipopolisacáridos/farmacología , Acetato de Tetradecanoilforbol/farmacología , Transfección

RESUMEN

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.

Asunto(s)

Apoptosis/inmunología , Antígenos CD2/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Transducción de Señal/inmunología , Compuestos de Sulfhidrilo/farmacología , Linfocitos T/efectos de los fármacos , Receptor fas/efectos de los fármacos , Acetilcisteína/farmacología , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Butionina Sulfoximina/farmacología , Antígenos CD2/fisiología , Células Cultivadas , Cicloheximida/farmacología , Dactinomicina/farmacología , Femenino , Glutatión/análogos & derivados , Glutatión/biosíntesis , Glutatión/efectos de los fármacos , Glutatión/farmacología , Humanos , Masculino , Transducción de Señal/efectos de los fármacos , Linfocitos T/inmunología , Receptor fas/fisiología

RESUMEN

Short-term activated peripheral T lymphocytes are susceptible to apoptotic cell death triggered by CD2 mAbs. The aim of this study was to examine whether the CD2-mediated pathway of apoptosis is linked to the Fas death pathway, as this is the case for CD3/TCR-triggered apoptosis in several models of T cells. Using T lymphocytes from patients harboring Fas gene mutations and displaying a profound defect in Fas signaling of cell death, we show that CD2- (but not CD3-) mediated apoptosis still proceeds normally. In normal activated T cells, CD3-mediated apoptosis is prevented by reagents that block the Fas/Fas-ligand interaction, namely soluble M3 (an antagonistic anti-Fas mAb) and soluble human Fas.Fc, a fusion protein able to bind released Fas-ligand. In contrast, CD2 signaling of apoptosis resists these blocking agents. Neither new protein synthesis nor the activation of calcineurin was required for CD2- and Fas-mediated apoptosis, suggesting that latent cytoplasmic "death" molecules were activated upon stimulation of the cells. In both cases, protein tyrosine kinases were transiently activated, as is exemplified by the autophosphorylation and exokinase activity of p56lck, yielding overlapping yet nonidentical profiles of protein tyrosine phosphorylation. Pretreating the cells with herbimycin A, before the addition of the apoptotic stimuli, almost completely inhibited CD2 transmembrane signaling of apoptosis, but left intact Fas-induced apoptosis. Our data suggest that CD2 is a Fas-independent cell death pathway that might contribute directly to the elimination of T cells expanding during an immune reaction.

Asunto(s)

Apoptosis/inmunología , Antígenos CD2/farmacología , Activación de Linfocitos/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Linfocitos T/inmunología , Receptor fas/fisiología , Anticuerpos Monoclonales/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Antígenos CD2/fisiología , Complejo CD3/fisiología , Femenino , Humanos , Activación de Linfocitos/genética , Masculino , Mutación/inmunología , Fosforilación , Proteínas Tirosina Quinasas/fisiología , Proteínas Recombinantes de Fusión/farmacología , Transducción de Señal/inmunología , Receptor fas/genética , Receptor fas/inmunología