RESUMO
The oncotropic phenotypes of several viruses correlate with tumor-associated deficiencies within interferon (IFN) signaling pathways. This observation formed the conceptual basis for developing oncolytic viruses deleted for viral proteins that inhibit the host IFN-dependent antiviral response, such as herpes simplex virus type-1 infected cell protein-0 (ICP0) and vesicular stomatitis virus matrix protein. Many viruses have evolved means to disrupt promyelocytic leukemia protein (PML) nuclear bodies. For example, ICP0 promotes PML degradation to inhibit the antiviral activities of this IFN-stimulated gene. As PML is downregulated in a variety of tumors, we hypothesized ICP0-null herpes simplex type-1 viruses are selectively oncolytic in tumors with impaired PML expression. We illustrate that ICP0-null herpes simplex type-1 viruses target tumor cells that either possess impaired PML signaling or cannot upregulate PML because of impaired IFN responsiveness. Disruption of PML signaling through overexpression of the dominant-negative protein PML-retinoic acid receptor alpha in PML-positive cells renders them sensitive to oncolysis by ICP0-null herpes simplex virus type-1 and vesicular stomatitis virus M protein mutant viruses, whereas PML overexpression reverses this phenomenon. Together, these data illustrate that PML mediates an antiviral mechanism that predicts the tropism of IFN-sensitive oncolytic viruses. To our knowledge, these viruses are the first examples of anti-cancer therapeutics capable of targeting deficiencies in PML expression.
Assuntos
Interferon-alfa/farmacologia , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/patogenicidade , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Biomarcadores Tumorais/metabolismo , Humanos , Proteínas Imediatamente Precoces/metabolismo , Neoplasias/patologia , Vírus Oncolíticos/efeitos dos fármacos , Vírus Oncolíticos/fisiologia , Proteína da Leucemia Promielocítica , Receptores do Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Simplexvirus/efeitos dos fármacos , Simplexvirus/patogenicidade , Simplexvirus/fisiologia , Células Tumorais Cultivadas , Ubiquitina-Proteína Ligases/metabolismo , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Vírus da Estomatite Vesicular Indiana/patogenicidade , Vírus da Estomatite Vesicular Indiana/fisiologia , Tropismo ViralRESUMO
Greater than 95% of acute promyelocytic leukemia (APL) cases are associated with the expression of PML-RARalpha. This chimeric protein has been strongly implicated in APL pathogenesis because of its interactions with growth suppressors (PML), retinoid signaling molecules (RXRalpha), and nuclear hormone transcriptional co-repressors (N-CoR and SMRT). A small number of variant APL translocations have also been shown to involve rearrangements that fuse RARalpha to partner genes other than PML, namely PLZF, NPM, and NuMA. We describe the molecular characterization of a t(5;17)(q35;q21) variant translocation involving the NPM gene, identified in a pediatric case of APL. RT-PCR, cloning, and sequence studies identified NPM as the RARalpha partner on chromosome 5, and both NPM-RARalpha and RARalpha-NPM fusion mRNAs were expressed in this patient. We further explored the effects of the NPM-RARalpha chimeric protein on the subcellular localization of PML, RXRalpha, NPM, and PLZF using immunofluorescent confocal microscopy. While PML remained localized to its normal 10-20 nuclear bodies, NPM nucleolar localization was disrupted and PLZF expression was upregulated in a microspeckled pattern in patient leukemic bone marrow cells. We also observed nuclear co-localization of NPM, RXRalpha, and NPM-RARalpha in these cells. Our data support the hypothesis that while deregulation of both the retinoid signaling pathway and RARalpha partner proteins are molecular consequences of APL translocations, APL pathogenesis is not dependent on disruption of PML nuclear bodies.
Assuntos
Cromossomos Humanos Par 17 , Cromossomos Humanos Par 5 , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Leucemia Promielocítica Aguda/genética , Fosfoproteínas/genética , Fatores de Transcrição/genética , Translocação Genética , Nucléolo Celular , Criança , Células HL-60 , Humanos , Fatores de Transcrição Kruppel-Like , Masculino , Proteína com Dedos de Zinco da Leucemia Promielocítica , RNA Mensageiro , Receptores do Ácido Retinoico/análise , Receptores do Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico , Receptores X de Retinoides , Fatores de Transcrição/análise , Células U937 , Regulação para CimaRESUMO
Chronic lymphocytic leukemia (CLL) is a heterogeneous disease of the elderly which can present in one of three stages; benign, intermediate or advanced. The molecular events governing the progression of CLL are poorly understood. In order to develop model systems for predicting the aggressiveness of leukemic clones in CLL, in vivo transplantation of SCID mice with CLL cells, and the in vitro growth of CLL cells on mouse and human stromal layers, were investigated. Bone marrow or peripheral blood cells from 40 patients at different stages of CLL were transplanted into 172 immune-deficient SCID mice. Thirty-five percent of SCID mice injected with CLL cells were positive for the presence of human DNA by Southern blot or PCR analysis. The most frequently involved sites were the spleen, lung, kidney and bone marrow, at levels corresponding from 0.1 to 10 percent human DNA. Thrice-weekly intraperitoneal injections of IL-2, alone or in combination with IL-7, did not increase the level of human cell engraftment. SCID mice developed endogenous thymic lymphomas at an incidence of 10-33 percent, a rate that was not increased by CLL cell transplantation. In vitro, CLL cells were able to proliferate for 9 weeks on human stromal layers supplemented with CM (conditioned media from a culture of the human bladder carcinoma cell line 5637), but failed to thrive on the murine stromal cell line MTE cultured either in CM or autologous serum. FACS analysis revealed that 81 percent of proliferating cells on human stromal layers carried the CD5 cell surface marker, identifying them as CLL cells. Previously EBV-negative CLL cells became EBV-positive after 9 to 12 weeks in culture. The results of this study provide a firm foundation for the development of in vivo and in vitro model systems for the study of human CLL.
Assuntos
Transplante de Medula Óssea , DNA de Neoplasias/análise , Leucemia Linfocítica Crônica de Células B/patologia , Transplante de Neoplasias , Neoplasias do Timo/patologia , Animais , Sequência de Bases , Primers do DNA , DNA Viral/análise , Herpesvirus Humano 4/isolamento & purificação , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Camundongos , Camundongos SCID , Dados de Sequência Molecular , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase , Neoplasias do Timo/genética , Fatores de Tempo , Transplante HeterólogoRESUMO
Translocation t(15;17)(q22;q21) is an acquired clonal cytogenetic change present in almost all cases of acute promelocytic leukemia (APL). The molecular genetic basis of the translocation supports its integral role in pathogenesis. We describe a patient with APL in whom the leukaemic clone was characterized by a true variant of the classical t(15;17). The patient whose disease had numerous atypical clinical features, had t(11;17)(q13;121). The chromosome 17 breakpoint was localized to intron 2 of RARA by Southern blotting, and there was no evidence at the molecular level for rearrangement at PML locus. These data, along with previous reports of rare variant translocations in APL, indicate that while dysregulation of RARA by gene fusion may be essential for the APL phenotype, the particular fusion partner may determine clinicopathological aspects, including presentation, response to treatment with all-trans retinoic acid (ATRA), and prognosis. This heterogeneity suggests that the variant fusion partners of RARA in APL encode factors with properties both common to and distinct from those of PML. Investigation of these factors promises to shed light on the complex development pathways involved in the regulation of haematopoiesis.