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Hong, Yong-Kil,Yung, W.K. Alfred 대한신경외과학회 1996 Journal of Korean neurosurgical society Vol.25 No.8
p53 종양억제유전자의 명확한 작용기전은 아직 밝혀지지 않았지만 WAFI/Cip1과 같은 유전자의 전사(transcription)를 조절하여 종양세포의 성장을 억제하는 것으로 알려졌다. 저자들은 p53 유전자 전달을 통한 유전자 요법의 가능성을 알아보기 위하여 증식력이 없는 아데노바이러스에 야생형 p53 유전자를 집어 넣어 만든 Ad5CMV-p53의 항종양효과를 사람교종세포주(U-251, LG)에서 검사하였다. 단층세포배야을 β-galactosidase 유전자를 지니는 아데노바이러스(Ad5CMV-β-gal) 25 plaque-forming units(PFU)/cell로 48시간 치료한 후 시행한 β-galactosidase 조직화학검사에서, U-251 세포와 LG 세포는 각각 90%, 42%의 유전자 전달효율을 보였다. Ad5CMV-p53 치료에 따른 외인성(exogenous0 p53 단백질의 발현과 WAFI/Cip1 단백질의 유도는 치료 후 24-72 시간에 최고치를 보였다. Ad5CMV-p53 치료는 U-251과 LG 세포의 성장을 각각 85%, 36% 억제하였고 뚜렷한 형태학적 변화를 유도하였다. 종양세포 성자억제에 필요한 Ad5CMV-p53의 적정용량은 10-40 PFU/cell 이었다. 이상의 결과는 Ad5CMV-p53가 사람교종세포에 대해 p53 유전자 전달효율이 높고 항종양 효과가 있음을 의미하며, 이를 이용한 뇌종양의 유전자요법의 가능성을 시사한다. The p53 tumor suppressor gene is one of the genes with greatest therapeutic potential for cancer treatment and its growth inhibitory mechanism is thought to be mediated through the activation of its downstream mediator WAFl/Cip1. In this study, we evaluated the effect of the replication-defective recombinant adenovirus expressing wild-type p53 gene(Ad5CMV-p53) in human glioma cell lines(U-251, LG) harboring mutant-type p53. β galactosidase histochemistry revealed that 90% of the U-251 and 42% the of LG cells are infected with the adenovirus at a multiplicity of infection(M01) of 25 plaque-forming units(PFU)/cell. Immunoblot analyses showed that endogenous p53 protein is expressed at a high level. and significant exogenous p53 protein expression and WAF1/Cip 1 induction peaked on day 1 and day 3 after Ad5CMV-p53 treatment Introduction of Ad5CMV-p53 inhibited the cell growth of U-251(85% inhibition) and LG cells(36% inhibition). and influenced cell morphology The optimal dose of Ad 5CMV p53 for the tumor cells growth inhibition was MOI of 10-40 PFU/cell These results suggest that Ad5CMV-p53 infects human glioma cells and transduces the p53 gene with high efficiency, and could be further developed for the gene therapy of human gliomas.
Tumor Suppressor Gene Therapy Strategies in Human Brain Tumor
Gomez-Manzano, Candeleria,Kyritsis, Athanassios,Alfred Yung, W. K.,Fueyo, Juan 가톨릭 의과학연구원 1997 가톨릭 의과학연구원 국제학술대회 Vol.1 No.-
Wild-type p53 is involved in several aspects of cell cycle control and suppression of transformation. inducing either apoptosis or G block in cell cycle progression. Using a recombinant adenovirus containting the wild-type p53 cDNA, the biological effects of the newly expressed wild-type p53 protein were examined in six human glioma cell lines. Three cell lines(U-251 MG. U0373 MG. and A-172) expressed endogenous mutant p53. and the other three(U-87 MG EFC-2, and D54 MG) expressed wild-type p53. The restoration of normal p53-encoded protein in the mutant cell lines induecd apoptosis as assessed by morphological studies using nuclear staining. electron microscopy. and flow cytometric assays. In wild-type p53 cell lines. However, the over expression of wild-type p53 did not result in apoptosis but inhibited cellular proliferation rather drastically and modified the neoplastic phenotype. This study shows that over-expression of wild-type p53 protein by an adenoviral vector induces apoptosis in mutant glioma cells as well as growth arrest in wild-type glioma cells.
Im, Seock-Ah,Mazano, Cankelaria Gomez,Fueyo, Juan,Liu, Ta-Jen,Ke, Li-Dao,Jeong Soo Kim,Lee, Ho-Young,Steck, Peter A.,Kyritsis, Athanassios P.,Yung, W.K. Alfred 가톨릭대학교 2000 Bulletin of The Catholic Research Institutes of Me Vol.28 No.-
Presently, there is no effective treatment for glioblastoma, the most malignant and common brain tumor. Angiogenic factors are potentially optimal targets for therapeutic strategies because they are essential for tumor growth and progression. In this study, we sought a strategy for efficiently delivering and antisinse cDNA molecule of the vascular endothelial growth factor(VEGF) to glioma cells. The recombinant adenoviral vector Ad5CMV-αVEGF carried the coding sequence of wild-type VEGF165 cDNA in an antisense orientation. Infection of U-87 MG malignant glioma cells with the Ad5CMV-αVEGF resulted in reduction of the level of the endogenous VEGF mRNA and drastically decreased the production of the targeted secretory form of the VEGF protein. Treatment of human glioma tumors established in nude mice with intralesional injection of Ad5CMV-αVEGF inhibited tumor growth. Taken together, these findings indicate that the efficient down-regulation of the VEGF produced by tumoral cells using antisense strategies has an antitumor effect in vivo, This is the first time that an adenoviral vector is used to transfer antisense VEGF sequence into glioma cells in an animal model, and our results suggest that this system may have a clinical and therapeutic utility. (Cancer Researcg 59:895-900, 1999)
Sano, Tetsuro,Lin, Huai,Chen, Xiashan,Langford, Lauren A.,Bondy, Dimpy Koul Melissa L.,Hess, Kenneth R.,Myers, Jeffery N.,Hong, Yong-Kil,Yung, W.K. Alfred,Steck, Peter A. 가톨릭대학교 2000 Bulletin of The Catholic Research Institutes of Me Vol.28 No.-
MMAC/PTEN, a tumor suppressor gene located on chromosome 10q, has recently been shown to act as a phosphatidylinositol 3,4,5-triphosphate phosphatase and to modulate cell growth and apoptosis. Somatic mutations of MMAC/PTEN have been reported in a number of human cancers, especially in glioblastoma multiforme (GBM), although the number of identified mutations (10-35%) is significantly lower than the frequency of LOH affecting the MMAC/PTEN locus in the specimens (75-95%). To further investigate the possible alterations that may affect MMAC/PTEN, we examined the expression of the gene by reverse transcription-PCR in a series of gliomas. A significant difference (P<0.001) was observed between the expression of MMAC/PTEN in GBMs versus lower grades of gliomas, thus mimicking the difference in allelic deletion associated with the locus in these tumors. Furthermore, Kaplan-Meier survival plots, adjusted for age and tumor grade, showed a significantly better prognosis for patients whose tumors expressed high level of MMAC/PTEN. Additionally, immunostaining of GBMs revealed little or no MMAC/PTEN expression in about two-thirds of the tumors, whereas the other approximately one-third of tumors had significantly higher levels of expression. However, in about two-thirds of the gigh-expressing specimens, a heterogeneous pattern of expression was observed, indicating that certain cells within the tumor failed to express MMAC/PTEN. The combination of these results suggest that, in addition to molecular alterations affecting the gene, altered expression of MMAC/PTEN may play a significant role in the progression of GBM and patient outcome. (Cancer Research 59:1820-1824, 1999)