Studies On Target Cells And Molecules Of Gliomagenesis, Malignant Progression, And Therapy

Part I Dedifferentiated cells: the targets of gliomagenesis, malignant progression, and therapy【Objective】To investigate in vitro differentiation reversion of brain tumor stem cells (BTSCs) for further study the inhibitory mechanisms of BTSCs differentiation.【Methods】CD133+ cells (BTSCs) were isolated from glioma specimens using the immunomagnetic bead separation system, and cultured in DMEM/F-12 medium supplemented with: (1) 10% fetal calf serum (FCS), (2) 10% FCS + sodium valproate (VPA) injection, (3) no FCS + growth factors, and (4) no FCS + growth factors + VPA injection, respectively. The morphological changes of cells were observed under phase contrast microscope at different time points; the cell surface markers relating with cell differentiation, cell cycles and changes in DNA ploids were detected with flow cytometry (FCM); co-expressions of cell surface markers relating with differentiation were analyzed with immunofluorescence staining and laser scanning confocal microscopy (LSCM).【Results】BTSCs cultured grew with a spherical shape in a suspended way in FCS-free medium and the expression levels of CD133 and nestin were high, but expressions of glial fibrillary acidic protein (GFAP) andβ-TubulinⅢwere negative. Cells in G0/G1 phase were the majority among these cells and in G2/M phase, nearly 0%. Besides, chromosomes of these cells were all heteroploids and the cells were insensitive to VPA. Having been cultured in FCS medium for 4 h, the suspended BTSCs spheres became adherent and turned to the round shape. Later on, the cells differentiated gradually into various shapes, until 7 d after in vitro culturing, the differentiated cells partially returned to the round shape and during the 10-21 d, some even regained the spherical shape and again grew in the suspended way. In addition, the results of FCM showed that the number of CD133 or Nestin positive cells decreased first and then increased after having been cultured in FCS medium for 3, 7, 10 and 21 days, but the number of GFAP+ or β-TubulinⅢ+ cells were at low levels. On the contrary, when VPA was added to the FCS medium, the phenomenon of reversion in cell morphology and changes of CD133 and nestin expression levels disappeared. The GFAP+ andβ-TubulinⅢ+ cells increased significantly after treatment with VPA for 7 d, but nestin was co-expressed with most of the GFAP+ orβ-TubulinⅢ+ cells. After cultured in FCS medium for 10 d, the majority of neural stem cells (NSCs) were GFAP+ andβ-TubulinⅢ+ cells with no CD133+ cells. Besides, the BTSCs cultured in FCS medium were heteroploids primarily including a few G2/M phase cells. After treatment with VPA, there were no significant differences in cell cycle and DNA ploids.【Conclusions】The phenotype of multi-directional differentiation of BTSCs in FCS medium was instable. The differentiated cells reversed frequently due to dedifferentiation. The differentiation inducer VPA inhibited differentiation reversion of BTSCs and facilitated the expressions of cell surface markers representing astrocytes and neurons, but the differentiation of BTSCs was incomplete for their co-expression with nestin, suggesting that the differentiation of BTSCs was still consistently inhibited.Part II Inactivation of p18INK4c gene was involved in gliomagenesis and malignant progression【Objective】To investigate the role of p18INK4c gene in gliomagenesis and malignant progression.【Methods】The p18INK4c expression in glioma cell lines and tissues was detected uing RT-PCR and immunohistochemistry. The methylation status of the p18INK4c promoter was analyzed by methylation-specific PCR. The p18INK4c mRNA was amplified and sequenced from 4 cell lines with p18INK4c expression, and then ligated into pcDNA3.1+ vector and transfected into glioma cell line SHG44 with Lipofectamine 2000. After transfection, the p18INK4c expression and cell cycle of SHG44 cell line were detected with RT-PCR and FCS, respectively.【Results】Five of 8 human glioma cell lines did not expressed p18INK4c, with promoter methylation in two. Three of 8 cell lines expressed p18INK4c, with all promoter hypomethylation. Twenty two of 68 glioma specimens did not express P18 protein, 46 of the specimens expressed. P18 mRNA and protein expression were negative in BTSCs. After transfected with pcDNA-p18INK4c, human glioma cell line SHG44 expressed p18INK4c significantly, the ratio of G0/G1 phase cells increased, S phase cells decreased compared with negative control.【Conclusions】Part of glioma cells and specimens did not express p18INK4c gene. Promoter methylation might contribute to p18INK4c silencing in some glioma cells. The p18INK4c gene inactivation might play a role in gliomagenesis and malignant progression.Part III Target cells being induced to differentiate by VPA in glioma【Objective】To investigate the target cells and molecules with sodium valproate induced differentiation of human glioma cells.【Methods】Nude mice bearing human glioma xenogenic graft subcutaneously were treated with VPA. The expressions of HDAC1 and Tob genes of xenografts were analyzed with semiquantitative RT-PCR. The CD133+ cells (BTSCs) were isolated from glioma specimens by immunomagnetic sorting, and cultured in the medium containing FCS or in the serum-free medium supplemented with growth factors, respectively, followed by treatment with VPA in vitro for 21days. The cell surface markers were detected with flow cytometry and confocal microscopy.【Results】VPA inhibited the growth of subcutaneous xenografs bearing on nude mice, and up-regulated the HDAC1 expression, down-regulated the Tob expression. The cell surface markers of BTSCs were detected by flow cytometry after VPA treatment for 21 days. In the FCS group, the GFAP orβ-tublinⅢpositive cells increased significantly, but in the growth factor group, no statistical differences were observed in the GFAP orβ-tublinⅢexpression. The results of confocal microscopy indicated that the GFAP+ orβ-tublinⅢ+ cells coexpressed with Nestin.【Conclusions】HDAC1 and Tob genes were the potential target molecules in reversion of the differential inhibition of human glioma cells with VPA. The BTSCs undergoing the processes of differentiation were the target cells for VPA. Part IV Promoter methylation of ABCG2 and MGMT gene in glioma cells【Objective】To analyze the correlations of ABCG2 and MGMT gene expression with the promoter methylation status in glioma cell lines.【Methods】The expressions of ABCG2 and MGMT in glioma cell lines were detected with RT-PCR. The promoter methylation status of ABCG2 and MGMT were analyzed by methylation-specific PCR. The expressions of ABCG2 and MGMT in U251 glioma cell line were analyzed after 5-Aza-dC treatment.【Results】Six of 8 glioma cell lines expressed ABCG2, with amplification only in U(unmethylated) reaction in five, and amplification in both U and M(methylated) reaction in one. Two of 8 cell lines did not expressed ABCG2, one displayed amplification bands in both the M and U reaction, and the other one failed to amplify either M or U bands. Five of 8 cell lines expressed MGMT, hypomethylation was detected in five, and hypermethylation in four.【Conclusions】Part of glioma cell lines expressed ABCG2 and/or MGMT, with promoter hypomethylation. These cell lines might be drug resistance. Promoter methyltion might contribute to inactivation of ABCG2 and MGMT in glioma cell lines.