Wild-type p53—dependent etoposide-induced apoptosis mediated by caspase-3 activation in human glioma cells

2000 ◽  
Vol 93 (2) ◽  
pp. 289-297 ◽  
Author(s):  
Dali Yin ◽  
Norihiko Tamaki ◽  
Takashi Kokunai
Keyword(s):  
Glioma Cell ◽  
Glioma Cells ◽  
Human Glioma ◽  
Wild Type ◽  
Human Glioma Cells ◽  
Content Type ◽  
Low Concentrations ◽  
Wild Type P53 ◽  
Induced Apoptosis ◽  
Fine Print

Object. In an attempt to understand the roles of several apoptosis-related genes in human glioma cells, the authors investigated the relationship of wild-type p53, interleukin-1β—converting enzyme (ICE), caspase-3 (CPP32), bax, and bcl-2 to the apoptotic response of three glioma cell lines after treatment with etoposide.Methods. A human glioma cell line (U-87MG) that expresses wild-type p53, one that expresses mutant p53 (T-98G), and a T-98G derivative (T-98G/p53) that was transfected with a wild-type p53 expression vector (pCDM8-p53/neo) were used. Cell growth inhibition in response to etoposide was quantified using a modified methylthiazol tetrazolium colorimetric assay. Induction of apoptosis was evaluated using Hoechst 33258 staining and a DNA fragmentation assay. To study the expression of the apoptosis-related proteins and messenger RNAs in the three glioma cell lines, Western blotting and polymerase chain reaction were performed. A caspase assay and Western blot analysis were used to assess CPP32 and ICE protease activity. A CPP32 inhibition assay was used to determine whether a specific CPP32 inhibitor, DEVD-CHO, affects the apoptosis induced by etoposide in malignant glioma cells. Etoposide significantly inhibited the growth of U-87MG and T-98G/p53 cells in a dose-dependent manner compared with the growth of the T-98G cells. Treatment with low concentrations of etoposide resulted in the increased expression of wild-type p53; it also initiated CPP32 activity and induced apoptosis in the U-87MG cells. Apoptosis was not induced in T-98G cells at low concentrations of etoposide, although it was induced at high concentrations. Furthermore, low concentrations of etoposide also induced apoptosis in the T-98G/p53 cells by enhancing the expression of transfected wild-type p53, decreasing the expression of bcl-2, and activating CPP32 activity. However, etoposide did not alter the expression of bax and did not initiate ICE activity in these three glioma cell lines. Etoposide-induced apoptosis can be suppressed by the CPP32 inhibitor DEVD-CHO.Conclusions. These findings indicate that wild-type p53, CPP32, and bcl-2 may mediate apoptosis induced by etoposide. Forced expression of wild-type p53 increases etoposide cytotoxicity in human glioma cells by inducing apoptosis and may have important therapeutic implications.

Differential effects of vincristine and phenytoin on the proliferation, migration, and invasion of human glioma cell lines

1995 ◽  
Vol 82 (6) ◽  
pp. 1035-1043 ◽  
Author(s):  
Jörg-Christian Tonn ◽  
Hans Kristian Haugland ◽  
Jaakko Saraste ◽  
Klaus Roosen ◽  
Ole Didrik Laerum
Keyword(s):  
Cell Lines ◽  
Glioma Cell ◽  
Glioma Cells ◽  
Migration And Invasion ◽  
Human Glioma ◽  
Human Glioma Cells ◽  
Content Type ◽  
Glioma Cell Lines ◽  
Dose Dependent ◽  
Fine Print

✓ The aim of this study was to investigate the antimigratory and antiinvasive potential of vincristine sulfate (VCR) on human glioma cells and to analyze whether phenytoin (5,5-diphenylhydantoin; DPH) might act synergistically with VCR. Vincristine affects the cytoplasmic microtubules; DPH has been reported to enhance VCR cytotoxicity in murine cells. In two human glioma cell lines, GaMG and D-37MG, we found VCR to reduce monolayer growth and colony formation in a dose-dependent fashion at concentrations of 10 ng/ml and above. Phenytoin increased the cytotoxic and cystostatic effects of VCR in monolayer cells but not in spheroids. Multicellular spheroids were used to investigate directional migration. A coculture system of GaMG and D-37MG spheroids with fetal rat brain aggregates was used to analyze and quantify tumor cell invasion. A dose-dependent inhibition of migration and invasion by VCR was observed in both cell lines without further enhancement by DPH. Immunofluorescence microscopy with antibodies against α-tubulin revealed dose-dependent morphological alterations in the microtubules when the cells were exposed to VCR but not after incubation with DPH. Based on the combination of standardized in vitro model systems currently in use and the present data, the authors strongly suggest that VCR inhibits migration and invasion of human glioma cells. This is not altered by DPH, which inhibits cell proliferation in combination with VCR.

Cell cycle arrest and astrocytic differentiation resulting from PTEN expression in glioma cells

1999 ◽  
Vol 91 (5) ◽  
pp. 822-830 ◽  
Author(s):  
Jun-ichi Adachi ◽  
Katsumi Ohbayashi ◽  
Tomonari Suzuki ◽  
Tomio Sasaki
Keyword(s):  
Expression System ◽  
Biological Significance ◽  
Glioma Cells ◽  
Genetic Alterations ◽  
Pten Expression ◽  
Human Glioma ◽  
Wild Type ◽  
Content Type ◽  
Astrocytic Differentiation ◽  
Fine Print

Object. Genetic alterations of the PTEN gene (also known as MMAC1 or TEP1) have frequently been identified in high-grade gliomas, indicating that inactivation of PTEN plays a crucial role in human glioma progression. The aim of this study was to assess the biological significance of PTEN inactivation in the development of glioma.Methods. The authors introduced wild-type PTEN complementary DNA into four human glioma cell lines (T98G, U-251MG, U-87MG, and A172) containing endogenous aberrant PTEN alleles. The number of colonies transfected with the wild-type PTEN was reduced to 15 to 32% of those found after transfection of a control vector, suggesting growth suppression by the exogenous PTEN. To analyze phenotypic alterations produced by PTEN expression, T98G-derived clones with inducible PTEN expression were further established using a tetracycline-regulated inducible gene expression system. Induction of PTEN expression suppressed the in vitro growth of T98G cells with accumulation of G1 phase cells. Furthermore, when cells were cultured in the presence of the extracellular matrix (ECM), PTEN expression caused distinct morphological changes, with multiple and elongated cytoplasmic processes similar to those of normal astrocytes. The level of glial fibrillary acidic protein, an intermediate protein specifically expressed in differentiated astrocytes, was upregulated concomitantly.Conclusions. These findings strongly indicate that exogenous PTEN expression inhibits the proliferation of glioma cells by inducing G1 arrest and elicits astrocytic differentiation in the presence of the ECM. Inactivation of PTEN would play an important role in the enhancement of unregulated growth of undifferentiated glioma cells.

Enhancement of radiosensitivity of wild-type p53 human glioma cells by adenovirus-mediated delivery of the p53 gene

1998 ◽  
Vol 89 (1) ◽  
pp. 125-132 ◽  
Author(s):  
Frederick F. Lang ◽  
W. K. Alfred Yung ◽  
Uma Raju ◽  
Floralyn Libunao ◽  
Nicholas H. A. Terry ◽  
...  
Keyword(s):  
Cell Line ◽  
Glioma Cell ◽  
Radiation Treatment ◽  
P53 Protein ◽  
Glioma Cells ◽  
Glioma Cell Line ◽  
Wild Type ◽  
Content Type ◽  
Infected Cells ◽  
Fine Print

Object. The authors sought to determine whether combining p53 gene transfer with radiation therapy would enhance the therapeutic killing of p53 wild-type glioma cells. It has been shown in several reports that adenovirus-mediated delivery of the p53 gene into p53 mutant gliomas results in dramatic apoptosis, but has little effect on gliomas containing wild-type p53 alleles. Therefore, p53 gene therapy alone may not be a clinically effective treatment for gliomas because most gliomas are composed of both p53 mutant and wild-type cell populations. One potential approach to overcome this problem is to exploit the role p53 plays as an important determinant in the cellular response to ionizing radiation. Methods. In vitro experiments were performed using the glioma cell line U87MG, which contains wild-type p53. Comparisons were made to the glioma cell line U251MG, which contains a mutant p53 allele. Monolayer cultures were infected with an adenovirus containing wild-type p53 (Ad5CMV-p53), a control vector (dl312), or Dulbecco's modified Eagle's medium (DMEM). Two days later, cultures were irradiated and colony-forming efficiency was determined. Transfection with p53 had only a minor effect on the plating efficiency of nonirradiated U87MG cells, reducing the plating efficiency from 0.23 ± 0.01 in DMEM to 0.22 ± 0.04 after addition of Ad5CMV-p53. However, p53 transfection significantly enhanced the radiosensitivity of these cells. The dose enhancement factor at a surviving fraction of 0.10 was 1.5, and the surviving fraction at 2 Gy was reduced from 0.61 in untransfected controls to 0.38 in p53-transfected cells. Transfection of the viral vector control (dl312) had no effect on U87MG radiosensitivity. In comparison, transfection of Ad5CMV-p53 into the p53 mutant cell line U251MG resulted in a significant decrease in the surviving fraction of these cells compared with controls, and no radiosensitization was detected. To determine whether Ad5CMV-p53—mediated radiosensitization of U87MG cells involved an increase in the propensity of these cells to undergo apoptosis, flow cytometric analysis of terminal deoxynucleotidyl transferase-mediated biotinylated-deoxyuridinetriphosphate nick-end labeling—stained cells was performed. Whereas the amount of radiation-induced apoptosis in uninfected and dl312-infected control cells was relatively small (2.1 ± 0.05% and 3.7 ± 0.5%, respectively), the combination of Ad5CMV-p53 infection and radiation treatment significantly increased the apoptotic frequency (18.6 ± 1.4%). To determine whether infection with Ad5CMV-p53 resulted in increased expression of functional exogenous p53 protein, Western blot analysis of p53 was performed on U87MG cells that were exposed to 9 Gy of radiation 2 days after exposure to Ad5CMV-p53, dl312, or DMEM. Infection with Ad5CMV-p53 alone increased p53 levels compared with DMEM- or dl312-treated cells. Irradiation of Ad5CMV-p53—infected cells resulted in a further increase in p53 that reached a maximum at 2 hours postirradiation. To determine whether exogenous p53 provided by Ad5CMV-p53 had transactivating activity, U87MG cells were treated as described earlier and p21 messenger RNA levels were determined. Infection of U87MG cells with Ad5CMV-p53 only resulted in an increase in p21 compared with DMEM- and dl312-treated cells. Irradiation of Ad5CMV-p53—infected cells resulted in an additional time-dependent increase in p21 expression. Conclusions. These data indicate that adenovirus-mediated delivery of p53 may enhance the radioresponse of brain tumor cells containing wild-type p53 and that this radiosensitization may involve converting from a clonogenic to the more sensitive apoptotic form of cell death. Although the mechanism underlying this enhanced apoptotic susceptibility is unknown, the Ad5CMV-p53—infected cells have a higher level of p53 protein, which increases further after irradiation, and this exogenous p53 is transcriptionally active. Thus, it is possible that the combination of Ad5CMV-p53 infection and radiation treatment increases p53 protein to a level that is sufficient to overcome at least partially the block in apoptosis existing in U87MG cells.

Enhancement of C2-ceramide antitumor activity by small interfering RNA on X chromosome—linked inhibitor of apoptosis protein in resistant human glioma cells

2004 ◽  
Vol 101 (1) ◽  
pp. 119-127 ◽  
Author(s):  
Manabu Hatano ◽  
Masaaki Mizuno ◽  
Jun Yoshida
Keyword(s):  
Small Interfering Rna ◽  
Glioma Cells ◽  
Inhibitor Of Apoptosis ◽  
Human Glioma ◽  
Human Glioma Cells ◽  
Content Type ◽  
Inhibitor Of Apoptosis Protein ◽  
Apoptosis Protein ◽  
Interfering Rna ◽  
Fine Print

Object. Many human glioma cells are resistant to ceramide. In this study the authors investigated the mechanisms of that resistance and considered ways to overcome it. Methods. The authors first administered C2-ceramide (N-acetylsphingosine) to human glioma cells from rare cell lines susceptible to C2-ceramide (SKMG1 and U87MG) and other cell lines resistant to it (U251SP, T98G, SKAO2, and U251MG). Following this, the authors analyzed the statuses of transduction signals such as cell viability, morphological changes, caspases, mitochondrial membrane potential, apoptosis-inducing factor, oligonucleosomal DNA fragmentation, and the inhibitor of apoptosis protein (IAP) family. Conclusions. Ceramide resistance was found to arise from the inhibition of caspase-7 induced by IAPs, especially X chromosome—linked IAP (XIAP). Small interfering RNA (siRNA) on XIAP quenched that resistance in ceramide-resistant human glioma cells (U251SP, T98G, SKAO2, U251MG), indicating that a siRNA for XIAP may be a useful tool for overcoming the resistance to ceramide in human glioma cells.

Enhanced radiosensitivity of malignant glioma cells after adenoviral p53 transduction

1999 ◽  
Vol 91 (6) ◽  
pp. 997-1004 ◽  
Author(s):  
William C. Broaddus ◽  
Yue Liu ◽  
Laura L. Steele ◽  
George T. Gillies ◽  
Peck-Sun Lin ◽  
...  
Keyword(s):  
Malignant Glioma ◽  
Glioma Cells ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Wild Type ◽  
Content Type ◽  
Cranial Radiation ◽  
Adenoviral Transduction ◽  
Wild Type P53 ◽  
Malignant Glioma Cells ◽  
Fine Print

Object. The goal of this study was to determine whether adenoviral vector—mediated expression of human wildtype p53 can enhance the radiosensitivity of malignant glioma cells that express native wild-type p53.The p53 gene is thought to function abnormally in the majority of malignant gliomas, although it has been demonstrated to be mutated in only approximately 30%. This has led to studies in which adenoviral transduction with wild-type human p53 has been investigated in an attempt to slow tumor cell growth. Recent studies suggest that reconstitution of wild-type p53 can render cells more susceptible to radiation-mediated death, primarily by p53-mediated apoptosis.Methods. Rat RT2 glioma cells were analyzed for native p53 status by reverse transcriptase—polymerase chain reaction and sequence analysis and for p53 expression by Western blot analysis. Clonogenic survival and the terminal deoxynucleotidyl transferase—mediated deoxyuridine triphosphate nick-end labeling assay were used to characterize RT2 cell radiosensitivity and apoptosis, respectively, with and without prior transduction with p53-containing and control adenoviral vectors. Animal survival length was monitored after intracerebral implantation with transduced and nontransduced RT2 cells, with and without cranial radiation.The RT2 cells were demonstrated to express native rat wild-type p53 and to markedly overexpress human p53 following adenoviral p53 transduction. The combination of p53 transduction followed by radiation resulted in marked decreases in RT2 cell survival and increases in apoptosis at radiation doses from 2 to 6 Gy. Animals receiving cranial radiation after intracerebral implantation with RT2 cells previously transduced with p53 survived significantly longer than control animals (p < 0.01).Conclusions. The ability to enhance the radiosensitivity of malignant glioma cells that express wild-type p53 by using adenoviral transduction to induce overexpression of p53 offers hope for this approach as a therapeutic strategy, not only in human gliomas that express mutant p53, but also in those that express wild-type p53.

Introduction of wild-type p53 enhances thrombospondin-1 expression in human glioma cells

2003 ◽  
Vol 191 (1) ◽  
pp. 109-119 ◽  
Author(s):  
Hironobu Harada ◽  
Kou Nakagawa ◽  
Masahiro Saito ◽  
Shohei Kohno ◽  
Shigeyuki Nagato ◽  
...  
Keyword(s):  
Glioma Cells ◽  
Human Glioma ◽  
Wild Type ◽  
Human Glioma Cells ◽  
Thrombospondin 1 ◽  
Wild Type P53

Inhibition of growth of established human glioma cell lines by modulators of the protein kinase-C system

1990 ◽  
Vol 73 (4) ◽  
pp. 594-600 ◽  
Author(s):  
William T. Couldwell ◽  
Jack P. Antel ◽  
Michael L. J. Apuzzo ◽  
Voon Wee Yong
Keyword(s):  
Glioma Cell ◽  
Glioma Cells ◽  
Thymidine Uptake ◽  
Human Glioma ◽  
Content Type ◽  
Inhibition Of Growth ◽  
Kinase C ◽  
Glioma Cell Lines ◽  
Fine Print

✓ The protein kinase-C (PKC) second messenger system contributes to regulation of cell growth and differentiation. This study was undertaken to examine the effects of modulators of the PKC enzyme system on the state of differentiation and proliferation rates of human gliomas in vitro. The administration of the PKC-activating phorbol esters 4-beta-phorbol-12,13-dibutyrate (PDB) and phorbol-12-myristate-13-acetate (PMA) resulted in a dose-related inhibition of growth of human glioma cell lines in vitro as measured by 3H-thymidine uptake. The synthetic nonphorbol PKC activator (SC-9) produced an even more pronounced decrease of 3H-thymidine uptake. Diacylglycerol, an endogenous activator of the system, applied externally, transiently decreased the proliferation, in concordance with its short-lived existence in vivo. Conversely, the administration of 4-alpha-phorbol-12,13-didecanoate (α-PDD), a phorbol ester that binds but does not activate the enzyme, had no effect on the proliferation rate. At the dosages that maximally decreased proliferation, there was no evidence of direct glioma cell lysis induced by these agents as measured by a chromium-release assay. Immunocytochemical analysis and cytofluorometric measurement of glial fibrillary acidic protein (GFAP) staining in the treated cultures revealed an increase in GFAP staining over control cultures. In contrast to the response of glioma cells, nonmalignant human adult astrocytes treated with the PKC activators responded by increasing their proliferation rate. The authors postulate that the diametrically opposed effects of PKC activators on nonmalignant astrocytes versus glioma growth may be due to a high intrinsic PKC activity in glioma cells, with resultant down-regulation of enzyme activity following the administration of the pharmacological activators.

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