Negative effects of wild-type p53 and s-Myc on cellular growth and tumorigenicity of glioma cells

ObjectThe authors sought to study the combined potential of wild-type p53 gene transfer and Gamma Knife surgery (GKS) for the treatment of glioblastomas multiforme. Modification of the radiation response in C6 glioma cells in vitro and in vivo by the wild-type p53 gene was investigated.MethodsStable expression of wild-type p53 in C6 cells was achieved by transduction of the cells with adenoviral p53. Two days later, some cells were treated with GKS. Forty-eight hours after irradiation, the comparative survival rate was assessed by monotetrazolium (MTT) assays. Treated and control C6 glioma cells (4 × 103 per well) were plated into a 96-well plate in octuplicate and tested every 24 hours. Meanwhile, immunohistopathological examination of proliferating cell nuclear antigen (PCNA) and terminal deoxynucleotidyl transferase—mediated deoxyuridine triphosphate (TUNEL) assays were performed. The MTT assays indicated the p53, GKS, and combined treated cells proliferated at a significantly lower rate than those of the control group (p < 0.01, Days 2–6) and the positive fraction of PCNA in p53-treated group and GKS-treated group was 70.18 ± 3.61 and 50.71 ± 2.61, respectively, whereas the percentage in the combined group was 30.68 ± 1.49 (p < 0.01).Fifty-six male Sprague–Dawley rats were anesthetized and inoculated with 106 cultured C6 glioma cells into the cerebrum. Forty-eight hours after transduction with adenoviral p53, some rats underwent GKS. A margin dose of 15 Gy was delivered to the 50% isodose line. Two days later, six rats in each group were killed. Their brains were removed and paraffin-embedded section were prepared for immunohistopathological examination and TUNEL assays. The remaining rats were observed for the duration of the survival period. The survival curve indicated that a modest but significant enhancement of survival duration was seen in the p53-treated or GKS alone groups, whereas a more marked and highly significant enhancement of survival duration was achieved when these two treatment modalities were combined. When PCNA expression was downregulated, apoptotic cells become obvious after TUNEL staining.Conclusions The findings of this study suggest that p53-based gene therapy in combination with GKS may be superior to single-modality treatment of C6 glioma.

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Enhanced radiosensitivity of malignant glioma cells after adenoviral p53 transduction

Journal of Neurosurgery ◽

10.3171/jns.1999.91.6.0997 ◽

1999 ◽

Vol 91 (6) ◽

pp. 997-1004 ◽

Cited By ~ 47

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.

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

Cancer Letters ◽

10.1016/s0304-3835(02)00592-x ◽

2003 ◽

Vol 191 (1) ◽

pp. 109-119 ◽

Cited By ~ 22

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

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The effects of wild-type p53 gene transfection on the growth and chemotherapeutic sensitivity of human glioma cells

Journal of Huazhong University of Science and Technology [Medical Sciences] ◽

10.1007/bf02904786 ◽

2002 ◽

Vol 22 (1) ◽

pp. 44-46 ◽

Cited By ~ 1

Author(s):

Xiang Wei ◽

Zhu Xianli ◽

Zhao Hongyang

Keyword(s):

Gene Transfection ◽

Glioma Cells ◽

P53 Gene ◽

Human Glioma ◽

Wild Type ◽

Human Glioma Cells ◽

Chemotherapeutic Sensitivity ◽

Wild Type P53

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

Journal of Neurosurgery ◽

10.3171/jns.2000.93.2.0289 ◽

2000 ◽

Vol 93 (2) ◽

pp. 289-297 ◽

Cited By ~ 23

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.

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Wild-type human p53 transactivates the human proliferating cell nuclear antigen promoter.

Molecular and Cellular Biology ◽

10.1128/mcb.15.12.6785 ◽

1995 ◽

Vol 15 (12) ◽

pp. 6785-6793 ◽

Cited By ~ 80

Author(s):

C V Shivakumar ◽

D R Brown ◽

S Deb ◽

S P Deb

Keyword(s):

Binding Site ◽

Proliferating Cell Nuclear Antigen ◽

P53 Protein ◽

Cellular Growth ◽

Nuclear Antigen ◽

Wild Type ◽

P53 Response ◽

Low Levels ◽

Wild Type P53 ◽

Proliferating Cell

The wild-type p53 protein is a transcriptional activator implicated in the control of cellular growth-related gene expression. Here, using a number of different cell lines and transient-transfection-transcription assays, we demonstrate that at low levels, wild-type p53 transactivates the human proliferating cell nuclear antigen (PCNA) promoter. When expressed at a similar level, the tumor-derived p53 mutants did not transactivate the PCNA promoter. We identified a p53-binding site on the human PCNA promoter with which p53 interacts sequence specifically. When placed on a heterologous synthetic promoter, the binding site functions as a wild-type p53 response element in either orientation. Deletion of the p53-binding site renders the PCNA promoter p53 nonresponsive, showing that wild-type p53 transactivates the PCNA promoter by binding to the site. At a higher concentration, wild-type p53 inhibits the PCNA promoter but p53 mutants activate. Transactivation by p53 mutants does not require the p53-binding site. These observations suggest that moderate elevation of the cellular wild-type p53 level induces PCNA production to help in DNA repair.

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