RESUMO
Loss of the fumarate hydratase (FH) tumor suppressor gene results in the development of benign tumors that rarely, but regrettably, progress to very aggressive cancers. Using mouse embryo fibroblasts (MEFs) to model transformation, we found that fh knockdown results in increased expression of the met oncogene-encoded tyrosine kinase receptor through hypoxia-inducible factor (hif) stabilization. MET-increased expression was alone able to stabilize hif, thus establishing a feed forward loop that might enforce tumor progression. The fh-defective MEFs showed increased motility and protection from apoptosis. Motility, but not survival, relied on hif-1alpha and was greatly enhanced by MET ligand hepatocyte growth factor. Met cooperated with a weakly oncogenic ras in making MEFs transformed and tumorigenic, as shown by in vitro and in vivo assays. Loss of fh was not equally effective by itself but enhanced the transformed and tumorigenic phenotype induced by ras and MET. Consistently, the rescue of fumarase expression abrogated the motogenic and transformed phenotype of fh-defective MEFs. In conclusion, the data suggest that the progression of tumors where FH is lost might be boosted by activation of the MET oncogene, which is able to drive cell-autonomous tumor progression and is a strong candidate for targeted therapy.
Assuntos
Transformação Celular Neoplásica , Fumarato Hidratase/fisiologia , Neoplasias/etiologia , Proteínas Proto-Oncogênicas c-met/fisiologia , Animais , Células Cultivadas , Fibroblastos , Fumarato Hidratase/deficiência , Fumarato Hidratase/genética , Fator de Crescimento de Hepatócito/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Camundongos , Proteínas Supressoras de Tumor/genéticaRESUMO
The ability to effectively transduce human hematopoietic stem cells (HSCs) and to ensure adequate but "physiological" levels of transgene expression in different hematopoietic lineages represents some primary features of a gene-transfer vector. The ability to carry, integrate, and efficiently sustain transgene expression in HSCs strongly depends on the vector. We have constructed lentiviral vectors (LV) containing fragments of different lengths of the hematopoietic-specific regulatory element of the Wiskott-Aldrich syndrome (WAS) gene-spanning approximately 1,600 and 170 bp-that direct enhanced green fluorescent protein (EGFP) expression. The performance of vectors carrying the 1,600 and 170 bp fragments of the WAS gene promoter was compared with that of a vector carrying the UbiquitinC promoter in human cord blood CD34(+) cells and their differentiated progeny both in vitro and in vivo in non-obese diabetic mice with severe combined immunodeficiency. All vectors displayed a similar transduction efficiency in CD34(+) cells and promoted long-term EGFP expression in different hematopoietic lineages, with an efficiency comparable to, and in some instances (for example, the 170-bp promoter) superior to, that of the UbiquitinC promoter. Our results clearly demonstrate that LV containing fragments of the WAS gene promoter/enhancer region can promote long-term transgene expression in different hematopoietic lineages in vitro and in vivo and represent suitable and highly efficient vectors for gene transfer in gene-therapy applications for different hematological diseases and for research purposes. In particular, the 170-bp carrying vector, for its reduced size, could significantly improve the transduction/expression of large-size genes.
Assuntos
Regulação da Expressão Gênica , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/metabolismo , Lentivirus/genética , Elementos Reguladores de Transcrição/genética , Proteína da Síndrome de Wiskott-Aldrich/genética , Animais , Antígenos CD34/metabolismo , Linhagem da Célula , Células Cultivadas , Citometria de Fluxo , Humanos , Imunofenotipagem , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Reação em Cadeia da Polimerase , Linfócitos T/metabolismoRESUMO
As mobilized peripheral blood (MPB) represents an attractive cell source for gene therapy, we investigated the ability of third-generation lentiviral vectors (LVs) to transfer the enhanced green fluorescent protein gene into MPB CD34(+) cells in culture conditions allowing expansion of transplantable human hematopoietic stem cells. To date, few studies have reported transduction of MPB cells with vesicular stomatitis virus G pseudotyped LVs. The critical issue remains whether primitive, hematopoietic repopulating cells have, indeed, been transduced. In vitro (5 weeks' culture in FLT3 ligand + thrombopoietin + stem cell factor + interleukin 6) and in vivo (serial transplantation in NOD/SCID mice) experiments show that MPB CD34(+) cells can be effectively long-term transduced by LV and maintain their proliferation, self-renewal, and multilineage differentiation potentials. We show that expansion following transduction improves the engraftment of transduced MPB CD34(+) (4.6-fold expansion of SCID repopulating cells by limiting dilution studies). We propose ex vivo expansion after transduction as an effective tool to improve gene therapy protocols with MPB. Disclosure of potential conflicts of interest is found at the end of this article.
Assuntos
Antígenos CD34/análise , Terapia Genética/métodos , HIV-1/genética , Animais , Células Sanguíneas/fisiologia , Células da Medula Óssea/fisiologia , Citometria de Fluxo , Genes Reporter , Vetores Genéticos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Proteínas de Fluorescência Verde/genética , Mobilização de Células-Tronco Hematopoéticas , Humanos , Imunofenotipagem , Interleucina-3/farmacologia , Camundongos , Camundongos SCID , Proteínas Recombinantes/farmacologia , Trombopoetina/farmacologiaRESUMO
Unresectable metastatic bone sarcoma and soft-tissue sarcomas (STS) are incurable due to the inability to eradicate chemoresistant cancer stem-like cells (sCSC) that are likely responsible for relapses and drug resistance. In this study, we investigated the preclinical activity of patient-derived cytokine-induced killer (CIK) cells against autologous bone sarcoma and STS, including against putative sCSCs. Tumor killing was evaluated both in vitro and within an immunodeficient mouse model of autologous sarcoma. To identify putative sCSCs, autologous bone sarcoma and STS cells were engineered with a CSC detector vector encoding eGFP under the control of the human promoter for OCT4, a stem cell gene activated in putative sCSCs. Using CIK cells expanded from 21 patients, we found that CIK cells efficiently killed allogeneic and autologous sarcoma cells in vitro. Intravenous infusion of CIK cells delayed autologous tumor growth in immunodeficient mice. Further in vivo analyses established that CIK cells could infiltrate tumors and that tumor growth inhibition occurred without an enrichment of sCSCs relative to control-treated animals. These results provide preclinical proof-of-concept for an effective strategy to attack autologous sarcomas, including putative sCSCs, supporting the clinical development of CIK cells as a novel class of immunotherapy for use in settings of untreatable metastatic disease.
Assuntos
Células Matadoras Induzidas por Citocinas/imunologia , Citocinas/farmacologia , Imunoterapia Adotiva/métodos , Sarcoma/imunologia , Sarcoma/terapia , Animais , Carcinogênese/imunologia , Carcinogênese/patologia , Linhagem Celular Tumoral , Células Matadoras Induzidas por Citocinas/efeitos dos fármacos , Citocinas/imunologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Sarcoma/patologiaRESUMO
Cytokine-induced killer cells (CIKs) are ex vivo expanded T-NK lymphocytes capable of HLA-unrestricted antitumor activity. CIKs are promising candidates for adoptive cancer immunotherapies; they can be generated and infused in autologous settings of cancer patients, or from donors, after allogeneic hematopoietic cell transplant. Ex vivo expansion rates of CIKs are greatly variable among patients, with consequent potential clinical limitations for "poor expanders." We compared the standard expansion protocol with a new one, which included the timed addition of irradiated allogeneic peripheral blood mononuclear cells. Our hypothesis is that allogeneic stimulation might provide CIK cells with a proliferative boost and simultaneously decrease their alloreactivity versus third parties, if HLA-mismatched from the allogeneic stimulators. Allo-stimulated CIKs (AS-CIK) reached significantly higher expansion rates compared with standard controls, regardless if generated form healthy donors (131- vs. 32-fold) or cancer patients (117- vs. 14-fold). The expansion of the CD3CD56 subset was 2243-fold for AS-CIKs compared with 362 for standard CIKs. AS-CIKs efficiently killed osteosarcoma targets in vitro, results were comparable with that of standard CIKs. Standard and AS-CIKs did not show differences in phenotype and telomere length. The alloreactivity of AS-CIKs against third party HLA-mismatched peripheral blood mononuclear cells was reduced compared with standard CIKs (37% vs. 23%). In conclusion, alloreactivity of CIK cells may be exploited enhancing their final ex vivo expansion. In clinical perspective these findings may facilitate the extension of CIK-based immunotherapy to larger numbers of patients and, translated into hematopoietic cell transplant settings, contribute to reduce the risk of graft versus host disease in the hypothesis of infusions across HLA barriers.
Assuntos
Neoplasias Ósseas/terapia , Células Matadoras Induzidas por Citocinas/imunologia , Imunoterapia Adotiva , Osteossarcoma/terapia , Neoplasias Ósseas/imunologia , Neoplasias Ósseas/patologia , Complexo CD3/metabolismo , Antígeno CD56/metabolismo , Técnicas de Cultura de Células/métodos , Proliferação de Células , Células Cultivadas , Células Matadoras Induzidas por Citocinas/transplante , Citotoxicidade Imunológica , Antígenos HLA/imunologia , Humanos , Imunização , Isoantígenos/imunologia , Leucócitos Mononucleares/imunologia , Osteossarcoma/imunologia , Osteossarcoma/patologiaRESUMO
The proteasome system restricts lentiviral transduction of stem cells. We exploited proteasome inhibition as a strategy to enhance transduction of both hematopoietic stem cells (HSC) and T lymphocytes with low dose or large-size lentiviral vectors (LV). HSC showed higher transduction efficiency if transiently exposed to proteasome inhibitor MG132 (41.8% vs 10.7%, p<0.0001). Treatment with MG132 (0.5 µM) retained its beneficial effect with 3 different LV of increasing size up to 10.9 Kb (p<0.01). We extended, for the first time, the application of proteasome inhibition to the transduction of T lymphocytes. A transient exposure to MG132 significantly improved lentiviral T-cell transduction. The mean percentage of transduced T cells progressively increased from 13.5% of untreated cells, to 21% (p=0.3), 30% (p=0.03) and 37% (p=0.01) of T lymphocytes that were pre-treated with MG132 at 0.1, 0.5 and 1 µM, respectively. MG132 did not affect viability or functionality of HSC or T cells, nor significantly increased the number of integrated vector copies. Transient proteasome inhibition appears as a new procedure to safely enhance lentiviral transduction of HSC and T lymphocytes with low viral doses. This approach could be useful in settings where the use of large size vectors may impair optimal viral production.