RESUMO
Therapeutic retroviral vector integration near the oncogene LMO2 is thought to be a cause of leukemia in X-SCID gene therapy trials. However, no published studies have evaluated the frequency of vector integrations near exon 1 of the LMO2 locus. We identified a high incidence region (HIR) of vector integration using PCR techniques in the upstream region close to the LMO2 transcription start site in the TPA-Mat T cell line. The integration frequency of the HIR was one per 4.46 x 10(4) cells. This HIR was also found in Jurkat T cells but was absent from HeLa cells. Furthermore, using human cord blood-derived CD34+ cells we identified a HIR in a similar region as the TPA-Mat T cell line. One of the X-linked severe combined immunodeficiency (X-SCID) patients that developed leukemia after gene therapy had a vector integration site in this HIR. Therefore, the descriptions of the location and the integration frequency of the HIR presented here may help us to better understand vector-induced leukemogenesis.
Assuntos
Proteínas de Ligação a DNA/genética , Éxons , Terapia Genética/efeitos adversos , Vetores Genéticos , Metaloproteínas/genética , Retroviridae/fisiologia , Linfócitos T/virologia , Integração Viral , Proteínas Adaptadoras de Transdução de Sinal , Linhagem Celular , Células Cultivadas , Terapia Genética/métodos , Humanos , Proteínas com Domínio LIM , Proteínas Proto-OncogênicasRESUMO
Several lines of evidence have revealed that ubiquitylation of membrane proteins serves as a signal for endosomal sorting into lysosomes or lytic vacuoles. The hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) interacts with ubiquitylated cargoes through its ubiquitin-interacting-motif domain (UIM domain), and plays an essential early role in endosomal sorting. Here, we show that the C-terminal region of Hrs, which does not contain the UIM domain, can bind to interleukin-2 receptor beta (IL-2Rbeta). We found a direct interaction between bacterially expressed IL-2Rbeta and Hrs in GST pull-down assays, indicating that their binding is independent of ubiquitin. Trafficking and degradation assays revealed that, similarly to wild-type IL-2Rbeta, an IL-2Rbeta mutant lacking all the cytoplasmic lysine residues is sorted from Hrs-positive early endosomes to LAMP1-positive late endosomes, resulting in degradation of the receptor. By contrast, an IL-2Rbeta mutant lacking the Hrs-binding region passes through early endosomes and is mis-sorted to compartments positive for the transferrin receptor. The latter mutant exhibits attenuated degradation. Taken together, these results indicate that precise sorting of IL-2Rbeta from early to late endosomes is mediated by Hrs, a known sorting component of the ubiquitin-dependent machinery, in a manner that is independent of UIM-ubiquitin binding.
Assuntos
Endossomos/metabolismo , Subunidade beta de Receptor de Interleucina-2/metabolismo , Proteínas de Membrana Lisossomal/metabolismo , Fosfoproteínas/metabolismo , Ubiquitina/metabolismo , Linhagem Celular , Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos/ultraestrutura , Humanos , Subunidade beta de Receptor de Interleucina-2/química , Subunidade beta de Receptor de Interleucina-2/genética , Fosfoproteínas/química , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Genomic analysis of integration will be important in evaluating the safety of human gene therapy with retroviral vectors. Here, we investigated MLV vector integration sites in human T-cells, since they are amenable to gene transfer studies, and have been used therapeutically in clinical trials. We mapped 340 MLV vector integration sites in the infected human T-cell clones we established. The data showed that MLV preferred integration near the transcription start sites (+/-5kb), near CpG islands (+/-1kb), and within the first intron of RefSeq genes. We also identified MLV integration hot spots that contained three or more integrations within a 100kb region. RT-PCR revealed that mRNA-levels of T-cell clones that contained MLV integrations near transcription start sites or introns were dysregulated compared to the uninfected cells. These studies help define the profile of MLV integration in T-cells and the risks associated with MLV-based gene therapy.
Assuntos
Vírus da Leucemia Murina/genética , Regiões Promotoras Genéticas , Linfócitos T/citologia , Linfócitos T/metabolismo , Integração Viral , Linhagem Celular , Clonagem Molecular , Ilhas de CpG , Terapia Genética/métodos , Vetores Genéticos , Humanos , Mutagênese , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/virologia , Sítio de Iniciação de TranscriçãoRESUMO
We tested the hypothesis that inhibition of Epstein-Barr virus (EBV) reactivation is controlled in part by nitric oxide (NO) generated from L-arginine (Arg). The spontaneous reactivation of EBV in the Burkitt's lymphoma (BL) cell line P3HR-1 was inhibited when the cells were cultured in L-Arg-supplemented medium. The expression of EBV early antigen (EA), immediate-early BZLF1 mRNA and the protein ZEBRA, and production of infectious virus were reduced by L-Arg supplementation in a dose-dependent manner. We demonstrated that inducible NO synthase (iNOS) mRNA was constitutively expressed in P3HR-1 cells, as quantitated by the reverse transcription-polymerase chain reaction. L-Arg supplementation enhanced iNOS and NOx expression in the cells. A specific NOS inhibitor, NG-monomethyl-L-Arg enhanced the expression of ZEBRA and early BMRF1 protein EA-D in the cells. L-Arg supplementation also inhibited the spontaneous EBV reactivation in another BL cell line EB1 and a B lymphoblastoid cell line OB. These results indicated that L-Arg induces iNOS and generates NO, which inhibits EBV reactivation in EBV-positive cells.
