RESUMEN
The HIV-1 virulence factor Nef promotes high-titer viral replication, immune escape, and pathogenicity. Nef interacts with interleukin-2-inducible T-cell kinase (Itk) and Bruton's tyrosine kinase (Btk), two Tec-family kinases expressed in HIV-1 target cells (CD4 T cells and macrophages, respectively). Using a cell-based bimolecular fluorescence complementation assay, here we demonstrate that Nef recruits both Itk and Btk to the cell membrane and induces constitutive kinase activation in transfected 293T cells. Nef homodimerization-defective mutants retained their interaction with both kinases but failed to induce activation, supporting a role for Nef homodimer formation in the activation mechanism. HIV-1 infection up-regulates endogenous Itk activity in SupT1 T cells and donor-derived peripheral blood mononuclear cells. However, HIV-1 strains expressing Nef variants with mutations in the dimerization interface replicated poorly and were significantly attenuated in Itk activation. We conclude that direct activation of Itk and Btk by Nef at the membrane in HIV-infected cells may override normal immune receptor control of Tec-family kinase activity to enhance the viral life cycle.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa/metabolismo , Membrana Celular/metabolismo , Infecciones por VIH/inmunología , VIH-1/inmunología , Proteínas Tirosina Quinasas/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/química , Agammaglobulinemia Tirosina Quinasa/genética , Antivirales/farmacología , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/virología , Multimerización de Proteína , Proteínas Tirosina Quinasas/genética , Transducción de Señal , Bibliotecas de Moléculas Pequeñas/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/virología , Replicación Viral , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Antiretroviral therapy has revolutionized the treatment of AIDS, turning a deadly disease into a manageable chronic condition. Life-long treatment is required because existing drugs do not eradicate HIV-infected cells. The emergence of drug-resistant viral strains and uncertain vaccine prospects highlight the pressing need for new therapeutic approaches with the potential to clear the virus. The HIV-1 accessory protein Nef is essential for viral pathogenesis, making it a promising target for antiretroviral drug discovery. Nef enhances viral replication and promotes immune escape of HIV-infected cells but lacks intrinsic enzymatic activity. Instead, Nef works through diverse interactions with host cell proteins primarily related to kinase signaling pathways and endosomal trafficking. This review emphasizes the structure, function, and biological relevance of Nef interactions with host cell protein-tyrosine kinases in the broader context of Nef functions related to enhancement of the viral life cycle and immune escape. Drug discovery targeting Nef-mediated kinase activation has allowed identification of promising inhibitors of multiple Nef functions. Pharmacological inhibitors of Nef-induced MHC-I down-regulation restore the adaptive immune response to HIV-infected cells in vitro and have the potential to enhance immune recognition of latent viral reservoirs as part of a strategy for HIV clearance.
Asunto(s)
Fármacos Anti-VIH/farmacología , VIH-1/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Antígenos CD4/metabolismo , Cristalografía por Rayos X , Regulación hacia Abajo , VIH-1/patogenicidad , Evasión Inmune , Complejo Mayor de Histocompatibilidad , Proteínas de la Membrana/metabolismo , Proteínas Quinasas/efectos de los fármacos , Transporte de Proteínas , Relación Estructura-Actividad , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
The HIV-1 Nef accessory factor enhances viral infectivity, immune evasion, and AIDS progression. Nef triggers rapid down-regulation of CD4 via the endocytic adaptor protein 2 (AP-2) complex, a process linked to enhanced viral infectivity and immune escape. Here, we describe a bimolecular fluorescence complementation (BiFC) assay to visualize the interaction of Nef with AP-2 and CD4 in living cells. Interacting protein pairs were fused to complementary non-fluorescent fragments of YFP and co-expressed in 293T cells. Nef interactions with both CD4 and AP-2 resulted in complementation of YFP and a bright fluorescent signal by confocal microcopy that localized to the cell periphery. Co-expression of the AP-2 α subunit enhanced the Nef·AP-2 σ2 subunit BiFC signal and vice versa, suggesting that the AP-2 α-σ2 hemicomplex interacts cooperatively with Nef. Mutagenesis of Nef amino acids Arg-134, Glu-174, and Asp-175, which stabilize Nef for AP-2 α-σ2 binding in a recent co-crystal structure, substantially reduced AP-2 interaction without affecting CD4 binding. A dimerization-defective mutant of Nef failed to interact with either CD4 or AP-2 in the BiFC assay, indicating that Nef quaternary structure is required for CD4 and AP-2 recruitment as well as CD4 down-regulation. A small molecule previously shown to bind the Nef dimerization interface also reduced Nef interactions with AP-2 and CD4 and restored CD4 expression to the surface of HIV-infected cells. Our findings provide a mechanistic explanation for previous observations that dimerization-defective Nef mutants fail to down-regulate CD4 and validate the Nef dimerization interface as a target site for antiretroviral drug development.
Asunto(s)
Antígenos CD4/metabolismo , Regulación hacia Abajo , VIH-1/metabolismo , Factor de Transcripción AP-2/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Línea Celular , Dimerización , Fluorescencia , Humanos , Modelos Moleculares , Unión Proteica , Fracciones Subcelulares/metabolismoRESUMEN
The HIV-1 Nef accessory factor enhances viral replication and promotes immune system evasion of HIV-infected cells, making it an attractive target for drug discovery. Recently we described a novel class of diphenylpyrazolodiazene compounds that bind directly to Nef in vitro and inhibit Nef-dependent HIV-1 infectivity and replication in cell culture. However, these first-generation Nef antagonists have several structural liabilities, including an azo linkage that led to poor oral bioavailability. The azo group was therefore replaced with either a one- or two-carbon linker. The resulting set of non-azo analogs retained nanomolar binding affinity for Nef by surface plasmon resonance, while inhibiting HIV-1 replication with micromolar potency in cell-based assays without cytotoxicity. Computational docking studies show that these non-azo analogs occupy the same predicted binding site within the HIV-1 Nef dimer interface as the original azo compound. Computational methods also identified a hot spot for inhibitor binding within this site that is defined by conserved HIV-1 Nef residues Asp108, Leu112, and Pro122. Pharmacokinetic evaluation of the non-azo B9 analogs in mice showed that replacement of the azo linkage dramatically enhanced oral bioavailability without substantially affecting plasma half-life or clearance. The improved oral bioavailability of non-azo diphenylpyrazolo Nef antagonists provides a starting point for further drug lead optimization in support of future efficacy testing in animal models of HIV/AIDS.
Asunto(s)
Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Administración Oral , Animales , Fármacos Anti-VIH/administración & dosificación , Sitios de Unión/efectos de los fármacos , Relación Dosis-Respuesta a Droga , VIH-1/efectos de los fármacos , Ratones , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Conformación Molecular , Simulación del Acoplamiento Molecular , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Bibliotecas de Moléculas Pequeñas/síntesis química , Relación Estructura-Actividad , Células Tumorales Cultivadas , Replicación Viral/efectos de los fármacosRESUMEN
The HIV-1 Nef accessory factor enhances the viral life cycle in vivo, promotes immune escape of HIV-infected cells, and represents an attractive antiretroviral drug target. However, Nef lacks enzymatic activity and an active site, complicating traditional occupancy-based drug development. Here we describe the development of proteolysis targeting chimeras (PROTACs) for the targeted degradation of Nef. Nef-binding compounds, based on an existing hydroxypyrazole core, were coupled to ligands for ubiquitin E3 ligases via flexible linkers. The resulting bivalent PROTACs induced formation of a ternary complex between Nef and the cereblon E3 ubiquitin ligase thalidomide-binding domain in vitro and triggered Nef degradation in a T cell expression system. Nef-directed PROTACs efficiently rescued Nef-mediated MHC-I and CD4 downregulation in T cells and suppressed HIV-1 replication in donor PBMCs. Targeted degradation is anticipated to reverse all HIV-1 Nef functions and may help restore adaptive immune responses against HIV-1 reservoir cells in vivo.
Asunto(s)
VIH-1 , Linfocitos T , Regulación hacia Abajo , Membrana Celular , Replicación Viral , Proteolisis , Ubiquitina-Proteína LigasasRESUMEN
In vitro studies have shown that deletion of nef and deleterious mutation in the Nef dimerization interface attenuates HIV replication and associated pathogenesis. Humanized rodents with human immune cells and lymphoid tissues are robust in vivo models for investigating the interactions between HIV and the human immune system. Here, we demonstrate that nef deletion impairs HIV replication and HIV-induced immune dysregulation in the blood and human secondary lymphoid tissue (human spleen) in bone marrow-liver-thymus-spleen (BLTS) humanized mice. Furthermore, we also show that nef defects (via deleterious mutations in the dimerization interface) impair HIV replication and HIV-induced immune dysregulation in the blood and human spleen in BLTS-humanized mice. We demonstrate that the reduced replication of nef-deleted and nef-defective HIV is associated with robust antiviral innate immune response, and T helper 1 response. Our results support the proposition that Nef may be a therapeutic target for adjuvants in HIV cure strategies.
Asunto(s)
Modelos Animales de Enfermedad , Infecciones por VIH , VIH-1 , Hígado , Bazo , Viremia , Replicación Viral , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Animales , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Ratones , Humanos , Viremia/inmunología , Bazo/inmunología , Bazo/virología , VIH-1/inmunología , VIH-1/genética , VIH-1/fisiología , Hígado/virología , Hígado/inmunología , Hígado/patología , Médula Ósea/virología , Médula Ósea/inmunología , Timo/inmunología , Timo/virología , Inmunidad InnataRESUMEN
Humanized mice have been used to study human immunodeficiency virus type 1 (HIV-1) transmission, pathogenesis, and treatment. The ability of pediatric thymus tissue implanted either in the leg (Leg PedThy) or under the renal capsule (Renal PedThy) with allogeneic CD34+ hematopoietic cells (HSCs) in NSG mice was evaluated for reconstitution of human immune cells and for rectal transmission of HIV-1. These mice were compared to traditional BLT mice implanted with fetal liver and thymus under the renal capsule and mice injected only with HSCs. Renal PedThy mice had similar immune reconstitution in the blood, spleen and intestine as BLT mice, while Leg PedThy mice had transient detection of immune cells, particularly CD4+ T cells and macrophages, the target cells for HIV-1 infection. Rectal transmission and replication of HIV-1 was efficient in BLT mice but lower and more variable in Renal PedThy mice. HIV-1 was poorly transmitted in HSC mice and not transmitted in Leg PedThy mice, which correlated with the frequencies of target cells in the spleen and intestine. Humanization of NSG mice with pediatric thymus was successful when implanted under the kidney capsule, but led to less efficient HIV-1 rectal transmission and replication compared to BLT mice.
Asunto(s)
Infecciones por VIH , VIH-1 , Ratones , Humanos , Animales , Niño , Modelos Animales de Enfermedad , Timo/patología , Linfocitos T CD4-Positivos , Ratones SCID , Ratones Endogámicos NODRESUMEN
The HIV-1 Nef accessory factor is critical to the viral life cycle in vivo where it promotes immune escape of HIV-infected cells and viral persistence. While these features identify Nef as an attractive antiretroviral drug target, Nef lacks enzymatic activity and an active site, complicating development of occupancy-based drugs. Here we describe the development of proteolysis targeting chimeras (PROTACs) for the targeted degradation of Nef. Nef-binding compounds, based on a previously reported hydroxypyrazole core, were coupled to ligands for ubiquitin E3 ligases via flexible linkers. The resulting bivalent PROTACs induced formation of a ternary complex between Nef and the Cereblon E3 ubiquitin ligase, resulting in ubiquitylation of Nef and proteolytic degradation. Nef-directed PROTACs efficiently rescued Nef-mediated MHC-I and CD4 downregulation in T cells and suppressed HIV-1 replication in donor PBMCs. Targeted degradation of Nef is anticipated to reverse all HIV-1 Nef functions and may help restore adaptive immune responses against HIV-1 reservoir cells in vivo .
RESUMEN
While antiretroviral drugs have transformed the lives of HIV-infected individuals, chronic treatment is required to prevent rebound from viral reservoir cells. People living with HIV also are at higher risk for cardiovascular and neurocognitive complications, as well as cancer. Finding a cure for HIV-1 infection is therefore an essential goal of current AIDS research. This review is focused on the discovery of pharmacological inhibitors of the HIV-1 Nef accessory protein. Nef is well known to enhance HIV-1 infectivity and replication, and to promote immune escape of HIV-infected cells by preventing cell surface MHC-I display of HIV-1 antigens. Recent progress shows that Nef inhibitors not only suppress HIV-1 replication, but also restore sufficient MHC-I to the surface of infected cells to trigger a cytotoxic T lymphocyte response. Combining Nef inhibitors with latency reversal agents and therapeutic vaccines may provide a path to clearance of viral reservoirs.
Asunto(s)
Infecciones por VIH , Seropositividad para VIH , VIH-1 , Antirretrovirales/uso terapéutico , Descubrimiento de Drogas , Infecciones por VIH/tratamiento farmacológico , VIH-1/fisiología , Humanos , Factores de Virulencia , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
BACKGROUND: Osteoblastic bone metastasis is the predominant phenotype observed in prostate cancer patients and is associated with high patient mortality and morbidity. However, the mechanisms determining the development of this phenotype are not well understood. Prostate cancer cells secrete several osteogenic factors including Wnt proteins, which are not only osteoinductive but also oncogenic. Therefore, the purpose of the study was to investigate the contribution of the Wnt signaling pathway in prostate cancer growth, incidence of bone metastases, and osteoblastic phenotype of bone metastases. The strategy involved overexpressing the Wnt antagonist, DKK-1, in the mixed osteoblastic and osteolytic Ace-1 prostate cancer cells. METHODS: Ace-1 prostate cancer cells stably expressing human DKK-1 or empty vector were established and transduced with lentiviral yellow fluorescent protein (YFP)-luciferase (Luc). The Ace-1/vector(YFP-LUC) and Ace-1/DKK-1(YFP-LUC) cells were injected subcutaneously, intratibially, or in the left cardiac ventricle in athymic mice. RESULTS: Unexpectedly, DKK-1 significantly increased Ace-1 subcutaneous tumor mass and the incidence of bone metastases after intracardiac injection of Ace-1 cells. DKK-1 increased Ace-1 tumor growth associated with increased phospho46 c-Jun amino-terminal kinase by the Wnt noncanonical pathway. As expected, DKK-1 decreased the Ace-1 osteoblastic phenotype of bone metastases, as confirmed by radiographic, histopathologic, and microcomputed tomographic analysis. DKK-1 decreased osteoblastic activity via the Wnt canonical pathway evidenced by an inhibition of T-cell factor activity in murine osteoblast precursor ST2 cells. CONCLUSION: The present study showed that DKK-1 is a potent inhibitor of bone growth in prostate cancer-induced osteoblastic metastases.
Asunto(s)
Neoplasias Óseas/secundario , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Neoplasias de la Próstata/patología , Proteínas Wnt/metabolismo , Animales , Neoplasias Óseas/metabolismo , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Perros , Histocitoquímica , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Ratones , Ratones Desnudos , Osteogénesis/fisiología , Neoplasias de la Próstata/metabolismo , Transducción de Señal , Estadísticas no Paramétricas , Tomografía Computarizada por Rayos X , Proteínas Wnt/antagonistas & inhibidoresRESUMEN
BACKGROUND: Prostate cancer in men has a high mortality and morbidity due to metastatic disease. The pathobiology of prostate cancer metastasis is not well understood and cell lines and animal models that recapitulate the complex nature of the disease are needed. Therefore, the goal of the study was to establish and characterize a new prostate cancer line derived from a dog with spontaneous prostate cancer. METHODS: A new cell line (Leo) was derived from a dog with spontaneous prostate cancer. Immunohistochemistry and PCR were used to characterize the primary prostate cancer and xenografts in nude mice. Subcutaneous tumor growth and metastases in nude mice were evaluated by bioluminescent imaging, radiography and histopathology. In vitro chemosensitivity of Leo cells to therapeutic agents was measured. RESULTS: Leo cells expressed the secretory epithelial cytokeratins (CK)8, 18, and ductal cell marker, CK7. The cell line grew in vitro (over 75 passages) and was tumorigenic in the subcutis of nude mice. Following intracardiac injection, Leo cells metastasized to the brain, spinal cord, bone, and adrenal gland. The incidence of metastases was greatest to the central nervous system (80%) with a lower incidence to bone (20%) and the adrenal glands (16%). In vitro chemosensitivity assays demonstrated that Leo cells were sensitive to Velcade and an HDAC-42 inhibitor with IC(50) concentrations of 1.9 nm and 0.95 µm, respectively. CONCLUSION: The new prostate cancer cell line (Leo) will be a valuable model to investigate the mechanisms of the brain and bone metastases.
Asunto(s)
Neoplasias Encefálicas/patología , Neoplasias Encefálicas/secundario , Carcinoma/patología , Carcinoma/secundario , Línea Celular Tumoral , Neoplasias de la Próstata/patología , Neoplasias de las Glándulas Suprarrenales/epidemiología , Neoplasias de las Glándulas Suprarrenales/secundario , Animales , Antineoplásicos/farmacología , Neoplasias Óseas/epidemiología , Neoplasias Óseas/secundario , Ácidos Borónicos/farmacología , Bortezomib , Neoplasias Encefálicas/epidemiología , Neoplasias Encefálicas/metabolismo , Pruebas de Carcinogenicidad , Carcinoma/epidemiología , Carcinoma/metabolismo , División Celular , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/patología , Perros , Inmunohistoquímica , Incidencia , Inyecciones Subcutáneas , Queratina-18/metabolismo , Queratina-7/metabolismo , Queratina-8/metabolismo , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias de Tejido Conjuntivo/secundario , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Fenilbutiratos/antagonistas & inhibidores , Pirazinas/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias de la Médula Espinal/epidemiología , Neoplasias de la Médula Espinal/secundario , Tejido Subcutáneo , Trasplante HeterólogoRESUMEN
The functions of parathyroid hormone-related protein (PTHrP) on morphogenesis, cell proliferation, apoptosis, and calcium homeostasis have been attributed to its N terminus. Evidence suggests that many of these effects are not mediated by the N terminus but by the midregion, a nuclear localization sequence (NLS), and C terminus of the protein. A knock-in mouse lacking the midregion, NLS, and C terminus of PTHrP (Pthrp(Delta/Delta)) was developed. Pthrp(Delta/Delta) mice had craniofacial dysplasia, chondrodysplasia, and kyphosis, with most mice dying by d 5 of age. In bone, there were fewer chondrocytes and osteoblasts per area, bone mass was decreased, and the marrow was less cellular, with erythroid hypoplasia. Cellular proliferation was impaired, and apoptosis was increased. Runx2, Ocn, Sox9, Crtl1, beta-catenin, Runx1, ephrin B2, cyclin D1, and Gata1 were underexpressed while P16/Ink4a, P21, GSK-3beta, Il-6, Ffg3, and Ihh were overexpressed. Mammary gland development was aberrant, and energy metabolism was deregulated. These results establish that the midregion, NLS, and C terminus of PTHrP are crucial for the commitment of osteogenic and hematopoietic precursors to their lineages, and for survival, and many of the effects of PTHrP on development are not mediated by its N terminus. The down-regulation of Runx1, Runx2, and Sox9 indicates that PTHrP is a modulator of transcriptional activation during stem cell commitment.
Asunto(s)
Apoptosis , Huesos/citología , Genes Letales , Hematopoyesis , Señales de Localización Nuclear/deficiencia , Proteína Relacionada con la Hormona Paratiroidea/fisiología , Animales , Western Blotting , Huesos/patología , Condrocitos/citología , Condrocitos/patología , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Técnicas de Sustitución del Gen , Técnicas para Inmunoenzimas , Masculino , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoblastos/citología , Osteoblastos/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de SupervivenciaRESUMEN
Non-receptor protein-tyrosine kinases regulate cellular responses to many external signals and are important drug discovery targets for cancer and infectious diseases. While many assays exist for the assessment of kinase activity in vitro, methods that report changes in tyrosine kinase activity in single cells have the potential to provide information about kinase responses at the cell population level. In this protocol, we combined bimolecular fluorescence complementation (BiFC), an established method for the assessment of protein-protein interactions, and immunofluorescence staining with phosphospecific antibodies to characterize changes in host cell tyrosine kinase activity in the presence of an HIV-1 virulence factor, Nef. Specifically, two Tec family kinases (Itk and Btk) as well as Nef were fused to complementary, non-fluorescent fragments of the Venus variant of YFP. Each kinase was expressed in 293T cells in the presence or absence of Nef and immunostained for protein expression and activity with anti-phosphotyrosine (pTyr) antibodies. Multi-color confocal microscopy revealed the interaction of Nef with each kinase (BiFC), kinase activity, and kinase protein expression. Strong BiFC signals were observed when Nef was co-expressed with both Itk and Btk, indicative of interaction, and a strong anti-pTyr immunoreactivity was also seen. The BiFC, pTyr, and kinase expression signals co-localized to the plasma membrane, consistent with Nef-mediated kinase activation in this subcellular compartment. Image analysis allowed calculation of pTyr-to-kinase protein ratios, which showed a range of responses in individual cells across the population that shifted upward in the presence of Nef and back down in the presence of a kinase inhibitor. This method has the potential to reveal changes in steady-state non-receptor tyrosine kinase activity and subcellular localization in a cell population in response to other protein-kinase interactions, information that is not attainable from immunoblotting or other in vitro methods.
RESUMEN
Parathyroid hormone-related protein (PTHrP) and macrophage inflammatory protein-1α (MIP-1α) are important factors that increase bone resorption and hypercalcemia in adult T-cell leukemia (ATL). We investigated the role of PTHrP and MIP-1α in the development of local osteolytic lesions in T-cell leukemia through overexpression in Jurkat T-cells. Injections of Jurkat-PTHrP and Jurkat-MIP-1α into the tibia and the left ventricle of NSG mice were performed to evaluate tumor growth and metastasis in vivo. Jurkat-pcDNA tibial neoplasms grew at a significantly greater rate and total tibial tumor burden was significantly greater than Jurkat-PTHrP neoplasms. Despite the lower tibial tumor burden, Jurkat-PTHrP bone neoplasms had significantly greater osteolysis than Jurkat-pcDNA and Jurkat-MIP-1α neoplasms. Jurkat-PTHrP and Jurkat-pcDNA cells preferentially metastasized to bone following intracardiac injection, though the overall metastatic burden was lower in Jurkat-PTHrP mice. These findings demonstrate that PTHrP induced pathologic osteolysis in T-cell leukemia but did not increase the incidence of skeletal metastasis.
Asunto(s)
Neoplasias Óseas , Hipercalcemia , Leucemia-Linfoma de Células T del Adulto , Osteólisis , Adulto , Animales , Humanos , Hipercalcemia/etiología , Ratones , Osteólisis/etiología , Proteína Relacionada con la Hormona Paratiroidea/genéticaRESUMEN
BACKGROUND: Adult T-cell leukemia/lymphoma (ATLL) is initiated by infection with human T-lymphotropic virus type-1 (HTLV-1); however, additional host factors are also required for T-cell transformation and development of ATLL. The HTLV-1 Tax protein plays an important role in the transformation of T-cells although the exact mechanisms remain unclear. Parathyroid hormone-related protein (PTHrP) plays an important role in the pathogenesis of humoral hypercalcemia of malignancy (HHM) that occurs in the majority of ATLL patients. However, PTHrP is also up-regulated in HTLV-1-carriers and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients without hypercalcemia, indicating that PTHrP is expressed before transformation of T-cells. The expression of PTHrP and the PTH/PTHrP receptor during immortalization or transformation of lymphocytes by HTLV-1 has not been investigated. RESULTS: We report that PTHrP was up-regulated during immortalization of lymphocytes from peripheral blood mononuclear cells by HTLV-1 infection in long-term co-culture assays. There was preferential utilization of the PTHrP-P2 promoter in the immortalized cells compared to the HTLV-1-transformed MT-2 cells. PTHrP expression did not correlate temporally with expression of HTLV-1 tax. HTLV-1 infection up-regulated the PTHrP receptor (PTH1R) in lymphocytes indicating a potential autocrine role for PTHrP. Furthermore, co-transfection of HTLV-1 expression plasmids and PTHrP P2/P3-promoter luciferase reporter plasmids demonstrated that HTLV-1 up-regulated PTHrP expression only mildly, indicating that other cellular factors and/or events are required for the very high PTHrP expression observed in ATLL cells. We also report that macrophage inflammatory protein-1alpha (MIP-1alpha), a cellular gene known to play an important role in the pathogenesis of HHM in ATLL patients, was highly expressed during early HTLV-1 infection indicating that, unlike PTHrP, its expression was enhanced due to activation of lymphocytes by HTLV-1 infection. CONCLUSION: These data demonstrate that PTHrP and its receptor are up-regulated specifically during immortalization of T-lymphocytes by HTLV-1 infection and may facilitate the transformation process.
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Transformación Celular Viral , Virus Linfotrópico T Tipo 1 Humano/crecimiento & desarrollo , Leucocitos Mononucleares/virología , Proteína Relacionada con la Hormona Paratiroidea/biosíntesis , Supervivencia Celular , Células Cultivadas , Quimiocina CCL3/biosíntesis , Técnicas de Cocultivo , Productos del Gen tax/biosíntesis , Humanos , Receptor de Hormona Paratiroídea Tipo 1/biosíntesis , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Acute myelogenous leukemia (AML) is the most common hematologic malignancy in adults and is often associated with constitutive tyrosine kinase signaling. These pathways involve the nonreceptor tyrosine kinases Fes, Syk, and the three Src-family kinases expressed in myeloid cells (Fgr, Hck, and Lyn). In this study, we report remarkable anti-AML efficacy of an N-phenylbenzamide kinase inhibitor, TL02-59. This compound potently suppressed the proliferation of bone marrow samples from 20 of 26 AML patients, with a striking correlation between inhibitor sensitivity and expression levels of the myeloid Src family kinases Fgr, Hck, and Lyn. No correlation was observed with Flt3 expression or mutational status, with the four most sensitive patient samples being wild-type for Flt3. Kinome-wide target specificity profiling coupled with in vitro kinase assays demonstrated a narrow overall target specificity profile for TL02-59, with picomolar potency against the myeloid Src-family member Fgr. In a mouse xenograft model of AML, oral administration of TL02-59 for 3 weeks at 10 mg/kg completely eliminated leukemic cells from the spleen and peripheral blood while significantly reducing bone marrow engraftment. These results identify Fgr as a previously unrecognized kinase inhibitor target in AML and TL02-59 as a possible lead compound for clinical development in AML cases that overexpress this kinase independent of Flt3 mutations.
Asunto(s)
Antineoplásicos/uso terapéutico , Benzamidas/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Quinazolinas/uso terapéutico , Familia-src Quinasas/antagonistas & inhibidores , Administración Oral , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Benzamidas/administración & dosificación , Línea Celular Tumoral , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Inhibidores de Proteínas Quinasas/administración & dosificación , Proteínas Proto-Oncogénicas c-hck/antagonistas & inhibidores , Quinazolinas/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
HIV-1 Nef, a critical AIDS progression factor, represents an important target protein for antiretroviral drug discovery. Because Nef lacks intrinsic enzymatic activity, we developed an assay that couples Nef to the activation of Hck, a Src family member and Nef effector protein. Using this assay, we screened a large, diverse chemical library and identified small molecules that block Nef-dependent Hck activity with low micromolar potency. Of these, a diphenylpyrazolo compound demonstrated submicromolar potency in HIV-1 replication assays against a broad range of primary Nef variants. This compound binds directly to Nef via a pocket formed by the Nef dimerization interface and disrupts Nef dimerization in cells. Coupling of nonenzymatic viral accessory factors to host cell effector proteins amenable to high-throughput screening may represent a general strategy for the discovery of new antimicrobial agents.
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Antirretrovirales/química , Antirretrovirales/farmacología , VIH-1/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Línea Celular , Infecciones por VIH/tratamiento farmacológico , VIH-1/fisiología , Humanos , Simulación del Acoplamiento Molecular , Multimerización de Proteína/efectos de los fármacos , Proteínas Proto-Oncogénicas c-hck/metabolismo , Pirazoles/química , Pirazoles/farmacología , Replicación Viral/efectos de los fármacos , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Parathyroid hormone-related protein (PTHrP) and macrophage inflammatory protein-1α (MIP-1α) have been implicated in the pathogenesis of adult T-cell leukemia/lymphoma, but their effects on T-cells have not been well studied. Here we analyzed the functions of PTHrP and MIP-1α on T-cell growth and death both in vitro and in vivo by overexpressing either factor in human Jurkat T-cells. PTHrP or MIP-1α did not affect Jurkat cell growth in vitro, but PTHrP increased their sensitivity to apoptosis. Importantly, PTHrP and MIP-1α decreased both tumor incidence and growth in vivo. To investigate possible mechanisms, polymerase chain reaction (PCR) arrays and real-time reverse transcription (RT)-PCR assays were performed. Both PTHrP and MIP-1α increased the expression of several factors including signal transducer and activator of transcription 4, tumor necrosis factor α, receptor activator of nuclear factor κB ligand and death-associated protein kinase 1, and decreased the expression of inhibitor of DNA binding 1, interferon γ and CD40 ligand in Jurkat cells. In addition, MIP-1α also increased the expression of transcription factor AP-2α and PTHrP increased expression of the vitamin D3 receptor. These data demonstrate that PTHrP and MIP-1α exert a profound antitumor effect presumably by increasing the sensitivity to apoptotic signals through modulation of transcription and apoptosis factors in T-cells.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Quimiocina CCL3/genética , Regulación Leucémica de la Expresión Génica , Leucemia Experimental/genética , Proteína Relacionada con la Hormona Paratiroidea/genética , Animales , Apoptosis/genética , Ligando de CD40/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Línea Celular Tumoral , Proteínas Quinasas Asociadas a Muerte Celular , Humanos , Interferón gamma/genética , Células Jurkat , Leucemia Experimental/patología , Leucemia de Células T/genética , Leucemia de Células T/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Análisis de Secuencia por Matrices de Oligonucleótidos , Ligando RANK/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT4/genética , Linfocitos T/metabolismo , Linfocitos T/patología , Trasplante Heterólogo , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
BACKGROUND: Nef is an HIV-1 accessory protein essential for viral replication and AIDS progression. Nef interacts with a multitude of host cell signaling partners, including members of the Src kinase family. Nef preferentially activates Hck, a Src-family kinase (SFK) strongly expressed in macrophages and other HIV target cells, by binding to its regulatory SH3 domain. Recently, we identified a series of kinase inhibitors that preferentially inhibit Hck in the presence of Nef. These compounds also block Nef-dependent HIV replication, validating the Nef-SFK signaling pathway as an antiretroviral drug target. Our findings also suggested that by binding to the Hck SH3 domain, Nef indirectly affects the conformation of the kinase active site to favor inhibitor association. RESULTS: To test this hypothesis, we engineered a "gatekeeper" mutant of Hck with enhanced sensitivity to the pyrazolopyrimidine tyrosine kinase inhibitor, NaPP1. We also modified the RT loop of the Hck SH3 domain to enhance interaction of the kinase with Nef. This modification stabilized Nef:Hck interaction in solution-based kinase assays, as a way to mimic the more stable association that likely occurs at cellular membranes. Introduction of the modified RT loop rendered Hck remarkably more sensitive to activation by Nef, and led to a significant decrease in the Km for ATP as well as enhanced inhibitor potency. CONCLUSIONS: These observations suggest that stable interaction with Nef may induce Src-family kinase active site conformations amenable to selective inhibitor targeting.
RESUMEN
Oral squamous cell carcinoma (OSCC) is the most commonly diagnosed oral malignancy in humans and cats and frequently invades bone. The objective of this study was to determine if feline OSCC serves as a relevant model of human OSCC in terms of osteolytic behavior and expression of bone resorption agonists. Novel feline OSCC cell lines (SCCF2 and SCCF3) were derived from spontaneous carcinomas. Gene expression and osteolytic behavior were compared to an established feline OSCC cell line (SCCF1) and three human OSCC cell lines (UMSCC-12, A253 and SCC25). Interaction of OSCC with bone and murine pre-osteoblasts (MC3T3) was investigated using in vitro co-culture techniques. In vivo bioluminescent imaging, Faxitron radiography and microscopy were used to measure xenograft growth and bone invasion in nude mice. Human and feline OSCC expressing the highest levels of parathyroid hormone-related protein (PTHrP) were associated with in vitro and in vivo bone resorption and osteoclastogenesis. MC3T3 cells had increased receptor activator of nuclear factor κB ligand (RANKL) expression and reduced osteoprotegerin (OPG) expression in conditioned medium from bone-invasive SCCF2 cells compared to minimally bone invasive SCCF3 cells, which was partially reversed with a neutralizing anti-PTHrP antibody. Human and feline OSCC cells cultured in bone-conditioned medium had increased PTHrP secretion and proliferation. Feline OSCC-induced bone resorption was associated with tumor cell secretion of PTHrP and with increased RANKL:OPG expression ratio in mouse preosteoblasts. Bone-CM increased OSCC proliferation and secretion of PTHrP. The preclinical models of feline OSCC recapitulated the bone-invasive phenotype characteristic of spontaneous OSCC and will be useful to future preclinical and mechanistic studies of bone invasive behavior.