Wild-type p53-dependent etoposide-induced apoptosis mediated by caspase-3 activation in human glioma cells.
OBJECTIVE
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-1beta-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.
Department of Neurosurgery, Kobe University School of Medicine, Japan.
D
D
Yin
N
N
Tamaki
T
T
Kokunai
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D001932:Brain Neoplasms / Q000473:pathology*; D020170:Caspase 1 / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005047:Etoposide / Q000494:pharmacology*; D019254:Genes, bcl-2 / Q000235:genetics; D016158:Genes, p53 / Q000235:genetics*; D005910:Glioma / Q000473:pathology*; D006801:Humans; D014407:Tumor Cells, Cultured
D000972:Antineoplastic Agents, Phytogenic; D005047:Etoposide; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D020170:Caspase 1