Steps Involved in Immortalization and Tumorigenesis in Human B-Lymphoblastoid Cell Lines Transformed by Epstein-Barr Virus

Sugimoto, Masanobu; Tahara, Hidetoshi; Ide, Toshinori; Furuichi, Yasuhiro

doi:10.1158/0008-5472.can-04-0079

Cited by 133 publications

(141 citation statements)

References 43 publications

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“…TP-transduced B-LCLs from P1 survived after a growth crisis and reached stable levels of TP activity for at least 17 weeks. In fact, the results depicted in Figures 6a and b indicate a clonal selection of p305-TP-transduced cells, probably related with the EBV-induced immortalization process discussed above, 29 although we cannot rule out the possibility that this proliferative advantage was induced by the increased TP activity or by a vector insertion enhancing cell proliferation. Alternatively, a biased transduction of rapidly proliferative cells that eventually predominate in the cultures could also account for this observation.…”

Section: Discussionmentioning

confidence: 85%

“…Several reports have demonstrated that true immortalization only occurs in some B-LCL clones that survive after long periods of proliferation in pre-immortal phases. 29 Our experiments to assess lentiviral transduction and TP expression were performed in B-LCLs with less than 100 culture passages; therefore, it is unlikely that cell lines achieved true immortalization. TP-transduced B-LCLs from P1 survived after a growth crisis and reached stable levels of TP activity for at least 17 weeks.…”

Section: Discussionmentioning

confidence: 99%

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Hematopoietic gene therapy restores thymidine phosphorylase activity in a cell culture and a murine model of MNGIE

et al. 2011

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Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in the TYMP gene, which encodes thymidine phosphorylase (TP). TP dysfunction results in systemic thymidine (dThd) and deoxyuridine (dUrd) overload, which selectively impair mitochondrial DNA replication. Allogeneic hematopoietic transplantation has been used to treat MNGIE patients; however, this approach has serious adverse effects, including the toxicity of myeloablative conditioning, graft rejection and graft-versus-host disease. With the aim of testing the feasibility of gene therapy for MNGIE, we transduced TP-deficient B-lymphoblastoid cells from two MNGIE patients, with lentiviral vectors carrying a functional copy of the human TYMP DNA coding sequence. This restored TP activity in the cells, which reduced the excretion of dThd and dUrd and their concentrations when added in excess. Additionally, lentiviral-mediated hematopoietic gene therapy was used in partially myeloablated double Tymp/Upp1 knockout mice. In spite of the relatively low levels of molecular chimerism achieved, high levels of TP activity were observed in the peripheral blood of the transplanted mice, with a concomitant reduction of nucleoside concentrations. Our results suggest that hematopoietic gene therapy could be an alternative treatment for this devastating disorder in the future.

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Section: Discussionmentioning

confidence: 85%

Section: Discussionmentioning

confidence: 99%

Hematopoietic gene therapy restores thymidine phosphorylase activity in a cell culture and a murine model of MNGIE

et al. 2011

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“…A possible explanation is that in lymphoblasts, immortalization by a virus may significantly influence the cell proliferation rate and apoptotic cell death. 24 Although baseline caspase-3 activity is important for normal cell survival in muscle, 25 in our muscle cell model, caspase-3 activity differed significantly in cells expressing mutated htt and control cells (Figure 6a and b).…”

Section: Discussionmentioning

confidence: 85%

Increased apoptosis, huntingtin inclusions and altered differentiation in muscle cell cultures from Huntington's disease subjects

et al. 2006

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Mutated huntingtin (htt) is ubiquitously expressed in tissues of Huntington's disease (HD) patients. In the brain, the mutated protein leads to neuronal cell dysfunction and death, associated with formation of htt-positive inclusions. Given increasing evidence of abnormalities in HD skeletal muscle, we extensively analyzed primary muscle cell cultures from seven HD subjects (including two unaffected mutation carriers). Myoblasts from presymptomatic and symptomatic HD subjects showed cellular abnormalities in vitro, namely mitochondrial depolarization, cytochrome c release, increased caspase-3, -8, and -9 activities, and defective cell differentiation. Another notable feature was the formation of htt inclusions in differentiated myotubes. This study helps to advance current knowledge about the downstream effects of the htt mutation in human tissues. Further applications may include drug screening using this human cellular model.

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“…10,11 Onehundred sixty population doublings would, however, provide an extremely large quantity of genetic material. Appropriate archiving of seed stocks during early expansion of the cultures should obviate this potential limitation in the use of EBV-transformed cell lines as reliable sources of control materials.…”

Section: Resultsmentioning

confidence: 99%

Establishment of Stably EBV-Transformed Cell Lines from Residual Clinical Blood Samples for Use in Performance Evaluation and Quality Assurance in Molecular Genetic Testing

Bernacki

Stanković

Williams

et al. 2003

The Journal of Molecular Diagnostics

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Positive control materials for clinical molecular genetic testing applications are currently in critically short supply or non-existent for many genetically based diseases of public health importance. Here we demonstrate that anonymous, residual, clinical blood samples are potential sources of viable lymphocytes for establishing Epstein-Barr virus (EBV)-transformed blood lymphocyte cell lines. We attempted to transform 34 residual blood samples, and analyzed transformation success with respect to sample age, anticoagulant, storage temperature, volume, hemolysis, and patient age and sex. In univariate analysis, sample age was significantly associated with transformation success (P ‫؍‬ 0.002). The success rate was 67% (6 of 9) for samples 1 to 7 days old, 38% (3 of 8) for samples 8 to 14 days old and 0% for samples 15 to 21 (0 of 11) days old. When we controlled for sample age in multivariate logistic regression, anticoagulant and storage temperature approached significance (P ‫؍‬ 0.070 and 0.087, respectively; samples in acid citrate dextrose (ACD) and refrigerated samples were more likely to transform). Based on these findings, we suggest that samples collected in either ACD or ethylene diamine tetraacetic acid, and up to 14 days old (refrigerated) or 7 days old (stored ambient), are reasonable candidates for EBV transformation. The transformation rate for samples that met these criteria was 63% (

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Steps Involved in Immortalization and Tumorigenesis in Human B-Lymphoblastoid Cell Lines Transformed by Epstein-Barr Virus

Cited by 133 publications

References 43 publications

Hematopoietic gene therapy restores thymidine phosphorylase activity in a cell culture and a murine model of MNGIE

Hematopoietic gene therapy restores thymidine phosphorylase activity in a cell culture and a murine model of MNGIE

Increased apoptosis, huntingtin inclusions and altered differentiation in muscle cell cultures from Huntington's disease subjects

Establishment of Stably EBV-Transformed Cell Lines from Residual Clinical Blood Samples for Use in Performance Evaluation and Quality Assurance in Molecular Genetic Testing

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