| Malignant gliomas are characterized by rapid growth and high invasiveness, which result in the difficulties in treatment. Recently, it has been found that cytoskeleton components and their relationship might be closely related to biologic characteristics of tumors. Our previous studies have shown that nordihydroguaiaretic acid (NDGA) possessed a marked differentiation inducement effect on human glioma cells, which outstandingly presented as an increase in intermediate filaments, up-regulation of GFAP expression, as well as changes of GFAP gene methylation pattern. However, the effects of NDGA on cytoskeleton system and the exact roles of GFAP gene in NDGA-induced differentiation remain obscure.In the present study, cytoskeletal components in malignant glioma cells and the effects of NDGA on them were observed. Meanwhile, antisense GFAP cDNA retrovirus expression vector pLXSN-asGFAP and sense GFAP cDNA fused with green fluorescent protein gene mammalian expression vector pIRGFP-GFAP were constructed. The effects of eliminating or reinforcing endogenous GFAP expression on the biological features were studied. Moreover, reversion of malignant phenotype in GFAP-deficient CHG-5 cells, which induced by NDGA simultaneously, was investigated in this experimental cell system. Our aim was to explore the possible roles of cytoskeleton proteins and GFAP gene in glioma cell proliferation, differentiation and NDGA-induced differentiation. The main results and conclusions are as follows:1. The localization and distribution of cytoskeletal components, including cytoplasmic cytoskeleton and nuclear cytoskeleton, in two glioma cells and itsrelationship with malignant grade were detected using immunofluorescence cytochemistry combined with confocal laser scanning microscopy and whole-mount embedded, selective extraction combined together high voltage transmission electron microscopy. The result showed that the localization and distribution of -tubulin, a subunit of microtubules, was identical in two glioma cell lines CHG-5 and SHG-44. In contrast, the structure and arrangement pattern of microfilaments, intermediate filaments as well as nuclear matrix in cell line CHG-5 were distinct from those of SHG-44, and these were also related to their degrees of differentiation and certain malignant phenotype. These results suggested that the difference of organization of cytoplasmic and nuclear cytoskeleton might provide inherent constructive foundation, and, might also provide an accessory evaluation in degrees of differentiation and malignant grade of glioma cells. It was also found that the skeleton protein expression and distribution could be altered by NDGA, along with its effects of growth inhibition and differentiation. This result suggested that enhancement of cytoskeletal protein synthesis and improvement of cytoplasmic and nuclear cytoskeleton organization might be one of the mechanisms of NDGA-induced differentiation in human glioma cells.2. Using gene recombination technique, antisense GFAP cDNA retrovirus expression vector pLXSN-asGFAP and sense GFAP cDNA fused with green fluorescent protein gene mammalian expression vector pIRGFP-GFAP were constructed respectively. The results of RT-PCR, in situ hybridization and immunocytochemistry showed that the expression of GFAP mRNA and its protein was reduced or even eliminated amongst CHG-5 cells infected with antisense GFAP retrovirus. GFAP-negative CHG-5 cells had marked cellular atypia and enhanced proliferative potential. In contrast with above, ithe expression of GFAP mRNA and its protein was markedly increased in SHG-44 cells after stably transfected with an expression vector containing GFAP cDNAin the sense orientation. The profound morphological changes in these cells, including exhibited stellate, abundant and thin cytoplasmic process formation, reduction of atypia occurred. And cell proliferation rate was markedly reduced. The contents of cytoskeletal proteins were also significantly increased. Cell cycle analysis results also revealed the differ...
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