Assuntos
Proteína 3 com Repetições IAP de Baculovírus , Leucemia Linfocítica Crônica de Células B , Linfocitose , Mutação , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/patologia , Linfocitose/genética , Linfocitose/diagnóstico , Linfocitose/patologia , Proteína 3 com Repetições IAP de Baculovírus/genética , Linfócitos B/patologia , Linfócitos B/metabolismo , Linfócitos B/imunologia , Masculino , Idoso , Evolução Clonal/genética , Pessoa de Meia-Idade , FemininoRESUMO
Multiple Endocrine Neoplasia Type 2B (MEN2B) is a cancer-predisposing syndrome that affects patients with germline RET mutations. The clinical spectrum of the syndrome includes medullary thyroid carcinoma (MTC) and pheochromocytoma. Currently, there is no satisfactory model recapitulating all the features of the disease especially at the level of stem cells. We generated induced pluripotent stem cells (iPSCs) from a patient with RET mutation at codon 918 who developed pheochromocytoma and MTC. These iPSC had normal karyotype, harboured the RETM918T mutation and expressed pluripotency hallmarks. A comprehensive pathological assessment of teratoma was performed after injection in immunodeficient mice.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes Induzidas/patologia , Neoplasia Endócrina Múltipla Tipo 2b/genética , Neoplasia Endócrina Múltipla Tipo 2b/patologia , Mutação/genética , Proteínas Proto-Oncogênicas c-ret/genética , Linhagem Celular , Humanos , Fatores de Risco , Adulto JovemRESUMO
Chronic myeloid leukemia (CML) is a clonal malignancy initiated by the occurrence of a t (9;22) translocation, generating Ph1 chromosome and BCR-ABL oncogene in a primitive hematopoietic stem cell (HSC). The resistance of HSC to targeted therapies using tyrosine kinase inhibitors remains a major obstacle towards the cure. We have generated an iPSC line from a patient with CML using leukemic CD34+ cells cryopreserved at diagnosis. Ph1+ CML cells were reprogrammed by non-integrative viral transduction. These iPSCs harboured Ph1 chromosome and expressed pluripotency hallmarks as well as BCR-ABL. Teratoma assays revealed normal differentiation after injection in immunodeficient mice.
Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucócitos Mononucleares/citologia , Adolescente , Animais , Antígenos CD34/metabolismo , Diferenciação Celular , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/transplante , Cariótipo , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucócitos Mononucleares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Teratoma/metabolismo , Teratoma/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Multiple Endocrine Neoplasia Type 2A (MEN2A) is a cancer-predisposing syndrome that affects patients with germline RET mutations. The clinical spectrum of the syndrome includes medullary thyroid carcinoma (MTC), pheochromocytoma, hyperparathyroidism and cutaneous lichen amyloidosis (CLA) and/or Hirschsprung disease in some variants. Currently, there is no satisfactory animal model recapitulating all the features of the disease especially at the level of stem cells. We generated induced pluripotent stem cells (iPSCs) from a patient with RET mutation at codon 634 who developed pheochromocytoma and MTC. RETC634Y-mutated cells were reprogrammed by non-integrative viral transduction. These iPSCs had normal karyotype, harboured the RETC634Y mutation and expressed pluripotency hallmarks as well as RET. A comprehensive pathological assessment of teratoma was performed after injection in immunodeficient mice.
RESUMO
Recent studies have demonstrated a relationship between the expression of stem cell-associated genes and relapses in glioblastoma (GBM), suggesting a key role for tumor stem cells in this process. Although there is increasing interest in this field, glioma stem cells (GSCs) are still poorly characterized, their 'stemness' state and factors maintaining these properties remain largely unknown. We performed an expression profiling analysis of pluripotency in gliomaspheres derived from 11 patients. Comparative analysis between GSCs and H1 and H9 human embryonic stem cells as well as H9-derived neural stem cells indicates major variations in gene expression of pluripotency factors Nanog and OCT4, but a stable pattern for SOX2 suggesting its important function in maintaining pluripotency in GSCs. Our results also showed that all GSC lines have the capacity to commit to neural differentiation and express mesenchymal or endothelial differentiation markers. In addition, hierarchical clustering analysis revealed two groups of GSCs reflecting their heterogeneity and identified COL1A1 and IFITM1 as the most discriminating genes. Similar patterns have been observed in tumors from which gliomaspheres have been established. To determine whether this heterogeneity could be clinically relevant, the expression of both genes was further analyzed in an independent cohort of 30 patients with GBM and revealed strong correlation with overall survival. In vitro silencing of COL1A1 and IFTM1 confirmed the effect of these mesenchymal-associated genes on cell invasion and gliomasphere initiation. Our results indicate that COL1A1 and IFITM1 genes could be considered for use in stratifying patients with GBM into subgroups for risk of recurrence at diagnosis, as well as for prognostic and therapeutic evolution.
RESUMO
Telomerase catalytic subunit (hTERT) exerts important cellular functions including telomere homeostasis, genetic stability, cell survival and perhaps differentiation. However, the nature of external or internal signals, which regulate hTERT expression in tissues, remains poorly understood. Thus, whereas it has been described that hTERT gene is regulated along the differentiation of primitive myeloid progenitors, the effect of specific cytokines on telomerase expression in each myeloid lineage is currently unknown. Based on these considerations, we have investigated hTERT expression in erythroid cells treated with erythropoietin (EPO) and transforming growth factor beta (TGFbeta), as putative positive and negative regulators, respectively. We describe here that EPO activates hTERT gene transcription in in vitro-expanded primary erythroid precursors as well as in UT7 erythroleukemia cells. In UT7 cells, this study shows also that EPO acts through a JAK2/STAT5/c-myc axis. In contrast, TGFbeta blocks EPO signaling downstream of c-myc induction through a Smad3-dependent mechanism. Finally, hTERT appears to be efficiently regulated by EPO and TGFbeta in an opposite way in erythropoietic cells, arguing for a role of telomerase in red blood cell production.
Assuntos
Células Precursoras Eritroides/metabolismo , Eritropoetina/metabolismo , Regulação Leucêmica da Expressão Gênica , Telomerase/biossíntese , Fator de Crescimento Transformador beta/metabolismo , Antígenos CD34/biossíntese , Apoptose , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Humanos , Modelos Biológicos , Plasmídeos/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
The hematopoietic system is one of the best characterized human cellular system in which a multipotent adult stem cell is at the origin of all the cells of a tissue. These last years, the use of stem cells has given rise to many hopes in regenerative medicine, especially in diseases without efficient therapies. However the hematopoietic system in which stem cell transplantation has entered in clinical practice for many years has also shown the limits of these approaches. Especially the in vitro manipulation of hematopoietic stem cells remains a challenge which requires more fundamental knowledge on the biology of stem cells including self renewal and homing. Knowledge for other tissue system is even more preliminary but the same experimental strategy used for hematopoietic stem cell can be translated and may accelerate their use in cell therapies. Characterization of human adult pluripotent stem cells and the generation of human ES cells capable to differentiate towards several tissues have led to new hopes but the road to their use in therapies may be long and will require a lot of investment in basic biology.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Adulto , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula , Terapia Baseada em Transplante de Células e Tecidos/métodos , Terapia Baseada em Transplante de Células e Tecidos/tendências , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/transplante , Citocinas/farmacologia , Substâncias de Crescimento/farmacologia , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Transplante de Células-Tronco/métodosRESUMO
To examine the role of the Notch ligand Delta-4 on hematopoietic stem cells, human CD34+CD38low cord blood cells were cocultured on S17 cells transduced with transmembrane Delta-4 (mbD4/S17) or an empty vector (C/S17). By the end of a 3-week culture, mbD4/S17 induced a 25-fold reduction in nucleated cell production, as compared to C/S17, by maintaining a higher proportion of cells in G0/G1 phase. A specific retention of a high proportion of CD34+ cells throughout the culture was observed with mbD4/S17, contrary to C/S17. Although mbD4/S17 promoted expansion of cells with the phenotype of committed lymphoid precursors (CD34+CD7+CD45RA+), these cells still retained their myeloid differentiation potential. mbD4/S17 maintained a higher LTC-IC frequency in output CD34+ cells, compared to C/S17, as in the subsets of cells having completed the same number of divisions on mbD4/S17. A Delta4-Fc protein (extracellular part of human Delta4 fused to Fc human IgG1 portion), immobilized on plastic, also reduced cell production and retained the LTC-IC potential. Transplantation of cells grown on mbD4/S17 into NOD/SCID mice showed no significant enhancement of the long-term repopulating ability. Thus, Delta4 appears to inhibit hematopoietic stem cell proliferation, in association with the maintenance of short-term lymphoid and myeloid repopulation capacity.
Assuntos
Proteínas Sanguíneas/genética , Proteínas Sanguíneas/fisiologia , Células-Tronco Hematopoéticas/citologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , ADP-Ribosil Ciclase/análise , ADP-Ribosil Ciclase 1 , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antígenos CD/análise , Antígenos CD34/análise , Proteínas de Ligação ao Cálcio , Diferenciação Celular , Divisão Celular , Técnicas de Cocultura , Sangue Fetal/citologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Glicoproteínas de Membrana , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos NOD , Fase de Repouso do Ciclo Celular , Transdução Genética , Transplante HeterólogoRESUMO
Mantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32), which is associated with cyclin D1 hyperexpression and a poor prognosis. MCL cases have been shown to progress to a more aggressive disease but the molecular events responsible of this phenomenon have not been determined. We have established two cell lines from the pleural effusions of two patients with MCL that we have used for further cytogenetic characterization to better define the incidence and nature of secondary chromosome abnormalities using multicolor fluorescence in situ hybridization, whole chromosome paint, and specific probes. Both cell lines grew independently without growth factors. Using CCND1/IGH-specific probes, patient UPN1 was found to have a masked t(11;14). Numerous and complex chromosomal abnormalities were found in both cell lines affecting chromosomes 2, 8, 13, 18, 22, X, and Y. These abnormalities included 8p losses, suggesting the presence of an anti-oncogene in this region, rearrangements of 8q24, MYC gene, and translocations involving 8, X, and Y chromosomes, which might be significant in the pathogenesis of MCL progression. The use of the cell lines (UPN1) allowed us to generate a mouse model of human MCL, mimicking a disseminated lymphoma and leading to the death of the animals in 4 weeks. This blastoid MCL model could be of major interest to determine molecular events involved in MCL progression, allowing isolation of involved genes and their functional characterization, and to study the effects of new chemotherapy regimens in mouse models.
Assuntos
Aberrações Cromossômicas , Modelos Animais de Doenças , Linfoma de Célula do Manto/genética , Translocação Genética , Animais , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 14 , Humanos , Hibridização in Situ Fluorescente , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Transplante de Neoplasias , Células Tumorais CultivadasRESUMO
Stem cell proliferation induced by potent cytokines usually leads to a loss of primitive potential through differentiation. In this study, the ability of cytokines and murine MS5 stromal cells to independently regulate the proliferation and long-term culture-initiating cell (LTC-IC) activity of primitive CD34(+)CD38(low/neg) human bone marrow cells was evaluated. To compare populations with identical proliferation histories, cells were labeled with carboxy fluorescein diacetate succinimidyl ester, and LTC-IC activity was assessed 4 days later in cells that had accomplished the same number of divisions with or without MS5 cells. MS5 cells counteracted dramatically the loss of LTC-IC activity observed in the presence of cytokines alone. Thus, in the presence of MS5 cells, means of 1233 (n = 5) and 355 (n = 9) LTC-IC-derived colony-forming cells (CFCs) were generated by 1000 cells that performed 3 and 4 divisions respectively, whereas 311 (n = 5) and 64 (n = 5) CFCs were generated by 1000 cells cultured without MS5 cells. Interestingly, MS5 cells had no detectable effect on the LTC-IC activity of cells that divided only twice in 4 days-1606 CFCs (n = 6) and 1993 (n = 6) CFCs, respectively, without and with MS5 cells-and a 48 additional hours of coculture were necessary to unmask changes in the LTC-IC activity mediated by stromal cells. These results indicate that cytokines and stroma-derived signals can regulate independently the proliferation and differentiation of primitive cells and that these stroma-derived extracellular factors act directly on their target cells.
Assuntos
Antígenos CD34/fisiologia , Antígenos CD , Diferenciação Celular/fisiologia , Citocinas/farmacologia , Células-Tronco/citologia , Células Estromais/fisiologia , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Animais , Antígenos de Diferenciação , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Comunicação Celular , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular , Técnicas de Cocultura , Ensaio de Unidades Formadoras de Colônias , Humanos , Glicoproteínas de Membrana , Camundongos , Mitose/fisiologia , NAD+ Nucleosidase , Células-Tronco/efeitos dos fármacos , Células-Tronco/imunologiaRESUMO
The objective of the present study was to evaluate the antitumor effect of a defective adenovirus expressing a secretable angiostatin-like molecule (AdK3) in combination with radiotherapy in rat C6 gliomas s.c. preestablished into athymic mice. In vitro, the combination regimen was significantly (P < 0.001) more cytotoxic for human microcapillary endothelial cells than either treatment alone, whereas survival of C6 glioma cells was not affected in the conditions used. Radiotherapy and AdK3 gene delivery was then studied on well established C6 xenografts (165 +/- 70 mm(3)). In these tumors, AdK3 intratumoral injections had only a marginal effect. Interestingly, when experimental radiotherapy was added, significantly higher (P < 0.005), and possibly synergistic, antitumoral effects were observed that tightly correlated a marked decrease of intratumoral vascularization. The combination of radiotherapy and AdK3 intratumoral injections also revealed a significant (P < 0.05) inhibition of tumor growth as compared with either treatment alone for larger tumors (467 +/- 120 mm(3)). Altogether, these data emphasize the potential of combining a destructive strategy directed against the tumor cells with an anti-angiogenic approach to fight cancer.
Assuntos
Terapia Genética , Glioma/terapia , Fragmentos de Peptídeos/genética , Plasminogênio/genética , Adenoviridae/genética , Angiostatinas , Animais , Terapia Combinada , Masculino , Camundongos , Camundongos Nus , Neovascularização Patológica/prevenção & controle , Dosagem Radioterapêutica , Ratos , Células Tumorais CultivadasAssuntos
Biomarcadores Tumorais/análise , Neoplasias/química , Previsões , Marcadores Genéticos/genética , Predisposição Genética para Doença/genética , Humanos , Proteínas de Neoplasias/análise , Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos/tendências , Antígeno Prostático Específico/análiseRESUMO
Evidence has been provided recently that shows that high concentrations of cytokines can fulfill functions previously attributed to stromal cells, such as promote the survival of, and led to a net increase in human primitive progenitors initiating long-term cultures in vitro (LTC-IC) or engrafting NOD-SCID (nonobese diabetic severe-combined immunodeficient) recipients in vivo. These data prompted us to re-evaluate whether stromal cells will further alter the properties of primitive progenitor cells exposed to cytokines. Single CD34(+)CD38(low) and CD38(neg) cells were incubated 10 days in serum-containing or serum-free medium in the presence or in the absence of murine marrow-derived stromal cells (MS-5). Recombinant human cytokines stem cell factor (SCF), pegylated-megakaryocyte growth and differentiation factor (PEG-MGDF), FLT3-L, Interleukin (IL)-3, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were systematically added at various concentrations (10 to 300 ng/mL). Cell proliferation and LTC-IC potential were evaluated in each clone after 10 days. A striking and consistent observation was the retention of a high LTC-IC potential in clones exposed to cytokines in the presence of stromal feeders, whereas clones exposed to cytokines alone in the absence of stromal feeders rapidly lost their LTC-IC potential as they proliferated. This was reflected both by the higher proportion of wells containing LTC-IC and by the high numbers of CFC produced after 5 weeks in clones grown with MS-5 during the first 10 days. We further showed by analyzing multiple replicates of a single clone at day 10 that MS-5 cells promoted a net increase in the LTC-IC compartment through self-renewal divisions. Interestingly, these primitive LTC-IC were equally distributed among small and large clones, as counted at day 10, indicating that active proliferation and loss of LTC-IC potential could be dissociated. These observations show that, in primitive cells, stromal cells counteract differentiation events triggered by cytokines and promoted self-renewal divisions. Furthermore, the almost identical distribution of the size of the clones with or without MS-5 suggests that proliferation and function of human primitive cells may be independently regulated by external signals, and that the former is primarily under the control of cytokines.
Assuntos
Antígenos CD , Células da Medula Óssea/fisiologia , Citocinas/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Animais , Antígenos CD34/análise , Antígenos de Diferenciação/análise , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Ensaio de Unidades Formadoras de Colônias , Meios de Cultura Livres de Soro/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células-Tronco Hematopoéticas/citologia , Humanos , Interleucina-3/farmacologia , Interleucina-6/farmacologia , Glicoproteínas de Membrana , Proteínas de Membrana/farmacologia , Camundongos , NAD+ Nucleosidase/análise , Polietilenoglicóis/farmacologia , Proteínas Recombinantes/farmacologia , Fator de Células-Tronco/farmacologia , Células Estromais/fisiologia , Trombopoetina/farmacologiaRESUMO
In contrast to myeloid and B-lymphoid differentiation, which take place in the marrow environment, development of T cells requires the presence of thymic stromal cells. We demonstrate in this study that human CD34+, CD34+ CD38+ and CD34+ CD38(low) cells from both cord blood and adult bone marrow reproducibly develop into CD4+ CD8+ T cells when introduced into NOD-SCID embryonic thymuses and further cultured in organotypic cultures. Such human/mouse FTOC fetal thymic organ culture) thus represents a reproducible and sensitive system to assess the T-cell potential of human primitive progenitor cells. The frequency of T-cell progenitors among cord-blood-derived CD34+ cells was estimated to be 1/500. Furthermore, the differentiation steps classically observed in human thymus were reproduced in NOD-SCID FTOC initiated with cord blood and human marrow CD34+ cells: immature human CD41(low) CD8- sCD3- TCR alphabeta- CD5+ CD1a+ T cells were mixed with CD4+ CD8+ cells and more mature CD4+ CD8- TCR alphabeta+ cells. However, in FTOC initiated with bone marrow T progenitors, <10% double-positive cells were observed, whereas this proportion increased to 50% when cord blood CD34+ cells were used, and most CD4+ cells were immature T cells. These differences may be explained by a lower frequency of T-cell progenitors in adult samples, but may also suggest differences in the thymic signals required by bone marrow versus cord blood T progenitors. Finally, since cytokine-stimulated CD34+ CD38(low) cells retained their ability to generate T cells, these FTOC assays will be of value to monitor, when combined with other biological assays, the influence of different expansion protocols on the potential of human stem cells.
Assuntos
Antígenos CD , Células da Medula Óssea/citologia , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Subpopulações de Linfócitos T/citologia , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Animais , Antígenos CD34 , Antígenos de Diferenciação , Diferenciação Celular , Células Cultivadas , Feminino , Humanos , Interleucina-3/farmacologia , Glicoproteínas de Membrana , Camundongos , Camundongos SCID , NAD+ Nucleosidase , Fenótipo , Timo/citologiaRESUMO
In the present study, we investigated the differentiation of human NK cells from bone marrow, cord blood and mobilized peripheral blood purified CD34+ stem cells using a potent culture system. Elutriated CD34+ stem cells were grown for several weeks in medium supplemented with stem cell factor (SCF) and IL-15 in the presence or absence of a murine stromal cell line (MS-5). Our data indicate that IL-15 induced the proliferation and maturation of highly positive CD56+ NK cells in both types of culture, although murine stromal cells slightly increased the proliferation of NK cells. NK cells differentiated in the presence of MS-5 were mostly CD56+ CD7 and a small subset expressed CD16. These in vitro differentiated CD56+ NK cells displayed cytolytic activity against the HLA class I- target K562. The CD56+ CD16+ subset also lysed NK-resistant Daudi cells. Neither of these NK subsets were shown to express Fas ligand. Total CD56+ cells expressed high amounts of transforming growth factor-beta and granulocyte-macrophage colony-stimulating factor, but no IFN-gamma. Investigation of NK receptor expression showed that most CD56+ cells expressed membrane CD94 and NKG2-A mRNA. PCR analysis revealed that p58 was also expressed in these cells. The role of CD94 in NK cell-mediated cytotoxicity was assessed on human HLA-B7-transfected murine L cells. While a low cytotoxic activity towards HLA-B7 cells was observed, the HLA-DR4 control cells were killed with high efficiency. These studies demonstrate that cytolytic and cytokine-producing NK cells may be derived from adult and fetal precursors by IL-15 and that these cells express a CD94 receptor which may influence their lytic potential.
Assuntos
Células Matadoras Naturais/fisiologia , Lectinas Tipo C , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Adulto , Antígenos CD/biossíntese , Antígenos CD/fisiologia , Antígenos CD34/imunologia , Antígenos CD7/imunologia , Antígenos de Diferenciação/imunologia , Células da Medula Óssea/citologia , Antígeno CD56/imunologia , Testes Imunológicos de Citotoxicidade , Proteína Ligante Fas , Sangue Fetal/citologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Imunofenotipagem , Interferon gama/genética , Interleucina-15/farmacologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Leucopoese , Linfotoxina-alfa/biossíntese , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/fisiologia , NAD+ Nucleosidase/imunologia , Subfamília D de Receptores Semelhantes a Lectina de Células NK , RNA Mensageiro , Receptores de IgG/imunologia , Fator de Crescimento Transformador beta/biossíntese , Células Tumorais CultivadasRESUMO
The antitumoral effects that follow the local delivery of the N-terminal fragment of human plasminogen (angiostatin K3) have been studied in two xenograft murine models. Angiostatin delivery was achieved by a defective adenovirus expressing a secretable angiostatin K3 molecule from the cytomegalovirus promoter (AdK3). In in vitro studies, AdK3 selectively inhibited endothelial cell proliferation and disrupted the G2/M transition induced by M-phase-promoting factors. AdK3-infected endothelial cells showed a marked mitosis arrest that correlated with the down-regulation of the M-phase phosphoproteins. A single intratumoral injection of AdK3 into preestablished rat C6 glioma or human MDA-MB-231 breast carcinoma grown in athymic mice was followed by a significant arrest of tumor growth, which was associated with a suppression of neovascularization within and at the vicinity of the tumors. AdK3 therapy also induced a 10-fold increase in apoptotic tumor cells as compared with a control adenovirus. Furthermore, we showed that systemic injection of AdK3 delayed C6 tumor establishment and growth, confirming that angiostatin can function in a paracrin manner. Our data support the concept that targeted antiangiogenesis, using adenovirus-mediated gene transfer, represents a promising alternative strategy for delivering antiangiogenic factors as their bolus injections present unsolved pharmacological problems.