ABSTRACT
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.
Subject(s)
Disease Models, Animal , HIV Infections , HIV-1 , Liver , Spleen , Viremia , Virus Replication , nef Gene Products, Human Immunodeficiency Virus , Animals , nef Gene Products, Human Immunodeficiency Virus/genetics , nef Gene Products, Human Immunodeficiency Virus/immunology , HIV Infections/immunology , HIV Infections/virology , Mice , Humans , Viremia/immunology , Spleen/immunology , Spleen/virology , HIV-1/immunology , HIV-1/genetics , HIV-1/physiology , Liver/virology , Liver/immunology , Liver/pathology , Bone Marrow/virology , Bone Marrow/immunology , Thymus Gland/immunology , Thymus Gland/virology , Immunity, InnateABSTRACT
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.
Subject(s)
HIV-1 , T-Lymphocytes , Down-Regulation , Cell Membrane , Virus Replication , Proteolysis , Ubiquitin-Protein LigasesABSTRACT
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.
Subject(s)
HIV Infections , HIV-1 , Mice , Humans , Animals , Child , Disease Models, Animal , Thymus Gland/pathology , CD4-Positive T-Lymphocytes , Mice, SCID , Mice, Inbred NODABSTRACT
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 .
ABSTRACT
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.
Subject(s)
HIV Infections , HIV Seropositivity , HIV-1 , Anti-Retroviral Agents/therapeutic use , Drug Discovery , HIV Infections/drug therapy , HIV-1/physiology , Humans , Virulence Factors , nef Gene Products, Human Immunodeficiency VirusABSTRACT
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.
ABSTRACT
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.
Subject(s)
Anti-HIV Agents/pharmacology , HIV-1/enzymology , Protein Kinase Inhibitors/pharmacology , Protein Kinases/metabolism , nef Gene Products, Human Immunodeficiency Virus/metabolism , CD4 Antigens/metabolism , Crystallography, X-Ray , Down-Regulation , HIV-1/pathogenicity , Immune Evasion , Major Histocompatibility Complex , Membrane Proteins/metabolism , Protein Kinases/drug effects , Protein Transport , Structure-Activity Relationship , nef Gene Products, Human Immunodeficiency Virus/chemistryABSTRACT
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.
Subject(s)
Agammaglobulinaemia Tyrosine Kinase/metabolism , Cell Membrane/metabolism , HIV Infections/immunology , HIV-1/immunology , Protein-Tyrosine Kinases/metabolism , nef Gene Products, Human Immunodeficiency Virus/chemistry , Agammaglobulinaemia Tyrosine Kinase/genetics , Antiviral Agents/pharmacology , HIV Infections/metabolism , HIV Infections/virology , Humans , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/virology , Protein Multimerization , Protein-Tyrosine Kinases/genetics , Signal Transduction , Small Molecule Libraries/pharmacology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/virology , Virus Replication , nef Gene Products, Human Immunodeficiency Virus/genetics , nef Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
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.
Subject(s)
Bone Neoplasms , Hypercalcemia , Leukemia-Lymphoma, Adult T-Cell , Osteolysis , Adult , Animals , Humans , Hypercalcemia/etiology , Mice , Osteolysis/etiology , Parathyroid Hormone-Related Protein/geneticsABSTRACT
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.
Subject(s)
Antineoplastic Agents/therapeutic use , Benzamides/therapeutic use , Leukemia, Myeloid, Acute/drug therapy , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins/antagonists & inhibitors , Quinazolines/therapeutic use , src-Family Kinases/antagonists & inhibitors , Administration, Oral , Adult , Aged , Aged, 80 and over , Animals , Antineoplastic Agents/administration & dosage , Apoptosis/drug effects , Benzamides/administration & dosage , Cell Line, Tumor , Female , Humans , Male , Mice , Middle Aged , Protein Kinase Inhibitors/administration & dosage , Proto-Oncogene Proteins c-hck/antagonists & inhibitors , Quinazolines/administration & dosage , Xenograft Model Antitumor AssaysABSTRACT
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.
Subject(s)
CD4 Antigens/metabolism , Down-Regulation , HIV-1/metabolism , Transcription Factor AP-2/metabolism , nef Gene Products, Human Immunodeficiency Virus/metabolism , Cell Line , Dimerization , Fluorescence , Humans , Models, Molecular , Protein Binding , Subcellular Fractions/metabolismABSTRACT
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.
Subject(s)
Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/pharmacology , Small Molecule Libraries/pharmacology , nef Gene Products, Human Immunodeficiency Virus/antagonists & inhibitors , Administration, Oral , Animals , Anti-HIV Agents/administration & dosage , Binding Sites/drug effects , Dose-Response Relationship, Drug , HIV-1/drug effects , Mice , Microbial Sensitivity Tests , Models, Molecular , Molecular Conformation , Molecular Docking Simulation , Small Molecule Libraries/administration & dosage , Small Molecule Libraries/chemical synthesis , Structure-Activity Relationship , Tumor Cells, Cultured , Virus Replication/drug effectsABSTRACT
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.
Subject(s)
Anti-Retroviral Agents/chemistry , Anti-Retroviral Agents/pharmacology , HIV-1/drug effects , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , nef Gene Products, Human Immunodeficiency Virus/antagonists & inhibitors , Cell Line , HIV Infections/drug therapy , HIV-1/physiology , Humans , Molecular Docking Simulation , Protein Multimerization/drug effects , Proto-Oncogene Proteins c-hck/metabolism , Pyrazoles/chemistry , Pyrazoles/pharmacology , Virus Replication/drug effects , nef Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
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.
ABSTRACT
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.
Subject(s)
Bone Resorption/metabolism , Carcinoma, Squamous Cell/metabolism , Mouth Neoplasms/metabolism , Animals , Bone Resorption/veterinary , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/veterinary , Cats , Cell Line, Tumor , Disease Models, Animal , Humans , Mice , Mouth Neoplasms/pathology , Mouth Neoplasms/veterinary , Osteoprotegerin/metabolism , Parathyroid Hormone-Related Protein/metabolism , RANK Ligand/metabolismABSTRACT
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.
Subject(s)
Apoptosis Regulatory Proteins/genetics , Chemokine CCL3/genetics , Gene Expression Regulation, Leukemic , Leukemia, Experimental/genetics , Parathyroid Hormone-Related Protein/genetics , Animals , Apoptosis/genetics , CD40 Ligand/genetics , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Cell Line, Tumor , Death-Associated Protein Kinases , Humans , Interferon-gamma/genetics , Jurkat Cells , Leukemia, Experimental/pathology , Leukemia, T-Cell/genetics , Leukemia, T-Cell/pathology , Male , Mice , Mice, Inbred NOD , Mice, SCID , Oligonucleotide Array Sequence Analysis , RANK Ligand/genetics , Reverse Transcriptase Polymerase Chain Reaction , STAT4 Transcription Factor/genetics , T-Lymphocytes/metabolism , T-Lymphocytes/pathology , Transplantation, Heterologous , Tumor Necrosis Factor-alpha/geneticsABSTRACT
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.
Subject(s)
Brain Neoplasms/pathology , Brain Neoplasms/secondary , Carcinoma/pathology , Carcinoma/secondary , Cell Line, Tumor , Prostatic Neoplasms/pathology , Adrenal Gland Neoplasms/epidemiology , Adrenal Gland Neoplasms/secondary , Animals , Antineoplastic Agents/pharmacology , Bone Neoplasms/epidemiology , Bone Neoplasms/secondary , Boronic Acids/pharmacology , Bortezomib , Brain Neoplasms/epidemiology , Brain Neoplasms/metabolism , Carcinogenicity Tests , Carcinoma/epidemiology , Carcinoma/metabolism , Cell Division , Cell Line, Tumor/drug effects , Cell Line, Tumor/pathology , Dogs , Immunohistochemistry , Incidence , Injections, Subcutaneous , Keratin-18/metabolism , Keratin-7/metabolism , Keratin-8/metabolism , Male , Mice , Mice, Nude , Neoplasm Transplantation , Neoplasms, Connective Tissue/secondary , Parathyroid Hormone-Related Protein/metabolism , Phenylbutyrates/antagonists & inhibitors , Pyrazines/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Spinal Cord Neoplasms/epidemiology , Spinal Cord Neoplasms/secondary , Subcutaneous Tissue , Transplantation, HeterologousABSTRACT
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.
Subject(s)
Bone Neoplasms/secondary , Intercellular Signaling Peptides and Proteins/metabolism , Prostatic Neoplasms/pathology , Wnt Proteins/metabolism , Animals , Bone Neoplasms/metabolism , Cell Growth Processes/physiology , Cell Line, Tumor , Dogs , Histocytochemistry , Intercellular Signaling Peptides and Proteins/genetics , Male , Mice , Mice, Nude , Osteogenesis/physiology , Prostatic Neoplasms/metabolism , Signal Transduction , Statistics, Nonparametric , Tomography, X-Ray Computed , Wnt Proteins/antagonists & inhibitorsABSTRACT
BACKGROUND: Protein phosphorylation is one of the essential steps in cell signaling, and aberrant phosphorylation is a common event in human cancer. The expression of receptor type protein tyrosine phosphatase gamma (PTPRG) in normal breast is found to be approximately 50-60% higher than that of breast tumor tissue. Overexpression of PTPRG inhibits anchorage-independent growth and proliferation of breast cancer cells. To understand the tumor suppression characteristics of PTPRG, we studied its tumor suppressive function in an athymic mouse model and evaluated factors that can potentially regulate its expression in breast cancer cells. MATERIALS AND METHODS: To investigate the function of PTPRG in vivo, athymic nude mice were implanted with MCF-7 cells overexpressing PTPRG. For in vitro study, protein levels of cell cycle regulators, cell cycle re-entry, and the phosphorylation levels of extracellular signal-regulated protein kinases 1/2 (ERK1/2) were examined. In addition, methylation assays were conducted to investigate the epigenetic modification on the promoter of PTPRG. RESULTS: Athymic nude mice bearing MCF-7 cells overexpressing PTPRG showed a reduction in tumor burden in comparison to animals implanted with MCF-7 cells transfected with vector alone. When these two cell lines were studied in an in vitro system, elevated mRNA and protein levels of cell cycle regulators, p21(cip) and p27(kip) were detected in MCF-7 cells overexpressing PTPRG compared to cells transfected with vector alone. Similarly, overexpression of PTPRG also delayed the re-entry of breast cancer cells into the cell cycle after serum starvation, and reduced the phosphorylation levels ERK1/2 in MCF-7 cells. In addition, methylation assays in PTPRG promoter in breast cancer cell lines (including SK-Br-3) revealed an aberrant methylation pattern. When SK-Br-3 and MCF-7 cells were treated with deoxy-5-azacytidine (DAC) and trichostatin A (TSA), these compounds reactivated the expression of PTPRG, suggesting an epigenetic control on its expression. CONCLUSION: Our results indicated that PTPRG inhibited breast tumor formation in vivo; PTPRG may up-regulate p21(cip) and p27(kip) proteins through the ERK1/2 pathway. This study also showed methylation-mediated silencing of PTPRG in breast cancer cell lines. These data indicate that PTPRG exhibits the characteristics of a breast tumor suppressor.
Subject(s)
Breast Neoplasms/prevention & control , Receptor-Like Protein Tyrosine Phosphatases, Class 5/physiology , Animals , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Breast Neoplasms/pathology , Cell Cycle , Cell Line, Tumor , CpG Islands , Cyclin-Dependent Kinase Inhibitor p21/analysis , Cyclin-Dependent Kinase Inhibitor p27 , DNA Methylation , Decitabine , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Hydroxamic Acids/pharmacology , Intracellular Signaling Peptides and Proteins/analysis , Mice , PhosphorylationABSTRACT
Adult T-cell leukemia/lymphoma (ATLL) is caused by human T lymphotropic virus type 1 (HTLV-1). Patients with ATLL frequently develop humoral hypercalcemia of malignancy (HHM) resulting from increased osteoclastic bone resorption. Our goal was to investigate the mechanisms of ATLL-induced osteoclastic bone resorption. Murine calvaria co-cultured with HTLV-1-infected cells directly or conditioned media from cell cultures had increased osteoclast activity that was dependent on RANKL, indicating that factors secreted from ATLL cells had a stimulatory effect on bone resorption. Factors released from resorbing bone stimulated proliferation of HTLV-1-infected T-cells. Parathyroid hormone-related protein (PTHrP) and macrophage inflammatory protein-1alpha (MIP-1alpha), both osteoclast stimulators, were expressed in HTLV-1-infected T-cell lines. Interestingly, when HTLV-1-infected T-cells were co-cultured with pre-osteoblasts, the expression of osteoprotegerin (OPG), an osteoclast inhibitory factor, was significantly down-regulated in the pre-osteoblasts. When OPG was added into the ex vivo osteoclastogenesis assay induced by HTLV-1-infected T-cells, osteoclastogenesis was strongly inhibited. In addition, HTLV-1-infected T-cells inhibited expression of early osteoblast genes and induced late genes. These regulators will serve as future therapeutic targets for the treatments of HHM in ATLL.