RESUMEN
The role of HIV-specific CD8 T cell activity in the course of HIV infection and the way it affects the virus that resides in the latent reservoir resting memory cells is debated. The PBMC of HIV-infected patients contain HIV-specific CD8 T cells and their potential targets, CD4 T cells latently infected by HIV. CD4 T cells and CD8 T cells procured from PBMC of HIV-infected patients were co-incubated and analyzed: Formation of CD8 T cells and HIV-infected CD4 T cell conjugates and apoptosis of these CD4 T cells were observed by fluorescence microscopy with in situ PCR of HIV LTR DNA. Furthermore, conjugation of CD8 T cells with CD4 T cells and apoptosis of CD4 T cells was observed and quantified by imaging flow cytometry using anti-human activated caspase 3 antibody and TUNEL assay. The conjugation activity and apoptosis were found to be much higher in patients with acute HIV infection or AIDS compared to patients in chronic infection on antiretroviral therapy (ART) or not. Patients on ART had low grade conjugation and apoptosis of isolated CD69, CD25, and HLA-DR-negative CD4 T cells (latent reservoir cells) by CD8 T cells. Using in situ PCR The latent reservoir CD4 T cells were shown to contain most of the HIV DNA. We demonstrate in HIV-infected patients, that CD8 T cells conjugate with and kill HIV-infected CD4 T cells, including HIV-infected resting memory CD4 T cells, throughout the course of HIV infection. We propose that in HIV-infected patients CD4 T cell annihilation is caused in part by ongoing activity of HIV-specific CD8 T cells. HIV Nef protein interacts with ASK 1 and inhibits its pro-apoptotic death signaling by Fas/FasL, thus protecting HIV-infected cells from CD8 T cells killing. A peptide that interrupts Nef-ASK1 interaction that had been delivered into CD4 T cells procured from patients on ART resulted in the increase of their apoptosis inflicted by autologous CD8 T cells. We suggest that elimination of the HIV-infected latent reservoir CD4 T cells can be achieved by Nef inhibition.
Asunto(s)
Apoptosis/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Inmunidad Celular , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/inmunología , Adulto , Apoptosis/efectos de los fármacos , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/patología , Linfocitos T CD8-positivos/virología , ADN Viral/inmunología , Proteína Ligando Fas/inmunología , Femenino , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/patología , Humanos , Memoria Inmunológica/efectos de los fármacos , MAP Quinasa Quinasa Quinasa 5/inmunología , Masculino , Persona de Mediana Edad , Péptidos/farmacología , Receptor fas/inmunologíaRESUMEN
Peripheral blood mononuclear cells (PBMC) of untreated, HIV-infected patients contain HIV-specific CD8 T cells as well as their corresponding targets, HIV-infected CD4 T cells. To determine if CD4 T-cell depletion in HIV-infected patients may result from autologous CD8-CD4 T-cell interaction, CD8 and CD4 T cells procured from PBMC of acute and chronic untreated HIV-infected patients were sorted and co-incubated. Formation of CD8-CD4 T-cell conjugates was observed by fluorescence microscopy. Apoptosis of CD4 T cells in conjugation was recorded by digitized images and was further observed and measured by FACS using Annexin staining. Perforin expression in the CD8 T cells was measured using intracellular monoclonal perforin antibody staining. HIV DNA in the conjugated CD4 T cells was detected by in situ PCR. We found that 6·1 ± 0·5% of CD4 T cells from acute HIV-infected patients and 3·0 ± 0·5% from chronic HIV-infected patients formed CD8-CD4 T-cell conjugates. Annexin binding and cell morphology typical of apoptosis were observed in the conjugated CD4 T cells. The majority of CD8 T cells that had conjugated to CD4 T cells expressed perforin. The conjugated CD4 T cells exhibited nuclear HIV DNA. CD8 T cells and HIV-infected CD4 T cells, both procured from the PBMC of untreated HIV-infected patients, form conjugates. Apoptotic lytic activity has been observed in the conjugated CD4 T cells. We propose that CD4 T-cell annihilation in HIV-infected patients results, at least in part, from the interactions of perforin-rich CD8 T cells with autologous, HIV-infected CD4 T cells.
RESUMEN
The reason(s) why individual cytotoxic T lymphocytes (CTL) possess a fast-acting, perforin/granzyme-mediated, as well as a much slower, Fas ligand (FasL) -driven killing mechanism is not clear, nor is the basis for wide variations in killing activity exhibited by individual CTL, ranging from minutes to hours. We show that perforin expression among individual, conjugated CTL varies widely, which can account for the heterogeneity in killing speeds exhibited by individual CTL. Despite a 2-hr lag in FasL-based killing, CTL lytic action is enhanced when the two mechanisms operate in concert. This is explained by finding that the two pathways in fact are jump-started simultaneously with the lag in FasL lytic action reflecting pre-lytic caspase-8 activation and BH3-interacting domain (BID) cleavage. The complementary action of the two lytic pathways, co-expressed at varying levels among individual CTL, facilitates the lytic action of late-stage poor perforin-expressing CTL, ensuring optimal cytocidal action throughout the CTL response.
Asunto(s)
Proteína Ligando Fas/metabolismo , Perforina/metabolismo , Linfocitos T Citotóxicos/inmunología , Animales , Western Blotting , Citotoxinas/metabolismo , Citometría de Flujo , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Perforina/genética , Subgrupos de Linfocitos T/inmunologíaRESUMEN
Advances in molecular cell biology, medical research, and drug development are driving a growing need for technologies that enable imaging the dynamics of molecular and physiological processes simultaneously in numerous non-adherent living cells. Here we describe a platform technology and software--the CKChip system--that enables continuous, fluorescence-based imaging of thousands of individual living cells, each held at a given position ("address") on the chip. The system allows for sequential monitoring, manipulation and kinetic analyses of the effects of drugs, biological response modifiers and gene expression in both adherent and non-adherent cells held on the chip. Here we present four specific applications that demonstrate the utility of the system including monitoring kinetics of reactive oxygen species generation, assessing the intracellular enzymatic activity, measuring calcium flux and the dynamics of target cell killing induced by conjugated cytotoxic T-lymphocytes. We found large variations among individual cells in the overall amplitude of their response to stimuli, as well as in kinetic parameters such as time of onset, initial rate and decay of the response, and frequency and amplitude of oscillations. These variations probably reflect the heterogeneity of even cloned cell populations that would have gone undetected in bulk cell measurements. We demonstrate the utility of the system in providing kinetic parameters of complex cellular processes such as Ca++ influx, transients and oscillations in numerous individual cells. The CKChip opens up new opportunities in cell-based research, in particular for acquiring fluorescence-based, kinetic data from multiple, individual non-adherent cells.
Asunto(s)
Técnicas de Cultivo de Célula/instrumentación , Técnicas Citológicas/instrumentación , Animales , Calcio/inmunología , Línea Celular Tumoral , Fenómenos Fisiológicos Celulares , Diseño de Equipo , Humanos , Inmunoglobulina E/inmunología , Células Jurkat , Ratones , Ratones Endogámicos C57BL , Especies Reactivas de Oxígeno/metabolismo , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Although CD8(+) cytotoxic T lymphocytes (CTL) exhibit both Fas ligand (FasL) -based and perforin-based lytic activities, the accepted hallmark of a fully active CTL remains its perforin killing machinery. Yet the origin, rationale for possessing both a slow-acting (FasL) and a fast-acting (perforin) killing mechanism has remained enigmatic. Here we have investigated perforin expression in CTL directly involved in acute tumour (i.e. leukaemias EL4 and L1210) allograft rejection occurring within the peritoneal cavity. We show that at the height of the immune response, the majority of conjugate-forming CD8(+) CTL express high levels of perforin messenger RNA and protein, and kill essentially via perforin. Later however, coinciding with complete rejection, fully cytocidal CTL emerge which exhibit a stark decrease in perforin and now kill preferentially via constitutively expressed FasL. Although late in emergence, and persistent, these powerful CTL are neither effector-memory nor memory CTL. This finding has implications for the monitoring of anti-transplant responses in clinical settings, based on assessing perforin expression in graft infiltrating CD8(+) T cells. The results show that as the immune response progresses in vivo, targeted cellular suicide mainly prunes high perforin-expressing CD8(+) cells, resulting in the gradual switch in effector CTL, from mostly perforin-based to largely Fas/FasL-based killers. Hence, two kinds of CD8(+) CTL have two killing strategies.
Asunto(s)
Leucemia Experimental/inmunología , Perforina/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Línea Celular Tumoral , Citotoxicidad Inmunológica/inmunología , Proteína Ligando Fas/inmunología , Rechazo de Injerto/inmunología , Memoria Inmunológica/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Perforina/deficienciaRESUMEN
Both the function and regulation of Fas expression in tumours is poorly understood. Our laboratory has reported that cultured, low Fas-expressing tumours undergo massive, yet reversible, up-regulation of cell surface Fas expression when injected into mice. The present study was aimed at determining what causes this enhanced Fas expression and whether the newly expressed Fas functions as a death receptor. Newly expressed Fas is indeed capable of inducing apoptosis. Based on our observation that Fas induction is reduced when tumour cells are injected into immune-deficient mice, we propose that Fas up-regulation in vivo involves the host's immune system. Accordingly, Fas up-regulation occurs in vitro when low Fas-expressing tumour cells are cocultured with lymphoid cells. Furthermore ascitic fluid extracted from tumour-bearing mice trigger Fas up-regulation in low Fas expressing tumours. This last finding suggests that a soluble factor(s) mediates induction of Fas expression. The best candidate for this soluble factor is nitric oxide (NO) based on the following observations: the factor in the ascites is unstable; Fas expression is induced to a lesser degree after injection into inducible NO synthase (NOS)-deficient (iNOS(-/-)) mice when compared to control mice; similarly, coculture with iNOS(-/-) splenocytes induces Fas less effectively than coculture with control splenocytes; and finally, the NO donor SNAP induces considerable Fas up-regulation in tumours in vitro. Our model is that host lymphoid cells in response to a tumour increase NO synthesis, which in turn causes enhanced Fas expression in the tumour.
Asunto(s)
Antígenos de Neoplasias/biosíntesis , Neoplasias Experimentales/inmunología , Regulación hacia Arriba/inmunología , Receptor fas/biosíntesis , Animales , Antígenos de Neoplasias/genética , Apoptosis , Citotoxicidad Inmunológica , Regulación Neoplásica de la Expresión Génica , Ratones , Ratones Endogámicos , Trasplante de Neoplasias , Óxido Nítrico/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Bazo/citología , Bazo/inmunología , Receptor fas/genéticaRESUMEN
Effector cells of the innate immune system have diverse functions that can result in tumour inhibition or tumour progression. Activation of macrophages by CD40 ligation has been shown to induce antitumour effects in vitro and in vivo. Here we investigated the role of nitric oxide (NO) and tumour necrosis factor-alpha (TNF-alpha) as mediators in the tumoristatic effects of murine peritoneal macrophages activated with agonistic anti-CD40 monoclonal antibody (alphaCD40) alone and following further stimulation with bacterial lipopolysaccharide (LPS). We found that macrophages activated in vivo by alphaCD40 exhibited tumoristatic activity in vitro against B16 melanoma cells; the tumoristatic effect correlated with the level of NO production and was enhanced by LPS. Use of the NO inhibitor L-nitro-arginine-methyl esterase (L-NAME) and evaluation of macrophages from inducible NO synthase (iNOS)-knockout (KO) mice following alphaCD40 activation showed reduced tumoristatic activity. CD40 ligation enhanced expression of TNF-alpha. Macrophage tumoristatic activity following alphaCD40 treatment was reduced by TNF-alpha mAb or use of macrophages from TNF-alpha-KO mice. However, further stimulation of alphaCD40-activated macrophages with LPS resulted in strong tumoristatic activity that was much less dependent on NO or TNF-alpha. Taken together, these results suggest that NO and TNF-alpha are involved in, but not solely responsible for, the antitumour effects of macrophages after activation by CD40 ligation.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos CD40/inmunología , Melanoma Experimental/inmunología , Óxido Nítrico/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Inhibidores Enzimáticos/farmacología , Femenino , Lipopolisacáridos/inmunología , Activación de Macrófagos/inmunología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/antagonistas & inhibidores , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
We have previously demonstrated T cell-independent antitumor and antimetastatic effects of CD40 ligation that involved natural killer (NK) cells. As CD40 molecules are expressed on the surface of macrophages (Mphi), we hypothesized that Mphi may also serve as antitumor effector cells when activated by CD40 ligation. Progression of subcutaneous NXS2 murine neuroblastomas was delayed significantly by agonistic CD40 monoclonal antibody (anti-CD40 mAb) therapy in immunocompetent A/J mice, as well as in T and B cell-deficient severe combined immunodeficiency (SCID) mice. Although NK cells can be activated by anti-CD40 mAb, anti-CD40 mAb treatment also induced a significant antitumor effect in SCID/beige mice in the absence of T and NK effector cells, even when noncytolytic NK cells and polymorphonuclear cells (PMN) were depleted. Furthermore, in vivo treatment with anti-CD40 mAb resulted in enhanced expression of cytokines and cell surface activation markers, as well as Mphi-mediated tumor inhibition in A/J mice, C57BL/6 mice, and SCID/beige mice, as measured in vitro. A role for Mphi was shown by reduction in the antitumor effect of anti-CD40 mAb when Mphi functions were inhibited in vivo by silica. In addition, activation of peritoneal Mphi by anti-CD40 mAb resulted in survival benefits in mice bearing intraperitoneal tumors. Taken together, our results show that anti-CD40 mAb immunotherapy of mice can inhibit tumor growth in the absence of T cells, NK cells, and PMN through the involvement of activated Mphi.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígenos CD40/fisiología , Inmunoterapia , Macrófagos/fisiología , Neuroblastoma/terapia , Animales , Anticuerpos Monoclonales/farmacología , Presentación de Antígeno , Línea Celular Tumoral/inmunología , Citocinas/fisiología , Citotoxicidad Inmunológica , Femenino , Células Asesinas Naturales/inmunología , Macrófagos Peritoneales/fisiología , Melanoma Experimental/inmunología , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos A , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones SCID , Trasplante de Neoplasias , Neuroblastoma/inmunología , Neutrófilos/inmunología , Ratas , Tejido Subcutáneo , Linfocitos T/inmunologíaRESUMEN
We have previously shown that macrophages (Mphi) can be activated by CD40 ligation to become cytotoxic against tumor cells in vitro. Here we show that treatment of mice with agonistic anti-CD40 mAb (anti-CD40) induced up-regulation of intracellular TLR9 in Mphi and primed them to respond to CpG-containing oligodeoxynucleotides (CpG), resulting in synergistic activation. The synergy between anti-CD40 and CpG was evidenced by increased production of IFN-gamma, IL-12, TNF-alpha, and NO by Mphi, as well as by augmented apoptogenic effects of Mphi against tumor cells in vitro. The activation of cytotoxic Mphi after anti-CD40 plus CpG treatment was dependent on IFN-gamma but not TNF-alpha or NO, and did not require T cells and NK cells. Anti-CD40 and CpG also synergized in vivo in retardation of tumor growth in both immunocompetent and immunodeficient mice. Inactivation of Mphi in SCID/beige mice by silica treatment abrogated the antitumor effect. Taken together, our results show that Mphi can be activated via CD40/TLR9 ligation to kill tumor cells in vitro and inhibit tumor growth in vivo even in immunocompromised tumor-bearing hosts, indicating that this Mphi-based immunotherapeutic strategy may be appropriate for clinical testing.
Asunto(s)
Antígenos CD40/metabolismo , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Neoplasias Experimentales/inmunología , Linfocitos T/inmunología , Receptor Toll-Like 9/metabolismo , Animales , Apoptosis/inmunología , Antígenos CD40/inmunología , Línea Celular Tumoral , Islas de CpG/inmunología , Citocinas/biosíntesis , Citocinas/inmunología , Citotoxicidad Inmunológica/inmunología , Citometría de Flujo , Humanos , Inmunofenotipificación , Activación de Linfocitos/inmunología , Ratones , Receptor Toll-Like 9/inmunologíaRESUMEN
We have shown previously that agonistic anti-CD40 mAb induced T cell-independent antitumor effects in vivo. In this study, we investigated mechanisms of macrophage activation with anti-CD40 mAb treatment, assessed by the antitumor action of macrophages in vitro. Intraperitoneal injection of anti-CD40 mAb into C57BL/6 mice resulted in activation of peritoneal macrophages capable of suppressing B16 melanoma cell proliferation in vitro, an effect that was greatly enhanced by LPS and observed against several murine and human tumor cell lines. Anti-CD40 mAb also primed macrophages in vitro to mediate cytostatic effects in the presence of LPS. The tumoristatic effect of CD40 ligation-activated macrophages was associated with apoptosis and killing of tumor cells. Activation of macrophages by anti-CD40 mAb required endogenous IFN-gamma because priming of macrophages by anti-CD40 mAb was abrogated in the presence of anti-IFN-gamma mAb, as well as in IFN-gamma-knockout mice. Macrophages obtained either from C57BL/6 mice depleted of T and NK cells by Ab treatment, or from scid/beige mice, were still activated by anti-CD40 mAb to mediate cytostatic activity. These results argued against the role of NK and T cells as the sole source of exogenous IFN-gamma for macrophage activation and suggested that anti-CD40 mAb-activated macrophages could produce IFN-gamma. We confirmed this hypothesis by detecting intracytoplasmic IFN-gamma in macrophages activated with anti-CD40 mAb in vivo or in vitro. IFN-gamma production by macrophages was dependent on IL-12. Taken together, the results show that murine macrophages are activated directly by anti-CD40 mAb to secrete IFN-gamma and mediate tumor cell destruction.
Asunto(s)
Antígenos CD40/inmunología , Antígenos CD40/metabolismo , Interferón gamma/fisiología , Activación de Macrófagos/inmunología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Melanoma Experimental/inmunología , Melanoma Experimental/prevención & control , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Apoptosis/genética , Apoptosis/inmunología , Antígenos CD40/genética , Línea Celular Tumoral , Técnicas de Cocultivo , Citoplasma/inmunología , Citoplasma/metabolismo , Femenino , Humanos , Interferón gamma/deficiencia , Interferón gamma/genética , Células Jurkat , Células Asesinas Naturales/inmunología , Leucemia L5178 , Ligandos , Activación de Macrófagos/genética , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Linfocitos T/inmunologíaRESUMEN
The RUNX transcription factors are important regulators of lineage-specific gene expression. RUNX are bifunctional, acting both as activators and repressors of tissue-specific target genes. Recently, we have demonstrated that Runx3 is a neurogenic transcription factor, which regulates development and survival of proprioceptive neurons in dorsal root ganglia. Here we report that Runx3 and Runx1 are highly expressed in thymic medulla and cortex, respectively, and function in development of CD8 T cells during thymopoiesis. Runx3-deficient (Runx3 KO) mice display abnormalities in CD4 expression during lineage decisions and impairment of CD8 T cell maturation in the thymus. A large proportion of Runx3 KO peripheral CD8 T cells also expressed CD4, and in contrast to wild-type, their proliferation ability was largely reduced. In addition, the in vitro cytotoxic activity of alloimmunized peritoneal exudate lymphocytes was significantly lower in Runx3 KO compared with WT mice. In a compound mutant mouse, null for Runx3 and heterozygous for Runx1 (Runx3-/-;Runx1+/-), all peripheral CD8 T cells also expressed CD4, resulting in a complete lack of single-positive CD8+ T cells in the spleen. The results provide information on the role of Runx3 and Runx1 in thymopoiesis and suggest that both act as transcriptional repressors of CD4 expression during T cell lineage decisions.
Asunto(s)
Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Proteínas de Unión al ADN/fisiología , Proteínas Proto-Oncogénicas , Timo/citología , Factores de Transcripción/fisiología , Animales , Antígenos CD4/biosíntesis , Linfocitos T CD4-Positivos/metabolismo , División Celular , Linaje de la Célula , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Subunidad alfa 3 del Factor de Unión al Sitio Principal , Citometría de Flujo , Inmunohistoquímica , Ratones , Ratones Noqueados , Bazo/metabolismo , Timo/metabolismoRESUMEN
The recent advent of peptide-MHC tetramers has provided a new and effective tool for studying antigen-specific T cell populations through monitoring tetramer binding to T cells by flow cytometry. Yet information regarding T cell activation induced by the bound tetramers cannot be deduced from binding studies alone; complementary methods are needed to bridge this gap. To this end, we have developed a new approach that now enables monitoring both binding to and activation of T cells by peptide-MHC tetramers at the single-cell level. For this purpose, we have employed the CellScan, a non-flow cytometer designed for repetitive measurements of optical parameters (e.g., fluorescence intensity and polarization) of individual living cells. A melanoma-specific MART1 CTL line and a gp100-specific CTL clone were incubated with specific and control single-chain peptide-MHC tetramers for 45 min. Subsequently, the fluorescence intensity and polarization were measured by the CellScan. Specific binding of fluorescently labeled peptide-MHC tetramers to CTLs, recorded by the CellScan, was comparable to that measured by flow cytometry. CellScan monitoring of the degree of fluorescence polarization of fluorescein diacetate-labeled CTLs that were reacted with tetramers revealed specific activation of the CTLs, which was confirmed by cytokine (INF gamma) production. These results provide a new means of monitoring both the binding to and activation of T lymphocytes by cognate peptide-MHC complexes at the single-cell level, which can now be applied to distinguish between cognate responding and anergic T cells.
Asunto(s)
Polarización de Fluorescencia/métodos , Complejo Mayor de Histocompatibilidad/inmunología , Linfocitos T/inmunología , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Polarización de Fluorescencia/instrumentación , Humanos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Cinética , Activación de Linfocitos/inmunología , Melanoma/inmunología , Melanoma/metabolismo , Péptidos/inmunología , Péptidos/metabolismo , Linfocitos T/metabolismo , Células Tumorales CultivadasRESUMEN
Our previous studies have shown that intercellular communication mediated by gap junctions is impaired in most tumors as well as in cancer cell lines. However, connexin genes that encode gap junction proteins are only rarely mutated in cancer cells. On the other hand, it was reported that mutated Connexin 37 (Cx37) is the origin of shared tumor-associated antigenic octa-peptides (MUT 1 and MUT 2) of two independently derived lung carcinomas 3LL and CMT 64 of mouse origin. Two Cx37 mutations have been implicated: a Cys-54-Gln substitution in FEQNTAQP (MUT 1) and FEQNTAQA (MUT 2); an additional Pro-59-Ala substitution has been proposed in MUT 2. A Cys-54-Gln mutation in both tumors requires three base changes (TGT-to-CAG) to have occurred twice in independently derived tumors. Another complication stems from the fact that Cys 54, which is located in the extra-cellular domain is conserved in all connexins. Due to the important implications that these findings may have regarding the role of gap junctional communication in lung carcinomas as well as in the origin of tumor-associated antigens, we decided to re-examine these mutations. Thus, we PCR-amplified genomic DNA from 3LL and CMT and sequenced the coding region of Cx37 encompassing codon 54. We then analyzed the PCR products by digestion with the restriction enzyme MaeIII, to discern the presence of the putative mutation. Here we have unambiguously demonstrated that clones K(b)39.5 (39.5) and D122 of 3LL, and C6 and E9 of CMT 64, previously employed, have only normal Cx37 sequences, including those of codon 54. Therefore, we concluded that Cx37 is not mutated in 3LL and CMT 64 carcinomas.
Asunto(s)
Carcinoma Pulmonar de Lewis/genética , Carcinoma/genética , Conexinas/genética , Neoplasias Pulmonares/genética , Proteínas de Neoplasias/genética , Sustitución de Aminoácidos , Animales , Carcinoma/patología , Carcinoma Pulmonar de Lewis/patología , Células Clonales/trasplante , Codón/genética , Análisis Mutacional de ADN , ADN de Neoplasias/genética , Uniones Comunicantes/fisiología , Neoplasias Pulmonares/patología , Linfoma/genética , Linfoma/patología , Linfoma de Células T/genética , Linfoma de Células T/patología , Ratones , Ratones Endogámicos C57BL , Mutación Missense , Trasplante de Neoplasias , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Estructura Terciaria de Proteína , Análisis de Secuencia de ADN , Células Tumorales Cultivadas/trasplante , Proteína alfa-4 de Unión ComunicanteRESUMEN
OBJECTIVE: Since apoptosis is an important contributor to heart diseases in which ischemia and hypoxia are key elements, we tested the hypothesis that hypoxia predisposes neonatal rat ventricular myocytes (NRVM) to Fas-mediated apoptosis, by shifting the balance between antiapoptotic and proapoptotic proteins towards the latter. METHODS: Normoxic or hypoxic (22 h, 1% O(2)) cultured NRVM were exposed to recombinant Fas L (rFasL) for 7 h, and apoptosis measured thereafter. RESULTS: Whereas in normoxic NRVM, rFasL did not cause apoptosis measured by the TUNEL assay (4.8+/-0.5% in control versus 4.5+/-0.9% in rFasL), in hypoxic cultures rFasL increased the background apoptosis level by 100%. That Fas was functional in normoxic NRVM, despite its inability to mediate apoptosis, was evidenced by the finding that Fas activation increased the diastolic [Ca(2+)](i) levels measured by Fura 2 fluorescence, and caused arrhythmias. In support of our working hypothesis, hypoxia increased Fas expression by 200% (measured by quantitative Western blot), and the expression of the proapoptotic proteins ARTS and FADD by 323 and 250%, respectively, and decreased the expression of the antiapoptotic proteins ARC and FLIP by 90 and 60%, respectively. CONCLUSION: By upregulating Fas expression and key proapoptotic proteins, and by downregulating antiapoptotic proteins, hypoxia predisposes ventricular myocytes to Fas-induced apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas de Arabidopsis , Hipoxia/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/farmacología , Miocardio/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Proteínas Portadoras/metabolismo , Células Cultivadas , Proteína Ligando Fas , Ácido Graso Desaturasas/metabolismo , Etiquetado Corte-Fin in Situ , Proteínas Musculares/metabolismo , Ratas , Proteínas Recombinantes/farmacología , Receptor fas/metabolismoRESUMEN
The theory that Fas ligand (FasL)-expressing tumours are immune-privileged and can directly counterattack Fas-expressing effector T lymphocytes has recently been questioned and several alternative mechanisms have been proposed. To address this controversial issue, we analysed the impact of FasL-expressing tumours on in vivo-primed cytotoxic T lymphocytes (CTLs) and the mechanisms involved. CTLs were obtained from the peritoneal cavity (PEL) after in vivo priming with syngeneic or allogeneic murine tumour cells. We have found that PEL populations undergo Fas-based apoptotic cell death when co-cultured with FasL-expressing tumour cells and that PEL destruction of cognate targets in a 51Cr-release assay was markedly inhibited by the pre-exposure to either cognate or non-cognate tumour cells expressing FasL. Furthermore, cytocidal function of PEL was markedly inhibited by preincubation with FasL-negative tumour cells, if and only if they were the cognate targets of the CTL; this CTL inhibition involved FasL-Fas interactions. The killing function of 'bystander' PELs, reactive to a third-party target cell, was inhibited by co-cultivation with PELs mixed with their cognate target. This activation-induced CTL fratricide was not influenced by the expression of FasL on the cognate target cells. These studies demonstrate the existence of two distinct pathways whereby FasL-expressing cells inhibit in vivo-primed FasL- and Fas-expressing CTLs: first, by FasL-based direct tumour counterattack, and second, by FasL-mediated activation-induced cell death of the CTLs, which is consistent with the concept that FasL expression in vivo could play a role in inducing immune privilege.
Asunto(s)
Tolerancia Inmunológica , Glicoproteínas de Membrana/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Apoptosis/inmunología , Líquido Ascítico/inmunología , Citotoxicidad Inmunológica , Proteína Ligando Fas , Ligandos , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos , Células Tumorales CultivadasRESUMEN
BACKGROUND: We used the CellScan, a novel static cytometer, to monitor changes induced by anti-neoplastic drugs in the fluorescence intensity and polarization of fluorescently-labeled tumor cells. MATERIALS AND METHODS: T47D and T80 human breast cancer cell lines were exposed to navelbine and to 5-fluorouracil and the fluorescence properties of the treated cells, stained with fluorescein diacetate and rhodamine 123, were measured by the CellScan. RESULTS: A strong correlation was found between the inhibition of cell growth induced by the two drugs, as estimated from cell counts, and the resulting changes in fluorescence intensity and polarization, as monitored by the CellScan. Fluorescence hyperpolarization of the labeled cells occurred in conjunction with AnnexinV binding and propidium iodide exclusion, indicating that such hyperpolarization, resulting from drug action, reflects an early stage of apoptosis, as previously proposed. CONCLUSION: The system presented here could serve as the basis for assessing drug sensitivity or resistance of cancer cells derived from small biopsies of solid human tumors, thus eliminating prior tumor culturing and time-consuming assays.