Retroviral transfer of the p16INK4a cDNA inhibits C6 glioma formation in Wistar rats

BACKGROUND: The p16(INK4A) gene product halts cell proliferation by preventing phosphorylation of the Rb protein. The p16INK4a gene is often deleted in human glioblastoma multiforme, contributing to unchecked Rb phosphorylation and rapid cell division. We show here that transduction of the human p16INK4a cDNA using the pCL retroviral system is an efficient means of stopping the proliferation of the rat-derrived glioma cell line, C6, both in tissue culture and in an animal model. C6 cells were transduced with pCL retrovirus encoding the p16INK4a, p53, or Rb genes. These cells were analyzed by a colony formation assay. Expression of p16INK4a was confirmed by immunohistochemistry and Western blot analysis. The altered morphology of the p16-expressing cells was further characterized by the senescence-associated β-galactosidase assay. C6 cells infected ex vivo were implanted by stereotaxic injection in order to assess tumor formation. RESULTS: The p16INK4a gene arrested C6 cells more efficiently than either p53 or Rb. Continued studies with the p16INK4a gene revealed that a large portion of infected cells expressed the p16INK4a protein and the morphology of these cells was altered. The enlarged, flat, and bi-polar shape indicated a senescence-like state, confirmed by the senescence-associated β-galactosidase assay. The animal model revealed that cells infected with the pCLp16 virus did not form tumors. CONCLUSION: Our results show that retrovirus mediated transfer of p16INK4a halts glioma formation in a rat model. These results corroborate the idea that retrovirus-mediated transfer of the p16INK4a gene may be an effective means to arrest human glioma and glioblastoma

Activation of endogenous p53 by combined p19Arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines B16 and C6

<p>Abstract</p> <p>Background</p> <p>Reactivation of p53 by either gene transfer or pharmacologic approaches may compensate for loss of p19Arf or excess mdm2 expression, common events in melanoma and glioma. In our previous work, we constructed the pCLPG retroviral vector where transgene expression is controlled by p53 through a p53-responsive promoter. The use of this vector to introduce p19Arf into tumor cells that harbor p53wt should yield viral expression of p19Arf which, in turn, would activate the endogenous p53 and result in enhanced vector expression and tumor suppression. Since nutlin-3 can activate p53 by blocking its interaction with mdm2, we explored the possibility that the combination of p19Arf gene transfer and nutlin-3 drug treatment may provide an additive benefit in stimulating p53 function.</p> <p>Methods</p> <p>B16 (mouse melanoma) and C6 (rat glioma) cell lines, which harbor p53wt, were transduced with pCLPGp19 and these were additionally treated with nutlin-3 or the DNA damaging agent, doxorubicin. Viral expression was confirmed by Western, Northern and immunofluorescence assays. p53 function was assessed by reporter gene activity provided by a p53-responsive construct. Alterations in proliferation and viability were measured by colony formation, growth curve, cell cycle and MTT assays. In an animal model, B16 cells were treated with the pCLPGp19 virus and/or drugs before subcutaneous injection in C57BL/6 mice, observation of tumor progression and histopathologic analyses.</p> <p>Results</p> <p>Here we show that the functional activation of endogenous p53wt in B16 was particularly challenging, but accomplished when combined gene transfer and drug treatments were applied, resulting in increased transactivation by p53, marked cell cycle alteration and reduced viability in culture. In an animal model, B16 cells treated with both p19Arf and nutlin-3 yielded increased necrosis and decreased BrdU marking. In comparison, C6 cells were quite susceptible to either treatment, yet p53 was further activated by the combination of p19Arf and nutlin-3.</p> <p>Conclusions</p> <p>To the best of our knowledge, this is the first study to apply both p19Arf and nutlin-3 for the stimulation of p53 activity. These results support the notion that a p53 responsive vector may prove to be an interesting gene transfer tool, especially when combined with p53-activating agents, for the treatment of tumors that retain wild-type p53.</p

Concurrent hypermethylation of DNMT1, MGMT and EGFR genes in progression of gliomas

<p>Abstract</p> <p>Background</p> <p>Gliomas are the most common neoplasm of the brain. High-grade gliomas often resist treatment even with aggressive surgical resection and adjuvant radiation and chemotherapy. Despite the combined treatment, they frequently recur with the same or higher-grade histology. Genetic instability is commonly associated with inactivation of the normal DNA repair function and tumour suppressor genes as well as activation of oncogenes resulting from alterations of promoter hypermethylation, but the molecular mechanisms of the histological and clinical progression of gliomas are still poorly understood.</p> <p>Methods</p> <p>This study involved longitudinal analysis samples of primary and recurrent gliomas to determine whether the progression of low- and high-grade gliomas is associated with the promoter methylation of the DNMT1, MGMT and EGFR genes by PCR-based restriction enzyme assay. Epigenetic inactivation of these three important glioma-associated genes was analyzed in paired biopsy samples from 18 patients with tumour recurrence.</p> <p>Results</p> <p>The methylation analysis of the CpG sites in the DNA methyltransferase (DNMT1) promoter revealed a total of 6 hypermethylations (6/18), the methylguanine-DNA methyltransferase (MGMT) promoter revealed a total of 10 hypermethylations (10/18) and the epithelial grow factor receptor (EGFR) promoter revealed a total of 12 (12/18) hypermethylations respectively in recurrent gliomas. The results demonstrated that DNMT1 promoter hypermethylation does not occur in low-grade gliomas, it was mainly observed in secondary glioblastomas. Additionally, the MGMT and EGFR promoter was hypermethylated in both low-and high-grade GLs and their corresponding histological transformed GLs.</p> <p>Conclusion</p> <p>This study has provided further evidence that the histological transformation and progression of gliomas may be associated with the inactivation of the EGFR and MGMT genes. It seems that EGFR and MGMT promoter hypermethylations are early events in the clonal evolution of gliomas and this gene inactivation has proved to be stable even in tumour recurrence. However, the DNMT hypermethylation is a late part of glioma progression.</p> <p>Virtual slides</p> <p>The virtual slide(s) for this article can be found here: <url>http://www.diagnosticpathology.diagnomx.eu/vs/1935054011612460</url></p

Combating oncogene activation associated with retrovirus-mediated gene therapy of X-linked severe combined immunodeficiency

A successful gene therapy clinical trial that also encountered serious adverse effects has sparked extensive study and debate about the future directions for retrovirus-mediated interventions. Treatment of X-linked severe combined immunodeficiency with an oncoretrovirus harboring a normal copy of the gc gene was applied in two clinical trials, essentially curing 13 of 16 infants, restoring a normal immune system without the need for additional immune-related therapies. Approximately 3 years after their gene therapy, tragically, 3 of these children, all from the same trial, developed leukemia as a result of this experimental treatment. The current understanding of the mechanism behind this leukemogenesis involves three critical and cooperating factors, i.e., viral integration, oncogene activation, and the function of the therapeutic gene. In this review, we will explore the causes of this unwanted event and some of the possibilities for reducing the risk of its reoccurrence

Perspectivas da terapia gênica

O conceito de terapia gênica é simples: introduzir material genético na célula com o objetivo de interceder na progressão de uma doença. Mesmo com uma definição de fácil entendimento, na verdade o campo de estudo da terapia gênica é rico em tecnologias inovadoras, conhecimentos e práticas interdisciplinares. O desenvolvimento e aplicação de terapia gênica encontram desafios significativos iguais como qualquer tratamento ainda em fase experimental, mas também enfrentam dificuldades próprias devidas ao uso de material biológico como agente farmacológico. Esta pequena revisão apresenta alguns conceitos básicos sobre a tecnologia e aplicação de terapia gênica e serve como base para uma discussão sobre o importante papel da associação entre pesquisa básica e clínica no desenvolvimento desta nova abordagem terapêutica. A contribuição dos conhecimentos dos especialistas em aspectos básicos e clínicos permite que tanto a tecnologia quanto a aplicação de terapia gênica em pacientes rapidamente recebam os benefícios do progresso da ciência. No Brasil, a terapia gênica se encontra em fase precoce quando comparado com vários países ao redor do mundo, porém nossos avanços são significativos e existem excelentes perspectivas de crescimento.The concept of gene therapy is really quite simple: delivery of genetic material to cells with the objective of impacting the progression of a disease. Even with this easy to understand definition, the field of gene therapy is actually rich in technologic innovations and interdisciplinary knowledge and practice. The development and application of gene therapy encounter significant challenges just as is seen with any experimental treatment, but also encounters additional difficulties that are particular to the use of biological material as a pharmacological agent. This short review will present a few basic concepts about the technology and application of gene therapy and will provide the basis for discussing the important role that is played by the association of basic and clinical researchers during the development of these new therapeutic approaches. The contribution of knowledge by specialists in the laboratory and clinical aspects permits the rapid progress of both technology and its application as gene therapy in patients. In Brazil, gene therapy is still at an early phase of development as compared to many countries around the world, yet our advances are significant and we have excellent perspectives for growth in this area

ESTERASE-D ANALYSIS in FAMILIAL RETINOMA and RETINOBLASTOMA

ESCOLA PAULISTA MED SCH,BIOCHEM GENET LAB,CP 20372,BR-04023 São Paulo,SP,BRAZILESCOLA PAULISTA MED SCH,BIOCHEM GENET LAB,CP 20372,BR-04023 São Paulo,SP,BRAZILWeb of Scienc