RESUMO
BACKGROUND AIMS: The T-cell suppressive property of bone marrow-derived mesenchymal stromal cells (MSCs) has been considered a major mode of action and basis for their utilization in a number of human clinical trials. However, there is no well-established reproducible assay to measure MSC-mediated T-cell suppression. METHODS: At the University of Wisconsin-Madison Production Assistance for Cellular Therapy (PACT) Center, we developed an in vitro quality control T-cell suppression immunopotency assay (IPA) that uses anti-CD3 and anti-CD28 antibodies to stimulate T-cell proliferation. We measured MSC-induced suppression of CD4+ T-cell proliferation at various effector-to-target cell ratios with the use of defined peripheral blood mononuclear cells and in parallel compared with a reference standard MSC product. We calculated an IPA value for suppression of CD4+ T cells for each MSC product. RESULTS: Eleven MSC products generated at three independent PACT centers were evaluated for cell surface phenotypic markers and T-cell suppressive properties. Flow cytometry results demonstrated typical MSC cell surface marker profiles. There was significant variability in the level of suppression of T-cell proliferation, with immunopotency assay values ranging from 27% to 88%. However, MSC suppression did not correlate with human leukocyte antigen-DR expression. CONCLUSIONS: We have developed a reproducible immunopotency assay to measure allogeneic MSC-mediated suppression of CD4+ T cells. Additional studies may be warranted to determine how these in vitro assay results may correlate with other immunomodulatory properties of MSCs, in addition to evaluating the ability of this assay to predict in vivo efficacy.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Imunoensaio , Imunomodulação/imunologia , Terapia de Imunossupressão/métodos , Células-Tronco Mesenquimais/imunologia , Anticorpos/imunologia , Antígenos de Superfície/imunologia , Células da Medula Óssea/citologia , Antígenos CD28/imunologia , Complexo CD3/imunologia , Proliferação de Células , Células Cultivadas , Humanos , Leucócitos Mononucleares/imunologia , Ativação Linfocitária/imunologia , Reprodutibilidade dos TestesRESUMO
Benefit from cytotoxic therapy in myeloma may be limited by the persistence of residual tumour cells within protective niches. We have previously shown that monocytes/macrophages acquire a proinflammatory transcriptional profile in the myeloma microenvironment. Here we report constitutive activation of MAP3K8 kinase-dependent pathways that regulate the magnitude and extent of inflammatory activity of monocytes/macrophages within myeloma niches. In myeloma tumour cells, MAP3K8 acts as mitogen-induced MAP3K in mitosis and is required for TNFα-mediated ERK activation. Pharmacological MAP3K8 inhibition results in dose-dependent, tumour cell-autonomous apoptosis despite contact with primary stroma. MAP3K8 blockade may disrupt crucial macrophage-tumour cell interactions within myeloma niches.
Assuntos
MAP Quinase Quinase Quinases/metabolismo , Macrófagos/enzimologia , Macrófagos/patologia , Monócitos/enzimologia , Monócitos/patologia , Mieloma Múltiplo/enzimologia , Mieloma Múltiplo/patologia , Proteínas Proto-Oncogênicas/metabolismo , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Mieloma Múltiplo/tratamento farmacológico , Neoplasia Residual , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Células Tumorais CultivadasRESUMO
Ex vivo gene correction with CRISPR-Cas9 and a recombinant adeno-associated virus serotype 6 (rAAV6) in autologous hematopoietic stem/progenitor cells (HSPCs) to treat sickle cell disease (SCD) has now entered early-phase clinical investigation. To facilitate the progress of CRISPR-Cas9/rAAV6 genome editing technology, we analyzed the molecular changes in key reagents and cellular responses during and after the genome editing procedure in human HSPCs. We demonstrated the high stability of rAAV6 to serve as the donor DNA template. We assessed the benefit of longer HSPC pre-stimulation in terms of increased numbers of edited cells. We observed that the p53 pathway was transiently activated, peaking at 6 h, and resolved over time. Notably, we revealed a strong correlation between p21 mRNA level and rAAV6 genome number in cells and beneficial effects of transient inhibition of p53 with siRNA on genome editing, cell proliferation, and cell survival. In terms of potential immunogenicity, we found that rAAV6 capsid protein was not detectable, while a trace amount of residual Cas9 protein was still detected at 48 h post-genome editing. We believe this information will provide important insights for future improvements of gene correction protocols in HSPCs.
RESUMO
BACKGROUND AIMS: Mesenchymal stromal cells (MSC) have now been shown to reside in numerous tissues throughout the body, including the pancreas. Ex vivo culture-expanded MSC derived from many tissues display important interactions with different types of immune cells in vitro and potentially play a significant role in tissue homeostasis in vivo. In this study, we investigated the biologic and immunomodulatory properties of human pancreatic islet-derived MSC. METHODS: We culture-expanded MSC from cadaveric human pancreatic islets and characterized them using flow cytometry, differentiation assays and nuclear magnetic resonance-based metabolomics. We also investigated the immunologic properties of pancreatic islet-derived MSC compared with bone marrow (BM) MSC. RESULTS: Pancreatic islet and BM-derived MSC expressed the same cell-surface markers by flow cytometry, and both could differentiate into bone, fat and cartilage. Metabolomics analysis of MSC from BM and pancreatic islets also showed a similar set of metabolic markers but quantitative polymerase chain reactions showed that pancreatic islet MSC expressed more interleukin(IL)-1b, IL-6, STAT3 and FGF9 compared with BM MSC, and less IL-10. However, similar to BM MSC, pancreatic islet MSC were able to suppress proliferation of allogeneic T lymphocytes stimulated with anti-CD3 and anti-CD28 antibodies. CONCLUSIONS: Our in vitro analysis shows pancreatic islet-derived MSC have phenotypic, biologic and immunomodulatory characteristics similar, but not identical, to BM-derived MSC. We propose that pancreatic islet-derived MSC could potentially play an important role in improving the outcome of pancreatic islet transplantation by promoting engraftment and creating a favorable immune environment for long-term survival of islet allografts.
Assuntos
Células da Medula Óssea , Ilhotas Pancreáticas , Células-Tronco Mesenquimais , Antígenos de Superfície/análise , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Cadáver , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Citometria de Fluxo , Expressão Gênica , Humanos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/metabolismoRESUMO
Although constitutive activation of beta-catenin/Tcf signalling is implicated in the development of human cancers, the mechanisms by which the beta-catenin/Tcf pathway promotes tumorigenesis are incompletely understood. Messenger RNA turnover has a major function in regulating gene expression and is responsive to developmental and environmental signals. mRNA decay rates are dictated by cis-acting elements within the mRNA and by trans-acting factors, such as RNA-binding proteins (reviewed in refs 2, 3). Here we show that beta-catenin stabilizes the mRNA encoding the F-box protein betaTrCP1, and identify the RNA-binding protein CRD-BP (coding region determinant-binding protein) as a previously unknown target of beta-catenin/Tcf transcription factor. CRD-BP binds to the coding region of betaTrCP1 mRNA. Overexpression of CRD-BP stabilizes betaTrCP1 mRNA and elevates betaTrCP1 levels (both in cells and in vivo), resulting in the activation of the Skp1-Cullin1-F-box protein (SCF)(betaTrCP) E3 ubiquitin ligase and in accelerated turnover of its substrates including IkappaB and beta-catenin. CRD-BP is essential for the induction of both betaTrCP1 and c-Myc by beta-catenin signalling in colorectal cancer cells. High levels of CRD-BP that are found in primary human colorectal tumours exhibiting active beta-catenin/Tcf signalling implicates CRD-BP induction in the upregulation of betaTrCP1, in the activation of dimeric transcription factor NF-kappaB and in the suppression of apoptosis in these cancers.
Assuntos
Genes myc/genética , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , beta Catenina/metabolismo , Proteínas Contendo Repetições de beta-Transducina/genética , Animais , Linhagem Celular , Humanos , Proteínas I-kappa B/metabolismo , Camundongos , NF-kappa B/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Fatores de Transcrição TCF/metabolismoRESUMO
The Class I MAGE proteins are normally expressed only in developing germ cells but are often aberrantly expressed in malignancies, particularly melanoma, making them good therapeutic targets. MAGE proteins promote tumor survival by binding to the RBCC region of KAP-1 and suppressing p53. Although, suppression of MAGE expression, by RNA interference, relieves p53 suppression and inhibits tumor growth, its therapeutic uses are limited by lack of methods for systemic delivery of small interfering RNA. To overcome this barrier, we sought to discover chemical compounds that inhibit binding between MAGE and KAP-1 proteins. Based on previously published effects of MAGE suppression, we developed a strategy for screening a small molecule library based on selective death of MAGE positive cells, activation of p53 and lack of caspase activity. We screened the Maybridge HitFinder library of compounds and eight compounds fulfilled these criteria. Seven of these compounds interfered with co-precipitation of MAGE and KAP-1, and three interfered with binding of MAGE and KAP-1 in a mammalian two hybrid assay. We now report identification of three potential compounds that interfere with MAGE/KAP-1 binding and can be developed as novel chemo-therapeutic agents for treatment of advanced melanoma and other cancers.
Assuntos
Antígenos de Neoplasias/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Proteínas Repressoras/metabolismo , Animais , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Humanos , Ligação Proteica/efeitos dos fármacos , Proteínas Repressoras/química , Proteína 28 com Motivo TripartidoRESUMO
Skin cancers are the most common cancers in the United States. Exposure to UVB radiation is a major risk factor for skin cancer induction. SCF(ß-TrCP) E3 ubiquitin ligase has been found to be involved in cell cycle, cell proliferation and transformation. Aberrant up-regulation of beta-transducin repeats-containing proteins (ß-TrCP) is often found in cancer cell lines and primary tumors. We have previously demonstrated that ß-TrCP2 is over-expressed in chemically induced mouse skin tumors. Various cellular stress stimuli, including UVB, induce an increase in ß-TrCP1 mRNA and protein levels in human cells. We have previously shown that inhibition of ß-TrCP function, by induction of dominant negative ß-TrCP2 (ß-TrCP2(ΔF)), in vitro in hTERT immortalized normal keratinocytes, results in increase in UVB induced apoptosis. We have generated transgenic mice with inducible, selective expression of dominant negative ß-TrCP2 in epidermis with the Keratin 5 promoter (K5-rTA x TRE-HA-ß-TrCP(ΔF)). Here we report that inhibition of ß-TrCP function in mouse epidermis results in decrease in UVB-induced edema, hyperplasia, and inflammatory response and increment in UVB-induced apoptosis in skin. Our results suggest that ß-TrCP may be an essential player in UVB induced responses in skin and can be a potential therapeutic target for skin cancer.
Assuntos
Pele/metabolismo , Pele/efeitos da radiação , Ubiquitina-Proteína Ligases/metabolismo , Raios Ultravioleta/efeitos adversos , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Animais , Apoptose/efeitos da radiação , Edema/etiologia , Edema/metabolismo , Edema/patologia , Células Epidérmicas , Epiderme/metabolismo , Epiderme/patologia , Epiderme/efeitos da radiação , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Hiperplasia/etiologia , Hiperplasia/metabolismo , Hiperplasia/patologia , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Interleucina-6/metabolismo , Queratina-5/genética , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Especificidade de Órgãos , Regiões Promotoras Genéticas/genética , Pele/citologia , Pele/patologiaRESUMO
Sickle cell disease (SCD) is the most common serious monogenic disease with 300,000 births annually worldwide. SCD is an autosomal recessive disease resulting from a single point mutation in codon six of the ß-globin gene (HBB). Ex vivo ß-globin gene correction in autologous patient-derived hematopoietic stem and progenitor cells (HSPCs) may potentially provide a curative treatment for SCD. We previously developed a CRISPR-Cas9 gene targeting strategy that uses high-fidelity Cas9 precomplexed with chemically modified guide RNAs to induce recombinant adeno-associated virus serotype 6 (rAAV6)-mediated HBB gene correction of the SCD-causing mutation in HSPCs. Here, we demonstrate the preclinical feasibility, efficacy, and toxicology of HBB gene correction in plerixafor-mobilized CD34+ cells from healthy and SCD patient donors (gcHBB-SCD). We achieved up to 60% HBB allelic correction in clinical-scale gcHBB-SCD manufacturing. After transplant into immunodeficient NSG mice, 20% gene correction was achieved with multilineage engraftment. The long-term safety, tumorigenicity, and toxicology study demonstrated no evidence of abnormal hematopoiesis, genotoxicity, or tumorigenicity from the engrafted gcHBB-SCD drug product. Together, these preclinical data support the safety, efficacy, and reproducibility of this gene correction strategy for initiation of a phase 1/2 clinical trial in patients with SCD.
Assuntos
Anemia Falciforme , Compostos Heterocíclicos , Anemia Falciforme/genética , Anemia Falciforme/terapia , Animais , Sistemas CRISPR-Cas/genética , Edição de Genes , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Humanos , Camundongos , Reprodutibilidade dos Testes , Globinas beta/genéticaRESUMO
Type 1 regulatory T (Tr1) cells are inducible, interleukin (IL)-10+FOXP3− regulatory T cells that can suppress graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We have optimized an in vitro protocol to generate a Tr1-enriched cell product called T-allo10, which is undergoing clinical evaluation in patients with hematological malignancies receiving a human leukocyte antigen (HLA)mismatched allo-HSCT. Donor-derived T-allo10 cells are specific for host alloantigens, are anergic, and mediate alloantigen-specific suppression. In this study, we determined the mechanism of action of T-allo10 cells and evaluated survival of adoptively transferred Tr1 cells in patients. We showed that Tr1 cells, in contrast to the non-Tr1 population, displayed a restricted T cell receptor (TCR) repertoire, indicating alloantigen-induced clonal expansion. Tr1 cells also had a distinct transcriptome, including high expression of cytotoxic T lymphocyteassociated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1). Blockade of CTLA-4 or PD-1/PD-L1 abrogated T-allo10mediated suppression, confirming that these proteins, in addition to IL-10, play key roles in Tr1-suppressive function and that Tr1 cells represent the active component of the T-allo10 product. Furthermore, T-allo10derived Tr1 cells were detectable in the peripheral blood of HSCT patients up to 1 year after T-allo10 transfer. Collectively, we revealed a distinct molecular phenotype, mechanisms of action, and in vivo persistence of alloantigen-specific Tr1 cells. These results further characterize Tr1 cell biology and provide essential knowledge for the design and tracking of Tr1-based cell therapies.
Assuntos
Isoantígenos , Receptor de Morte Celular Programada 1 , Linfócitos T CD4-Positivos , Antígeno CTLA-4 , Humanos , Linfócitos T ReguladoresRESUMO
BACKGROUND: Chimeric antigen receptor (CAR) therapy and hematopoietic stem cell transplantation (HSCT) are therapeutics for relapsed acute lymphocytic leukemia (ALL) that are increasingly being used in tandem. We identified a non-physiologic CD19+/CD3+ T-cell population in the leukapheresis product of a patient undergoing CAR T-cell manufacturing who previously received a haploidentical HSCT, followed by infusion of a genetically engineered T-cell addback product. We confirm and report the origin of these CD19+/CD3+ T cells that have not previously been described in context of CAR T-cell manufacturing. We additionally interrogate the fate of these CD19-expressing cells as they undergo transduction to express CD19-specific CARs. MAIN BODY: We describe the case of a preteen male with multiply relapsed B-ALL who was treated with sequential cellular therapies. He received an αß T-cell depleted haploidentical HSCT followed by addback of donor-derived T cells genetically modified with a suicide gene for iCaspase9 and truncated CD19 for cell tracking (RivoCel). He relapsed 6 months following HSCT and underwent leukapheresis and CAR T-cell manufacturing. During manufacturing, we identified an aberrant T-cell population dually expressing CD19 and CD3. We hypothesized that these cells were RivoCel cells and confirmed using flow cytometry and PCR that the identified cells were in fact RivoCel cells and were eliminated with iCaspase9 activation. We additionally tracked these cells through CD19-specific CAR transduction and notably did not detect T cells dually positive for CD19 and CD19-directed CARs. The most likely rationale for this is in vitro fratricide of the CD19+ 'artificial' T-cell population by the CD19-specific CAR+ T cells in culture. CONCLUSIONS: We report the identification of CD19+/CD3+ cells in an apheresis product undergoing CAR transduction derived from a patient previously treated with a haploidentical transplant followed by RivoCel addback. We aim to bring attention to this cell phenotype that may be recognized with greater frequency as CAR therapy and engineered αßhaplo-HSCT are increasingly coupled. We additionally suggest consideration towards using alternative markers to CD19 as a synthetic identifier for post-transplant addback products, as CD19-expression on effector T cells may complicate subsequent treatment using CD19-directed therapy.
Assuntos
Antígenos CD19/metabolismo , Leucaférese/métodos , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Criança , Humanos , MasculinoRESUMO
Chronic skin exposure to UV radiation manifests in a score of biochemical events, DNA damage and mutations which can potentially cause skin cancer. The ubiquitin proteasome pathway controls the degradation of a majority of regulatory eukaryotic proteins including those which play a key role in tumorigenesis. SCFbetaTrCP E3 ubiquitin ligases mediate ubiquitination and proteasomal degradation of phosphorylated substrates that play a key role in signal transduction. Activation of several signaling pathways involved in tumorigenesis was shown to elevate expression and activity of beta-TrCP1/2. In this study, we established and characterized human neonatal foreskin keratinocytes, rendered immortal by retroviral introduction of human telomerase reverse transcriptase (hTERT). These skin hTERT immortalized normal keratinocytes (STINKs) maintain characteristic traits of keratinocytes, such as expression of keratins, cytoplasmic localization of basonuclin and susceptibility to high concentration of calcium. We analyzed the response of STINKs to UVB radiation and its classical markers, such as p53 and nuclear factor (NF)-kappaB. We also demonstrate that inhibition of beta-TrCP2 function, by induction of dominant negative beta-TrCP2 (beta-TrCP2DeltaN), accentuates UVB induced apoptosis, and this phenomenon is independent of NF-kappaB and p53 pathways.
Assuntos
Apoptose/efeitos da radiação , Queratinócitos/efeitos da radiação , Telomerase/metabolismo , Raios Ultravioleta , Proteínas Contendo Repetições de beta-Transducina/antagonistas & inibidores , Linhagem Celular Transformada , Humanos , Queratinócitos/citologiaRESUMO
The restriction of energy intake has documented beneficial effects on numerous diseases including cancer, yet the mechanism(s) that accounts for these effects is unknown. Recently, we showed that the inhibitory activity against mammary carcinogenesis mediated by energy restriction (ER) is accompanied by an increase in the secretion of adrenal cortical steroids. However, ER caused a concomitant reduction in circulating levels of insulin-like growth factor-1, which also may be involved in inhibiting carcinogenesis. To determine what cellular and molecular effects may be because of corticosterone per se, detailed mechanistic studies were performed in vitro using a mouse mammary hyperplastic cell line (TM10). The following questions were addressed: (a) is corticosterone-mediated growth inhibition accounted for by disruption of cell cycle machinery; (b) is growth inhibition accompanied by the induction of apoptosis; and (c) is growth inhibition reversible? At doses of corticosterone (50-200 micro M for 24-72 h) that resulted in inhibition (up to 76%; P < 0.001) of growth, a dose- and time-dependent G(1) arrest in cell cycle progression was observed. In the studies analyzing cell cycle regulatory molecules, corticosterone treatment of cells resulted in a strong induction (up to approximately 10-fold over control; P < 0.01) of KIP1/P27 together with a decrease (up to 98%; P < 0.01) in cyclin-dependent kinase 4 (CDK4) and cyclin D1 protein levels. Cells treated with corticosterone also showed an increased binding (up to 2.6-fold over control; P < 0.01) of KIP1/P27 with CDK4, together with a strong decrease (up to 89%; P < 0.01) in the kinase activity of the CDK4-cyclin D1 complex. Treatment of cells with KIP1/P27 antisense oligonucleotides reversed the growth inhibitory effects of corticosterone. Treatment of cells with RU 486, a glucocorticoid receptor blocker, reversed the effects of corticosterone on cell growth and KIP/P27 protein levels suggesting the involvement of the glucocorticoid receptor in accounting for these effects. Additional studies assessing the biological fate of cells after corticosterone treatment showed that corticosterone exerted reversible growth inhibitory effects with limited apoptotic cell death. Together, these findings show a reversible cytostatic effect of corticosterone via perturbations in cell cycle regulators causing a G(1) arrest in the absence of increased levels of apoptosis. These data provide evidence for a role of corticosterone on some but not all of the cellular activities associated with ER-mediated inhibition of mammary carcinogenesis.
Assuntos
Corticosterona/farmacologia , Ingestão de Energia/fisiologia , Glândulas Mamárias Animais/patologia , Proteínas Proto-Oncogênicas , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/metabolismo , Hiperplasia/metabolismo , Hiperplasia/patologia , Fator de Crescimento Insulin-Like I/metabolismo , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/prevenção & controle , Camundongos , Mifepristona/farmacologia , Proteínas Supressoras de Tumor/metabolismoRESUMO
NF-kappaB transcription factor is activated upon ubiquitination and subsequent proteolysis of its inhibitor IkappaB. The phosphorylation-dependent ubiquitination is mediated by SCF E3 ubiquitin ligase. In this study, we identified a novel murine F-box/WD40 repeat-containing protein, mHOS (a homologue of HOS/betaTrCP2). mHOS efficiently binds Skp1 protein (a 'core' component of SCF ubiquitin ligase), and phosphorylated IkappaB(alpha). We found that mHOS associates with SCF-ROC1 E3 ubiquitin ligase activity. We have also observed that mHOS is overexpressed in chemically-induced mouse skin tumors, and its overexpression (but not accelerated IkappaB phosphorylation) coincides with the accelerated degradation of IkappaB in vivo. The role of mHOS in the constitutive activation of NF-kappaB in skin carcinogenesis is discussed.
Assuntos
Proteínas de Transporte/biossíntese , Proteínas de Transporte/metabolismo , Proteínas I-kappa B , NF-kappa B/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/metabolismo , Neoplasias Cutâneas/metabolismo , Sequência de Aminoácidos , Animais , Carcinoma/induzido quimicamente , Carcinoma/genética , Carcinoma/metabolismo , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Feminino , Proteínas de Ligação ao GTP/genética , Imuno-Histoquímica , Camundongos , Dados de Sequência Molecular , Inibidor de NF-kappaB alfa , NF-kappa B/imunologia , Proteínas de Neoplasias/genética , Papiloma/induzido quimicamente , Papiloma/genética , Papiloma/metabolismo , Peptídeo Sintases/metabolismo , Fosforilação , RNA Neoplásico/biossíntese , Proteínas Quinases Associadas a Fase S , Proteínas Ligases SKP Culina F-Box , Homologia de Sequência de Aminoácidos , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/genética , Regulação para Cima , Proteínas Contendo Repetições de beta-TransducinaRESUMO
Melanoma-associated antigen-encoding (MAGE) genes are expressed in melanoma and other cancers but not in normal somatic cells. MAGE expression is associated with aggressive tumor growth, poor clinical outcome, and resistance to chemotherapy, but the mechanisms have not been completely elucidated. In this study, we show that downregulation of MAGE-C2 in A375 melanoma cells and low-passage cultures from human metastatic melanomas (MRA cells) results in increased apoptosis and decreased growth of tumor xenografts in athymic nude mice. Previously, we showed that MAGE-C2 binds KAP1, a scaffolding protein that regulates DNA repair. Phosphorylation of KAP1-Serine 824 (Ser824) by ataxia-telangiectasia-mutated (ATM) kinase is necessary for repair of DNA double-strand breaks (DSBs); now we show that MAGE-C2 knockdown reduces, whereas MAGE-C2 overexpression increases, ATM kinase-dependent phosphorylation of KAP1-Ser824. We demonstrate that MAGE-C2 increases co-precipitation of KAP1 with ATM and that binding of MAGE-C2 to KAP1 is necessary for increased KAP1-Ser824 phosphorylation. Furthermore, ectopic expression of MAGE-C2 enhances repair of I-SceI endonuclease-induced DSBs in U-2OS cells. As phosphorylation of KAP1-Ser824 facilitates relaxation of heterochromatin, which is necessary for DNA repair and cellular proliferation, our results suggest that MAGE-C2 can promote tumor growth by phosphorylation of KAP1-Ser824 and by enhancement of DNA damage repair.
Assuntos
Antígenos de Neoplasias/metabolismo , Proliferação de Células , Transformação Celular Neoplásica/patologia , Reparo do DNA/fisiologia , Melanoma/patologia , Proteínas de Neoplasias/metabolismo , Proteínas Repressoras/metabolismo , Neoplasias Cutâneas/patologia , Animais , Antígenos de Neoplasias/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Células Cultivadas , Reparo do DNA/efeitos dos fármacos , DNA de Neoplasias/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Regulação para Baixo/efeitos dos fármacos , Células HEK293 , Humanos , Melanoma/genética , Melanoma/metabolismo , Camundongos , Camundongos Nus , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Transplante Heterólogo , Proteína 28 com Motivo TripartidoRESUMO
Class I MAGE proteins (MAGE I) are normally expressed only in developing germ cells but are aberrantly expressed in many cancers. They have been shown to promote tumor survival, aggressive growth, and chemoresistance but the underlying mechanisms and MAGE I functions have not been fully elucidated. KRAB domain zinc finger transcription factors (KZNFs) are the largest group of vertebrate transcription factors and regulate neoplastic transformation, tumor suppression, cellular proliferation, and apoptosis. KZNFs bind the KAP1 protein and direct KAP1 to specific DNA sequences where it suppresses gene expression by inducing localized heterochromatin characterized by histone 3 lysine 9 trimethylation (H3me3K9). Discovery that MAGE I proteins also bind to KAP1 prompted us to investigate whether MAGE I can affect KZNF and KAP1 mediated gene regulation. We found that expression of MAGE I proteins, MAGE-A3 or MAGE-C2, relieved repression of a reporter gene by ZNF382, a KZNF with tumor suppressor activity. ChIP of MAGE I (-) HEK293T cells showed KAP1 and H3me3K9 are normally bound to the ID1 gene, a target of ZNF382, but that binding is greatly reduced in the presence of MAGE I proteins. MAGE I expression relieved KAP1 mediated ID1 repression, causing increased expression of ID1 mRNA and ID1 chromatin relaxation characterized by loss of H3me3K9. MAGE I binding to KAP1 also induced ZNF382 poly-ubiquitination and degradation, consistent with loss of ZNF382 leading to decreased KAP1 binding to ID1. In contrast, MAGE I expression caused increased KAP1 binding to Ki67, another KAP1 target gene, with increased H3me3K9 and decreased Ki67 mRNA expression. Since KZNFs are required to direct KAP1 to specific genes, these results show that MAGE I proteins can differentially regulate members of the KZNF family and KAP1 mediated gene repression.
Assuntos
Antígenos de Neoplasias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Proteínas de Neoplasias/metabolismo , Proteínas Repressoras/genética , Fatores de Transcrição/metabolismo , Dedos de Zinco , Sítios de Ligação , Genes Reporter , Células HEK293 , Histonas/metabolismo , Humanos , Proteína 1 Inibidora de Diferenciação/metabolismo , Lisina/metabolismo , Metilação , Modelos Biológicos , Proteínas Mutantes/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Repressoras/metabolismo , Reprodutibilidade dos Testes , Proteína 28 com Motivo Tripartido , UbiquitinaçãoRESUMO
Aberrant activation of the Hedgehog (Hh) signaling pathway has been reported in various cancer types including prostate cancer. The GLI2 transcription factor is a primary mediator of Hh signaling. However, its relative contribution to development of prostate tumors is poorly understood. To establish the role of GLI2 in maintaining the tumorigenic properties of prostate cancer cells, we developed GLI2-specific small hairpin RNA. Knockdown of GLI2 in these cells resulted in significant down-regulation of the Hh signaling pathway, followed by inhibition of colony formation, anchorage-independent growth, and growth of xenografts in vivo. Conversely, ectopic expression of Gli2 in nontumorigenic prostate epithelial cells resulted in accelerated cell cycle progression, especially transition through G(2)-M, and augmented proliferation. Altogether, our findings suggest that GLI2 plays a critical role in the malignant phenotype of prostate cancer cells, and GLI2 may potentially become an attractive therapeutic target for the treatment of prostate cancer.
Assuntos
Fatores de Transcrição Kruppel-Like/fisiologia , Proteínas Nucleares/fisiologia , Próstata/metabolismo , Neoplasias da Próstata/patologia , Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica , Proteínas Hedgehog/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Transplante de Neoplasias , Proteínas Nucleares/metabolismo , Fenótipo , Transdução de Sinais , Proteína Gli2 com Dedos de ZincoRESUMO
The Hedgehog (Hh) signaling pathway plays a crucial role in embryogenesis and has been linked to the development of several human malignancies. The transcription factor Gli2 plays a key role in the transduction of Hh signals by modulating transcription of some Hh target genes, yet the mechanisms that control Gli2 protein expression are largely unknown. Here we report that beta-transducin repeat-containing protein (beta-TrCP) E3 ubiquitin ligase is required for Gli2 degradation. beta-TrCP2 directly binds wild type Gli2 and promotes its ubiquitination. Single amino acid substitution in Gli2 putative binding site inhibits its interaction with beta-TrCP2, its ubiquitination, and stabilizes the Gli2 protein. Stable Gli2 mutant is expressed in higher levels and is more potent in the activation of Gli-dependent transcription as compared with wild type Gli2. We also found that GLI2 protein is expressed highly in prostate cancer cell lines and primary tumors, whereas the level of GLI2 mRNA is not appreciably different in normal and neoplastic prostate. These data identify beta-TrCP2 as a pivotal regulator of Gli2 expression and point to an important role for posttranslational modulation of GLI2 protein levels in Hh pathway-associated human prostate cancer.
Assuntos
Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição Kruppel-Like/fisiologia , Proteínas Nucleares/fisiologia , Proteínas Ligases SKP Culina F-Box/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Transducina/química , Ubiquitina/metabolismo , Proteína Gli2 com Dedos de ZincoRESUMO
Although Wnt/beta-catenin/Tcf signaling pathway has been shown to be an important factor in the development of many malignancies including colorectal, ovarian, prostate, and many other cancers, little is known about its role in non-melanoma skin cancers. Here, we report the first evidence that beta-catenin/Tcf signaling pathway is constitutively activated in non-melanocytic skin tumors induced by two stage chemical carcinogenesis protocol. Mouse skin tumors showed cytoplasmic and nuclear accumulation of beta-catenin, and upregulation of beta-catenin/Tcf target genes (c-myc and c-jun). We found high levels of skin-expressed Wnt proteins (Wnt 3, 4, and 10b) in different parts of the tumors, likely representing key upstream events in beta-catenin/Tcf activation during mouse skin carcinogenesis. Inhibition of beta-catenin/Tcf signaling by ectopic expression of dominant negative Tcf4 resulted in significant inhibition of growth in squamous cell carcinoma cells. A role of the constitutive activation of beta-catenin/Tcf signaling in skin carcinogenesis is discussed.
Assuntos
Carcinoma de Células Escamosas/genética , Proteínas do Citoesqueleto/fisiologia , Proteínas de Ligação a DNA/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Neoplasias Cutâneas/genética , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Animais , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/genética , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Peptídeos e Proteínas de Sinalização Intercelular/genética , Fator 1 de Ligação ao Facilitador Linfoide , Camundongos , Camundongos Endogâmicos , Papiloma/genética , Papiloma/patologia , RNA/genética , RNA/isolamento & purificação , Transdução de Sinais , Neoplasias Cutâneas/patologia , Transativadores/genética , Fatores de Transcrição/genética , Proteínas Wnt , beta CateninaRESUMO
BACKGROUND: The tremendous impact of prostate cancer (PCA) on the US male population has led to an increased attention on its prevention and on therapeutic intervention. Short-term models are needed to quickly screen the efficacy of promising agents against PCA. We have established recently several rat PCA cell lines from primary PCA in rats induced by a MNU-testosterone protocol, but their usefulness as a model for screening PCA preventive and therapeutic agents remains to be established. With the rationale that agents found effective in these cells could be promising for efficacy testing in long-term in vivo experiments, e.g., with MNU-testosterone-induced PCA in rats, the major goal of our study was to assess the antiproliferative and apoptotic efficacy in rat PCA cell lines of silibinin, a major active flavonoid component of silymarin, which consists of a group of flavonoid antioxidants occurring in milk thistle (Silybum marianum). METHODS: Three rat PCA cell lines, namely H-7, I-8, and I-26, were treated with silibinin or silymarin, a crude silibinin-containing preparation, at various doses for varying lengths of time. Cell growth and viability studies were carried out by using hemocytometer and Trypan blue dye exclusion methods. Cell cycle distribution studies were conducted by using PI staining and flow cytometry analysis, and DNA synthesis was assessed by bromodeoxyuridine incorporation. Apoptotic cell death was assessed as DNA damage by using an enzyme-linked immunosorbent assay method and by annexin V and PI staining followed by flow cytometry analysis. RESULTS: Silibinin resulted in a significant growth inhibition and reduction in cell viability in each cell line studied in both a dose- and a time-dependent manner. Silibinin treatment of H-7 and I-8 cells at 100 microM dose for 12 and 24 hr resulted in a G1 arrest but caused S phase arrest after a 48-hr treatment period in each cell line studied. Similar silibinin treatment of I-26 cells resulted in a slight S phase arrest at all time points studied. Consistent with these findings, silibinin showed a strong inhibition of DNA synthesis. Silibinin also induced a substantial apoptotic death in each cell line studied. Similar to silibinin, silymarin induced growth inhibition and reduced viability in a dose- and time-dependent manner. CONCLUSION: This study demonstrates that silibinin as well as silymarin induce growth inhibition and apoptosis in rat PCA cells. These results form a strong rationale for PCA prevention and therapeutic intervention studies with silibinin and silymarin in animal models, such as the MNU-testosterone rat PCA model, to establish their efficacy and to further define their mechanisms of action under in vivo conditions.