RESUMEN
Epidermal growth factor receptor (EGFR) regulates multiple signaling cascades essential for cell proliferation, growth and differentiation. Using a genetic approach, we found that Drosophila FERM and PDZ domain-containing protein tyrosine phosphatase, dPtpmeg, negatively regulates border cell migration and inhibits the EGFR/Ras/mitogen-activated protein kinase signaling pathway during wing morphogenesis. We further identified EGFR pathway substrate 15 (Eps15) as a target of dPtpmeg and its human homolog PTPN3. Eps15 is a scaffolding adaptor protein known to be involved in EGFR endocytosis and trafficking. Interestingly, PTPN3-mediated tyrosine dephosphorylation of Eps15 promotes EGFR for lipid raft-mediated endocytosis and lysosomal degradation. PTPN3 and the Eps15 tyrosine phosphorylation-deficient mutant suppress non-small-cell lung cancer cell growth and migration in vitro and reduce lung tumor xenograft growth in vivo. Moreover, depletion of PTPN3 impairs the degradation of EGFR and enhances proliferation and tumorigenicity of lung cancer cells. Taken together, these results indicate that PTPN3 may act as a tumor suppressor in lung cancer through its modulation of EGFR signaling.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Movimiento Celular , Proliferación Celular , Receptores ErbB/metabolismo , Neoplasias Pulmonares/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 3/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Animales Modificados Genéticamente , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular , Línea Celular Tumoral , Endocitosis , Femenino , Células HEK293 , Humanos , Immunoblotting , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Microdominios de Membrana/metabolismo , Ratones Endogámicos BALB C , Ratones Desnudos , Microscopía Confocal , Mutación , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 3/genética , Interferencia de ARN , Trasplante HeterólogoRESUMEN
BACKGROUND: Resiquimod, a toll-like receptor 7 and 8 agonist, may be effective as a topical treatment of actinic keratosis (AK). OBJECTIVES: To evaluate the effect of resiquimod gel concentration on lesion clearance. METHODS: Patients with AK lesions on the face or balding scalp were randomly assigned to resiquimod 0.01%, 0.03%, 0.06% or 0.1% gel applied once daily three times a week for 4 weeks to a contiguous 25-cm(2) area with four to eight lesions. Patients with persistent lesions received a second course after an 8-week treatment-free interval. Complete and partial lesion clearance was assessed 8 weeks after treatment for each course. RESULTS: For the 132 patients randomized, overall complete clearance rates were 77.1% (27/35), 90.3% (28/31), 78.1% (25/32) and 85.3% (29/34) and complete clearance rates after course 1 only were 40.0%, 74.2%, 56.3% and 70.6%, respectively, for the resiquimod 0.01%, 0.03%, 0.06% and 0.1% groups. During course 1, respectively 0%, 13%, 31% and 38% of patients discontinued treatment for adverse events or local skin reactions, for the resiquimod 0.01%, 0.03%, 0.06% and 0.1% groups. Possibly or probably related nonapplication site adverse events of severe intensity, including influenza-like symptoms, were reported by 0%, 3%, 13% and 12% of patients, respectively, for the resiquimod 0.01%, 0.03%, 0.06% and 0.1% groups. CONCLUSIONS: Efficacy in clearing AK lesions was similar between the resiquimod concentrations evaluated, but resiquimod 0.01% and 0.03% were better tolerated than the higher concentrations.
Asunto(s)
Imidazoles/uso terapéutico , Queratosis/tratamiento farmacológico , Lesiones Precancerosas/prevención & control , Neoplasias Cutáneas/prevención & control , Receptor Toll-Like 7/metabolismo , Administración Tópica , Anciano , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Geles , Humanos , Masculino , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , Regulación hacia Arriba/inmunologíaRESUMEN
Treatment failures for leishmaniasis with pentavalent antimonials, including meglumine antimonate, are increasingly common in many endemic areas. Imiquimod (Aldara; 3M Pharmaceuticals) is a novel immune response-activating compound, approved by the United States Food and Drug Administration, that is currently used to treat cervical warts and has been shown to activate macrophage killing of Leishmania species. Therefore, an open-label, prospective study was conducted of combined imiquimod plus meglumine antimonate therapy in 12 patients with cutaneous leishmaniasis who had previously not responded to meglumine antimonate therapy. All of the patients responded well to this combination therapy, and 90% were found to be cured at the 6-month follow-up period.
Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Aminoquinolinas/uso terapéutico , Antiprotozoarios/uso terapéutico , Resistencia a Medicamentos , Leishmaniasis Cutánea/tratamiento farmacológico , Meglumina/uso terapéutico , Compuestos Organometálicos/uso terapéutico , Adolescente , Adulto , Niño , Preescolar , Quimioterapia Combinada , Femenino , Humanos , Imiquimod , Lactante , Leishmaniasis Cutánea/patología , Masculino , Antimoniato de Meglumina , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
PURPOSE: Understanding cell proliferation regulation in hormone refractory prostate cancer may provide answers for novel solutions. Protein tyrosine phosphatases have been thought to have key roles in regulating cell proliferation and be involved in oncogenesis, although to our knowledge their functional roles in human prostate cancer remain unknown. Human prostatic acid phosphatase (PAcP), a major phosphatase in prostate epithelium, has been shown to function as a neutral protein tyrosine phosphatase in these cells. We evaluated the biological significance of cellular prostatic acid phosphatase expression in human prostate cancer cells. MATERIALS AND METHODS: Immunohistochemical testing of human prostate cancer archival specimens was done to evaluate the expression of cellular PAcP. Immunoprecipitation and immunoblotting were performed to determine cellular PAcP and SH2 domain-bearing tyrosine phosphatase-1 levels as well as tyrosine phosphorylation of c-ErbB-2/neu in different human prostate cancer cells. The biological behavior of LNCaP derivative sublines was characterized in vitro and in vivo by soft agar analysis and xenograft animal inoculation. RESULTS: Immunohistochemical staining of human prostate clearly showed that cellular levels of PAcP significantly decreases in prostate cancer cells (p <0.001). The results of biochemical characterization revealed that the cellular level of PAcP but not SHP-1, another differentiation associated protein tyrosine phosphatase, consistently correlated negatively with the growth of several human prostate cancer cell lines. Reintroducing cellular PAcP activity in prostate cancer cells by PAcP complementary DNA transfection resulted in decreased tyrosine phosphorylation of c-ErbB-2/neu, decreased proliferation rates in culture as well as decreased anchorage independent growth in soft agar. The xenograft animal model demonstrated that a higher tumor growth rate as well as larger size is associated with a lower level of cellular PAcP. CONCLUSIONS: Cellular PAcP can down-regulate prostate cancer cell growth, at least partially by dephosphorylating c-ErbB-2/neu. Therefore, decreased cellular PAcP expression in cancer cells may be involved in prostate cancer progression.
Asunto(s)
Neoplasias de la Próstata/enzimología , Proteínas Tirosina Fosfatasas/metabolismo , Receptor ErbB-2/metabolismo , Fosfatasa Ácida , Andrógenos/farmacología , Animales , Western Blotting , Regulación hacia Abajo , Femenino , Humanos , Inmunohistoquímica , Masculino , Ratones , Fosforilación , Próstata/citología , Próstata/enzimología , Proteína Fosfatasa 1 , Trasplante HeterólogoRESUMEN
Resiquimod (R-848), a topically active immune response modifier, induced production of interferon-alpha and interleukin-12 in cultured blood mononuclear cells and decreased genital herpes recurrences in an animal model. In this study, 52 patients with frequently recurrent genital herpes applied topical resiquimod gel 0.01% (twice or thrice weekly) or 0.05% (once or twice weekly) or vehicle gel to herpes lesions for 3 weeks. During the 6-month observation period after treatment, median days to first recurrence in the pooled resiquimod group was 169 days, compared with 57 days for the vehicle group (P=.0058). In all, 32% of resiquimod-treated patients completed the observation period without a recurrence, compared with 6% of vehicle-treated patients (P=.039). Resiquimod 0.05% twice weekly produced dose-limiting inflammation at the lesion sites, but the other regimens were well tolerated. Application of resiquimod to genital herpes lesions appeared to reduce the frequency of recurrences.
Asunto(s)
Herpes Genital/tratamiento farmacológico , Herpes Genital/prevención & control , Imidazoles/administración & dosificación , Factores Inmunológicos/administración & dosificación , Adulto , Método Doble Ciego , Femenino , Geles , Humanos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Modelos de Riesgos Proporcionales , Prevención Secundaria , Resultado del TratamientoRESUMEN
Steroid hormones play key roles in regulating cell proliferation and differentiation in targeting tissues. However, in advanced cancers, the steroid hormone regulation is frequently attenuated through a yet unknown mechanism even in the presence of functional steroid hormone receptors. We investigate the functional role of tyrosine phosphorylation signaling in the hormone-refractory growth of human prostate tumors. Initial studies demonstrate that the androgen-responsive phenotype of human prostate cancer cells associates with a low phosphotyrosine (p-Tyr) level of ErbB-2, which is regulated by cellular prostatic acid phosphatase (PAcP), a protein tyrosine phosphatase. In prostate cancer cells, the p-Tyr level, but not the protein level, of ErbB-2 inversely correlates with the androgen-responsiveness of cell proliferation. Androgen-stimulated cell growth concurs with a down-regulation of cellular PAcP, an elevated p-Tyr level of ErbB-2, and the activation of mitogen-activated protein kinases. Furthermore, only the ErbB-2 inhibitor AG 879, but not the EGFR inhibitor AG 1478, abolishes androgen-induced cell proliferation. Forced expression of ErbB-2 can also attenuate androgen promotion of cell growth. Data taken collectively conclude that in human prostate cancer cells, the tyrosine phosphorylation of ErbB-2 regulated by cellular PAcP plays a key role in regulating androgen-mediated proliferation signaling. Oncogene (2000).
Asunto(s)
Andrógenos/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Andrógenos/farmacología , División Celular/efectos de los fármacos , Humanos , Masculino , Fosforilación , Receptor ErbB-2/metabolismo , Transducción de Señal/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Human prostatic acid phosphatase (PAcP) is a prostate epithelium-specific differentiation antigen. In prostate carcinomas, the cellular PAcP is decreased. We investigated its functional role in these cells. Several lines of evidence support the hypothesis that cellular PAcP functions as a neutral protein-tyrosine phosphatase and is involved in regulating prostate cell growth. In this study, we identify its in vivo substrate. Our results demonstrated that, in different human prostate cancer cell lines, the phosphotyrosine (Tyr(P)) level of a 185-kDa phosphoprotein (pp185) inversely correlates with the cellular activity of PAcP. On SDS-PAGE, this pp185 co-migrates with the c-ErbB-2 oncoprotein. Immunodepletion experiments revealed that c-ErbB-2 protein is the major pp185 in cells. Results from subclones of LNCaP cells indicated the lower the cellular PAcP activity, the higher the Tyr(P) levels of c-ErbB-2. This inverse correlation was further observed in PAcP cDNA-transfected cells. In clone 33 LNCaP cells, L-(+)-tartrate suppresses the cellular PAcP activity and causes an elevated Tyr(P) level of c-ErbB-2 protein. Epidermal growth factor stimulates the proliferation of LNCaP cells, which concurs with a decreased cellular PAcP activity as well as an increased Tyr(P) level of c-ErbB-2. Biochemically, PAcP dephosphorylates c-ErbB-2 at pH 7.0. The results thus suggest that cellular PAcP down-regulates prostate cell growth by dephosphorylating Tyr(P) on c-ErbB-2 oncoprotein in those cells.
Asunto(s)
Fosfatasa Ácida/metabolismo , Próstata/enzimología , Neoplasias de la Próstata/enzimología , Receptor ErbB-2/metabolismo , Tirosina/metabolismo , Humanos , Masculino , Peso Molecular , Fosforilación , Tartratos/farmacología , Células Tumorales CultivadasRESUMEN
Excystation of Giardia lamblia entails differentiation of dormant cysts into parasitic trophozoites. Despite its importance for infection, this transformation is not understood at the cellular or molecular levels. In these studies, we report that excystation entails detection of environmental stimuli across the tough extracellular cyst wall leading to highly coordinated physiological, structural, and molecular responses. We found that novel cytoplasmic rearrangements and changes in individual species of mRNA and in cytoplasmic pH occur within the cyst wall in the earliest stage of excystation, in response to conditions modeling cyst ingestion and passage into the human stomach. This suggests that cysts do not contain all the mRNA needed for excystation and emergence and supports our hypothesis that external stimuli, including hydrogen ions, may penetrate or be perceived across the cyst wall. In contrast, changes in cyst wall structure or proteins were detected only later in excystation, in the stage that models passage into the human small intestine, where trophozoites can emerge and survive. These findings show that excystation of G. lamblia is a highly complex and active process and provide important insights into its cellular and molecular components.
Asunto(s)
Giardia lamblia/fisiología , Transcripción Genética , Animales , Western Blotting , Giardia lamblia/genética , Giardia lamblia/ultraestructura , Glicoproteínas/química , Glicoproteínas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Intestino Delgado/química , Microscopía Electrónica , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , ARN Mensajero/metabolismo , Estómago/química , Regulación hacia ArribaRESUMEN
Androgen plays a critical role in regulating the growth and differentiation of normal prostate epithelia, as well as the initial growth of prostate cancer cells. Nevertheless, prostate carcinomas eventually become androgen-unresponsive, and the cancer is refractory to hormonal therapy. To gain insight into the mechanism involved in this hormone-refractory phenomenon, we have examined the potential role of the androgen receptor (AR) in that process. We have investigated the expression of AR and two prostate-specific androgen-responsive antigens, prostatic acid phosphatase (PAcP) and prostate-specific antigen (PSA), for the functional activity of AR in LNCaP and PC-3 human prostate carcinoma cells. Our results are as follows. (i) Clone 33 LNCaP cells express AR, PAcP, and PSA, and cell growth is stimulated by 5alpha-dihydrotestosterone (DHT). Stimulation of cell growth correlates with decreased cellular PAcP activity. (ii) In clone 81 LNCaP cells, the expression of PAcP decreases with a concurrent decrease in the degree of androgen stimulation of cell growth, whereas the expression of PSA mRNA level is up-regulated by DHT, as in clone 33 cells. Conversely, in PAcP cDNA-transfected clone 81 cells, an additional expression of cellular PAcP correlates with an increased stimulation by androgen, higher than the corresponding control cells. (iii) PC-3 cells express a low level of functional AR with no detectable PAcP or PSA, and the growth of PC-3 cells is not affected by DHT treatment. Nevertheless, in two PAcP cDNA-transfected PC-3 sublines, the expression of exogenous cellular PAcP correlates with androgen stimulation. This androgen stimulation of cell growth concurs with an increased tyrosine phosphorylation of a phosphoprotein of 185 kDa. In summary, the data indicate that the expression of AR alone is not sufficient for androgen stimulation of cell growth. Furthermore, in AR-expressing prostate cancer cells, the expression of cellular PAcP correlates with androgen stimulation of cell proliferation.
Asunto(s)
Fosfatasa Ácida/metabolismo , Andrógenos/farmacología , Regulación Neoplásica de la Expresión Génica/genética , Neoplasias de la Próstata/enzimología , Receptores Androgénicos/fisiología , División Celular/efectos de los fármacos , Humanos , Masculino , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Fosfotirosina/análisis , Antígeno Prostático Específico/metabolismo , ARN Mensajero/metabolismo , Testosterona/análogos & derivados , Testosterona/farmacología , Transfección/genética , Células Tumorales Cultivadas , Regulación hacia Arriba/fisiologíaRESUMEN
Encystation of Giardia lamblia is required for survival outside the host, whereas excystation initiates infection. The dormant cyst was considered an adaptation to external survival and passage through the stomach. However, we found previously that trophozoites which had recovered after completion of the life cycle had switched their major variant surface protein (VSP), called TSA 417, but neither the timing nor the molecular mechanism of switching had been elucidated. Here we demonstrate that TSA 417 predominates in cysts, but is downregulated during the stage of excystation that models cyst arrival in the small intestine. Transcripts of new VSPs appear late in encystation, and during and after excystation. Trophozoites appear to prepare for switching during encystation, when the major VSP on the cell surface diminishes and is internalized in lysosome-like vacuoles. As short-range DNA rearrangements were not detected, giardial VSP switching during differentiation appears to resemble the in situ switching of surface glycoproteins in African trypanosomes. We also report a unique extended 15 nucleotide polyadenylation signal in all VSP transcripts, but not in other known giardial genes. Antigenic variation during encystation-excystation may be a novel form of immune evasion that could help explain the common occurrence of reinfection by Giardia and other parasites with similar life cycles.
Asunto(s)
Variación Antigénica , Antígenos de Protozoos/genética , Antígenos de Superficie/genética , Giardia lamblia/genética , Giardia lamblia/inmunología , Proteínas Protozoarias , Animales , Compartimento Celular , ADN Protozoario , Células Eucariotas , Reordenamiento Génico , Lisosomas , ARN ProtozoarioRESUMEN
We conducted a three-arm, randomized, phase II study to evaluate the combination of zidovudine (600 mg/day) and zalcitabine (2.25 mg/day) alone or with one of two interferon-alpha2a doses (1 mIU or 6 mIU daily). Primary study endpoints included toxicity and changes from baseline for plasma HIV-1 RNA, CD4 cells, and quantitative microculture at weeks 8 and 24. Sixty-three patients with HIV infection and <400 CD4 cells/mm3 were enrolled; four patients discontinued therapy within 2 weeks. Adverse event rates were 37%, 32%, and 60%, respectively, for the nucleoside, 1-mIU interferon, and 6-mIU interferon combination groups. Increasing doses of interferon resulted in significantly greater hematologic toxicity (p = 0.03) and peripheral neuropathy (p = 0.02). Plasma HIV-1 RNA reductions were noted across all treatment groups at week 8 (p < 0.001) but only for the nucleoside and 1-mIU interferon combination groups at week 24 (p < 0.001). Mean reductions in HIV-1 RNA at week 8 were 0.94, 1.29, and 1.40 log10, respectively, for the nucleoside, 1-mIU interferon, and 6-mIU interferon combination groups (p = 0.05); no differences were noted at week 24. No differences in CD4 cell counts were seen. The addition of interferon-alpha2a to zidovudine and zalcitabine resulted in transient enhanced decreases in viral load and increased toxicity.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Adulto , Fármacos Anti-VIH/efectos adversos , Esquema de Medicación , Quimioterapia Combinada , Femenino , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Humanos , Inyecciones Subcutáneas , Interferón alfa-2 , Interferón-alfa/administración & dosificación , Interferón-alfa/efectos adversos , Interferón-alfa/uso terapéutico , Masculino , Persona de Mediana Edad , Proteínas Recombinantes , Replicación Viral/efectos de los fármacos , Zalcitabina/efectos adversos , Zalcitabina/uso terapéutico , Zidovudina/efectos adversos , Zidovudina/uso terapéuticoRESUMEN
Although encystation and excystation are crucial to transmission of Giardia lamblia, little is known about the regulation of these very distinct differentiation processes. Fingerprinting of giardial mRNA populations throughout the time course of differentiation demonstrated complex patterns in mRNA differential display. Certain transcripts appeared or increased, while others decreased or disappeared at specific times, in response to physiologic stimuli that mimic key stages in parasite descent through the host gastrointestinal tract. This approach has allowed the direct identification of critical stages in differentiation, as well as isolation of genes which may be crucial to the development of G. lamblia. One stage-specific single copy gene (ENC6) whose transcript is greatly upregulated during encystation was analyzed further. Partial sequence analysis revealed no correspondence with known genes. 3'-rapid amplification of cDNA ends (3'-RACE) analysis of ENC6 transcripts at various times of encystation revealed two polyadenylation sites. The more proximal site, 10 nucleotides past the single classic AGTAAA sequence, was utilized only during encystation and its transcript increased approximately 16-fold during the first 24 h of encystation. In contrast, a slightly divergent polyadenylation site 288 nucleotides downstream from the open reading frame (ORF) was used during both vegetative growth and encystation, although its transcript was present at low levels. These studies are the first evidence of differential mRNA processing in G. lamblia and suggest a potential role of the 3'-untranslated region (3'-UTR) in modulating gene expression during differentiation of this primitive eukaryote.
Asunto(s)
Giardia lamblia/genética , Giardia lamblia/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Protozoario/genética , ARN Protozoario/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN/genética , ADN Protozoario/genética , Regulación del Desarrollo de la Expresión Génica , Genes Protozoarios , Giardia lamblia/crecimiento & desarrollo , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Procesamiento Postranscripcional del ARN , ARN Mensajero/química , ARN Protozoario/químicaRESUMEN
Human prostatic acid phosphatase (PAcP), a prostate epithelium-specific differentiation antigen, was determined to exhibit the endogenous protein tyrosine phosphatase activity. We investigated the phosphoprotein(s) that might be dephosphorylated by PAcP in human prostate carcinoma cells. Several lines of evidence were presented to show that the tyrosine phosphorylation level of a 185 kDa phosphoprotein (pp185) is negatively correlated with the cellular activity of PAcP. (i) In DU145, PC-3 and high passaged LNCaP prostate carcinoma cells that have no or low PAcP expression, the phosphotyrosine (p-tyr) level of pp185 was higher than that in low passaged LNCaP cells that express an endogenous PAcP. (ii) In LNCaP cells grown in the presence of L(+)-tartrate, an inhibitor of PAcP, the tyrosine phosphorylation of pp185 was increased. (iii) Mediated by Lipofectin, a cationic liposome, the incorporation of purified PAcP protein into DU145 cells resulted in the decreased phosphorylation of pp185. Thus, the results taken collectively demonstrated that the p-tyr level of pp185 is inversely correlated with the cellular activity of PAcP and indicated that the pp185 may be a putative substrate of PAcP in prostate carcinoma cells.
Asunto(s)
Fosfatasa Ácida/metabolismo , Neoplasias de la Próstata/enzimología , Tirosina/metabolismo , Fosfatasa Ácida/antagonistas & inhibidores , Humanos , Masculino , Fosforilación , Neoplasias de la Próstata/patología , Tartratos/farmacología , Células Tumorales CultivadasRESUMEN
Cellular oncogenes have been shown to play crucial roles in the cell death process induced by cytotoxic agents. In this study, we have demonstrated that v-H-ras transformed NIH 3T3 cells but not other transformants (v-raf, v-src, v-erbB-2, v-fes and v-mos) exhibited a survival advantage to treatment by a DNA-damaging agent, methylmethanesulfonate (MMS). Subsequently, the biochemical and morphologic criteria of MMS-treated cells were examined. It was found that MMS induced v-H-ras transformants to go through necrosis, but it induced other transformed cells to undergo apoptosis. The levels of glutathione (GSH) within each transformant as well as in NIH 3T3 cells, were determined. The results showed that GSH levels within ras transformants were 2- to 7-fold higher than the levels in other transformants and normal NIH 3T3 cells. By using the GSH synthesis inhibitor buthionine sulfoximine, GSH levels were artificially reduced. This depletion, however, made ras transformed cells more sensitive to MMS killing, but the mode of cell death was still necrosis. Western blot analysis demonstrated that the anti-apoptotic protein Bcl-2 was constitutively expressed in ras transformed cells but not in NIH 3T3 or other transformed cells. The level of Bcl-2 was correlated with the resistant phenotype of ras transformants during MMS treatment. These observations suggest that GSH and Bcl-2 levels may cooperatively confer the resistant phenotype of ras transformants in response to MMS. In addition, the mode of cell death may possibly be determined at least in part by Bcl-2 protein.
Asunto(s)
Células 3T3/efectos de los fármacos , Muerte Celular/efectos de los fármacos , ADN/efectos de los fármacos , Mesilatos/farmacología , Animales , Apoptosis , Células Cultivadas/efectos de los fármacos , Citometría de Flujo , RatonesAsunto(s)
Proteínas de Unión al Calcio/genética , Proteínas Cromosómicas no Histona , Genes Protozoarios , Giardia lamblia/genética , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Animales , Evolución Biológica , Proteínas de Unión al Calcio/aislamiento & purificación , Células Eucariotas , Flagelos/ultraestructura , Técnica del Anticuerpo Fluorescente , Giardia lamblia/ultraestructura , Datos de Secuencia Molecular , Proteínas Protozoarias/aislamiento & purificación , Homología de Secuencia de AminoácidoRESUMEN
Although excystation is crucial to the initiation of infection by Giardia lamblia, little is known about the regulation of this important process. We have been able to reliably induce excystation in vitro by mimicking cyst passage through the stomach and upper small intestine by the exposure of in vitro-derived cysts to an acidic, reducing environment (stage I) followed by protease treatment at a slightly alkaline pH (stage II). Preexposure of cysts to polyclonal rabbit antiserum against purified cyst walls (PCWs) or to wheat germ agglutinin (WGA) inhibited excystation by > 90%. Adsorption of either ligand with PCWs eliminated inhibition, demonstrating specificity for cyst wall epitopes. Inhibition by WGA was reversed by either chitotriose or sialic acid, while inhibition by polyclonal antibodies against PCWs (anti-PCW) was reversed only by sialic acid, which also inhibited binding of both ligands to intact cysts and to cyst wall antigens in immunoblots. Binding of anti-PCW did not affect acidification of cyst cytoplasm during stage I. Exposure of cysts to anti-PCW and WGA prior to, but not after, stage II was sufficient to inhibit excystation, and inhibition could be partially reversed by increasing the protease concentration during stage II. A 7- to 10-fold higher proportion of WGA- and anti-PCW-treated cysts than control cysts remained intact after stage II. Our results suggest that these ligands, which bind cyst wall epitopes, inhibit excystation, most likely by interfering with proteolysis of cyst wall glycoproteins during stage II.
Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Giardia lamblia/inmunología , Aglutininas del Germen de Trigo/farmacología , Animales , Giardia lamblia/fisiología , Concentración de Iones de Hidrógeno , ConejosRESUMEN
The c-jun proto-oncogene plays a vital role in the carcinogenic process. Although numerous works have extensively investigated the induction mechanisms of c-jun by UV, hydrogen peroxide or 12-O-tetradecanoylphorbol-13-acetate, the mechanism induced by alkylating agents has received little attention. In this study, NIH 3T3 cells were exposed to methylmethanesulfonate (MMS), revealing that the agent clearly induced c-jun expression with a peak at 2 h. Pretreatment of cells with various kinase inhibitors, e.g. H7, genistein, herbimycin A and tyrphostin, did not show any significant effects on the MMS-induced c-jun expression. Benzamide, an inhibitor of poly(ADP)-ribosylation, enhanced the MMS-induced DNA breakages, but did not potentiate that agent which elicited c-jun expression. Another experiment showed that this agent transfected and overexpressed an activated v-H-ras gene in NIH 3T3 cells, which became more resistant to MMS-induced DNA damage but expressed the same level of c-jun transcript as compared with NIH 3T3 cells in response to MMS. If intracellular glutathione (GSH) was completely depleted by buthionine sulfoximine (BSO), the MMS-elicited c-jun expression was blocked. Subsequently, re-elevating intracellular GSH by washing off BSO caused the expression of c-jun by MMS to increase proportionately. Based on these findings, we can conclude that the mechanism by which MMS induced c-jun expression does not occur through activation of protein tyrosine kinases or initiation of DNA damage, but is closely related to the intracellular GSH.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Glutatión/metabolismo , Metilmetanosulfonato/farmacología , Células 3T3 , Animales , Daño del ADN/genética , Inhibidores Enzimáticos/farmacología , Peróxido de Hidrógeno/farmacología , Cinética , Ratones , Inhibidores de Proteínas Quinasas , ARN Mensajero/genética , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
BACKGROUND: The clinical benefits of zidovudine remain unproved in patients with asymptomatic human immunodeficiency virus (HIV) infection when CD4 cell counts exceed 500 per cubic millimeter. We compared zidovudine therapy given immediately with deferred therapy in such subjects. METHODS: Beginning in 1987, subjects with asymptomatic HIV infection and 500 or more CD4 cells per cubic millimeter were randomly assigned to receive placebo or zidovudine (either 500 or 1500 mg per day, starting immediately). In 1989, the study was modified so that open-label treatment with 500 mg of zidovudine per day (deferred therapy) was offered when CD4 cell counts fell below 500 per cubic millimeter. The study end points included overall survival, survival free of the acquired immunodeficiency syndrome (AIDS), toxic effects, and changes in CD4 cell counts. RESULTS: There were 1637 subjects who could be evaluated: 547 in the deferred-therapy group, 549 in the group receiving 500 mg of zidovudine immediately, and 541 in the 1500-mg group. The subjects were followed for up to 6.5 years (group medians, 4.8, 4.8, and 4.9, respectively). There was no significant difference in AIDS-free survival in the deferred-therapy group as compared with the low-dose or high-dose groups (81 cases of progression to AIDS or death vs. 81 and 74, respectively; P = 0.95 and P = 0.13) or in overall survival (51 deaths vs. 47 and 46; P = 0.25 and P = 0.16). The decline in CD4 cells was slower in both immediate-therapy groups than in the deferred-therapy group (P < 0.001 for both). Adverse effects were uncommon, and before the study modification their incidence was similar among the treatment groups, but severe anemia and granulocytopenia were more frequent in the 1500-mg group than in the deferred-therapy group (P < 0.001). CONCLUSIONS: In asymptomatic, HIV-infected adults with 500 or more CD4 cells per cubic millimeter, treatment with zidovudine slows the decline in the CD4 cell count but does not significantly prolong either AIDS-free or overall survival. These results do not encourage the routine use of zidovudine monotherapy in this population.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/prevención & control , Infecciones por VIH/tratamiento farmacológico , Zidovudina/uso terapéutico , Adulto , Recuento de Linfocito CD4/efectos de los fármacos , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Método Doble Ciego , Estudios de Seguimiento , Infecciones por VIH/inmunología , Infecciones por VIH/mortalidad , Humanos , Masculino , Modelos de Riesgos Proporcionales , Análisis de Supervivencia , Factores de Tiempo , Zidovudina/administración & dosificación , Zidovudina/efectos adversosRESUMEN
To determine the effect of zidovudine (ZDV) on the pharmacokinetic disposition of recombinant soluble CD4 immunoglobulin G (rCD4-IgG) and to evaluate the safety and preliminary activity of concurrent administration of ZDV with rCD4-IgG, we undertook an open-label, dose-escalating, 12-week study. The regimens of intravenous rCD4-IgG and oral ZDV we used were (a) 300 micrograms/kg rCD4-IgG twice per week and 300 mg ZDV per day, (b) 300 micrograms/kg rCD4-IgG twice per week and 600 mg ZDV per day, (c) 1,000 micrograms/kg rCD4-IgG twice per week and 300 mg ZDV per day, (d) 1,000 micrograms/kg rCD4-IgG twice per week and 600 mg ZDV per day, and (e) 3,000 micrograms/kg rCD4-IgG twice per week and 300 mg ZDV per day. Subjects were recruited from three AIDS clinical trials units. Forty-one patients with HIV infection who had CD4 cell counts < or = 500 cells/mm3 and < 120 days of previous ZDV therapy participated. Pharmacokinetic interactions were assessed with the second regimen. Mean calculated peak serum rCD4-IgG concentrations were 5.47 micrograms/ml with ZDV and 8.28 micrograms/ml without ZDV, with serum half-lives of 34.2 and 32.0 h, respectively. Antibodies to rCD4-IgG were not detected. Seven episodes of severe adverse events occurred in five patients: one episode each of severe nausea, fever, or abnormal liver function tests and four episodes of severe neutropenia. Mean hemoglobin and neutrophil counts decreased, and mean platelet counts increased in all regimens, but there were no significant differences among regimens, rCD4-IgG dose, or ZDV dose.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Inmunoadhesinas CD4/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Zidovudina/uso terapéutico , Adulto , Inmunoadhesinas CD4/efectos adversos , Recuento de Linfocito CD4 , Interacciones Farmacológicas , Quimioterapia Combinada , Femenino , Proteína p24 del Núcleo del VIH/sangre , Infecciones por VIH/sangre , Infecciones por VIH/inmunología , Humanos , Masculino , Persona de Mediana Edad , Seguridad , Estados Unidos , Viremia , Zidovudina/efectos adversos , Zidovudina/farmacocinéticaRESUMEN
To ascertain whether combination therapy with zidovudine (AZT) and zalcitabine (ddC) delayed the emergence of AZT resistance, isolates of human immunodeficiency virus (HIV) were evaluated from 15 previously untreated patients with advanced HIV disease who received combination therapy. Eighteen sequential viral isolates were available from 15 patients who received > or = 6 months of combination therapy. Isolates from eight (73%) of 11 patients obtained between 24 and 48 weeks of therapy were AZT resistant [50% inhibitory concentration (IC50) > or = 0.45 microM; range, 0.45-2.0 microM]. Four of these eight patients yielded virus isolates that were highly AZT resistant (IC50 > 1.0 microM). No changes in ddC susceptibility were discerned. The median IC50 for ddC was 0.2 microM and ranged from 0.07 to 0.5 microM. The CD4 cell counts of patients with AZT-sensitive virus tended to have higher median areas under the curve (AUC) and greater increases compared with patients who had AZT-resistant virus. They also had higher means and ranges of the average CD4 cell counts at week 36 (p = 0.01) and week 48 (p = 0.04). These data would indicate that the previously described more sustained CD4 cell responses conferred by the addition of ddC to AZT therapy cannot be ascribed to delayed emergence of AZT resistance with combination therapy.