Phosphatase and tensin homolog deleted on chromosome ten gene induces apoptosis and down-regulation of B cell lymphoma/leukemia-2 protein in human brain glioma SHG44 cells

Abstract

Objective To investigate the effect of phosphatase and tensin homolog deleted on chromosome ten (PTEN) gene on the apoptosis and expression of apoptosis related gene B cell lymphoma/leukemia-2 (bcl-2) in human glioma SHG44 cells, and to elucidate the mechanism of PTEN gene to inhibit tumor cell proliferation. Methods The SHG44 cells were cultured in RPMI 1640 medium supplemented with 10% calf serum. pBP-PTEN and vector pBP were transfected into SHG44 cells in logarithmic phase by Lipofectamine. Positive cell clones were screened, and SHG44 cells successfully transfected with pBP-PTEN and vector pBP served as SHG44-pBP-PTEN cells and SHG44-pBP cells respectively. The SHG44-pBP-PTEN, SHG44-pBP and SHC44 cells were detected by in situ hybridization. The immunohistochemistry was used to detect the expression of PTEN gene in three kinds of cells. Transmission electron microscopy, flow cytometry and nuclear DNA agarose gel electrophoresis were applied to examine the apoptosis, and immunofluorescence was used to detect the expression of B cell lymphoma/leukemia-2 (bcl-2) gene. Results Expression of PTEN gene and protein in SHG44 cells transfected by PTEN gene. Under the transmission electron microscope, SHC44-pBP-PTEN nuclear chromatin condensation, edge set, nuclear fragmentation, cytoplasmic condensation, and obvious apoptotic bodies were detected, which showed the typical apoptosis of tumor cells. The structure of SHG44 and SHG44-pBP cells was not found abnormal. Flow cytometry showed that cell cycle from G1 phase to S phase inhibition, and apoptosis peak appeared before G1 peak, SHG44 cell G1 phase, S phase, G2 phase and apoptotic peak (AP) phase proportion was 61.2%, 28.2%, 8.3% and 2.3%, SHG44-pBP cells were 64.1% 24.6%, 8.9% and 2.4%, SHG44-pBP-PTEN cells were 75.3%, 9.6%, 2.2% and 12.9%. The growth rate of SHG44-pBP-PTEN cells was significantly lower than that of the other two cells. Nuclear DNA agarose gel electrophoresis showed a typical apoptotic cell ladder; bcl-2 expression was also significantly reduced. The mean intensity of fluroscence (MIF) of SHG44-pBP cells was 100.4% (P>0.05) of control cell, while the MIF of SHG44-pBP-PTEN cells was 40.2%, and the difference between the 2 groups was statistically significant (P<0.01). Conclusion PTEN gene can induce apoptosis of SHG44 cells and down regulate the expression of bcl-2 protein, which may be one of the molecular mechanisms of PTEN gene inhibiting the proliferation of tumor cells. Key words: Brain glioma; Phosphatase and tensin homolog deleted on chromosome ten gene; Apoptosis; B cell lymphoma/leukemia-2