Effect of Human Cytomegalovirus Infection on Nerve Growth Factor Expression in Human Glioma U251 Cells

Objective To explore the change of endogenic nerve growth factor （NGF） expression in human glioma cells infected with human cytomegalovirus （HCMV）. Methods U251 cells were cultured in RPMI 1640 culture medium and infected with HCMV AD 169 strain in vitro to establish a cell model of viral infection. Morphologic changes of U251 cells were observed under inverted microscope before and after infection with HCMV. Expression of NGF gene and protein of cells was detected by RT-PCR and Western blotting before and after infection with HCMV. Results The cytopathic effects of HCMV-infected cells appeared on day 5 after infection. However, differential NGF expression was evident on day 7. NGF expression was decreased significantly in U251 cells on day 7 after infection in comparison with control group （P〈0.05）. Conclusion HCMV can down-regulate endogenous NGF levels in human glioma cell line U251.

Apoptosis of Glioblastoma U251 Cells Induced by Carmustine Combined All-trans Retinoic Acid via Regulating Cyclin E and p27kip 1

The effect and mechanism of carmustine（BCNU） combined with all-trans retinoic acid（ATRA） on the apoptosis of human glioblastoma U251 cells were investigated by means of 3-（4,5-dimethylthiazol-2-yl）-2,5-diphe- nyltetrazolium bromide（MTT） assay, flow cytometry, reverse transcription-polymerase chain reaction（RT-PCR） and Western blot analysis. The results show that BCNU or ATRA shows time- and dose-dependent inhibition effects on human glioblastoma U251 cells and the combination of BCNU with ATRA shows an synergistic inhibition effect on human glioblastoma U251 cells, and the combined BCNU and ATRA can significantly inhibit the proliferation of human glioblastoma U251 cells, and induce the apoptosis of them, making the cells arrest in the stage of G1 phase, the stage of S and G2 phases decline, the rate of the apoptosis of human glioblastoma U251 cells increase, the corresponding mRNA expression of cyclin E and cyclin-dependent kinase 2（CDK2） downregulated and the correspon- ding mRNA expression of p27kip 1 unregulated. In addition, the combined BCNU and ATRA reduced the protein expression of nuclear factor kappa B（NF-κB）. Taken together, these results suggest that the treatment of human glioblastoma U251 cells with a combination application of ATRA and BCNU can exert synergistic effect, the course of this kind of combination chemotherapy may likely be associated with multiple molecular mechanisms for apoptosis, furthermore, the cyclin E and p27kip 1 should be considered as novel targets for controlling the growth of glioblastoma cells.

HCMV Infection Depress NGF Expression in Human Glioma Cells

Human cytomegalovirus （HCMV） is the most common cause of congenital infection, resulting in birth defects such as microcephaly. In this study, RT-PCR and Western Blotting were performed to quantify the regulation of endogenic nerve growth factor expression in neuroglia ceils by HCMV infection. The results showed that basal, endogenous NGF expression in U251 was unchanged during early HCMV infection. NGF expression is strongly down-regulated during the latent phase of infection. These results suggest that HCMV can depress the NGF expression in U251 cells.

Proteomics of the differential protein expressions in human glioma cell line U251 cells infected with human cytomegalovirus,as demonstrated by the surface enhanced laser desorption/ionization(SELDI)protein chip system

The proteomics of the differential protein expressions in human glioma cell line U251 cells infected with human cytomegalovirus （HCMV） was investigated at the protein level by using the surface enhanced laser desorption/ionization （SELDI） protein chip system in order to develop a method of study for the pathogenesis of HCMV infection. In this study, the cultured U251 cells were infected with HCMV in good condition and the supernatants of lysates and the extracellular fluids of the cultivated infected cells were quantitatively defined for the expressed proteins. The proteomics of the differential protein expression in cells before and after infection was analyzed by WCX2 arrays on the protein chip reader. It was demonstrated that the eytopathic effects of infected cells appeared on the 5th day after infection, however, the differential protein expression was evident at 6 h after infection as revealed by RT-PCR and mass spectrometry. The protein peaks captured from different batches of samples, from the same sample detected with different arrays or for the different times were all equivalent. With the molecular weight range from 2000 Da to 3000 Da, chip captured 82 peaks from the intracellular fluids and 11 protein peak from the cellular fluid in which compared with the control group, the protein peaks with molecular weight of 13 536.3 Da, 10 046.1 Da and 17 106.2 Da were close to those of β-amyloid protein, caspase-1 precursor and LPS-induced TNF-α factor respectively, which showed brief up-regulation 4 h after infection, and continued to raise 48 h later. These results infer that these proteins may be related to the apoptosis induced by HCMV infection, thus suggesting that the apoptosis induced by HCMV infection may play a role in the pathogenesis of HCMV infection.