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Wild-type p53-dependent etoposide-induced apoptosis mediated by caspase-3 activation in human glioma cells
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-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.
Insights
Wild-type p53, caspase-3 (CPP32), and bcl-2 mediate etoposide-induced apoptosis in human glioma cells. Forced expression of wild-type p53 enhances etoposide
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- Apoptosis plays a crucial role in regulating cell death and is implicated in cancer development.
- Understanding the molecular mechanisms of apoptosis in glioma is vital for developing effective cancer therapies.
- The roles of specific apoptosis-related genes, including p53 and caspases, are critical in cancer cell response to chemotherapy.
Purpose of the Study:
- To investigate the involvement of wild-type p53, interleukin-1beta-converting enzyme (ICE), caspase-3 (CPP32), bax, and bcl-2 in the apoptotic response of human glioma cells to etoposide.
- To elucidate the relationship between p53 status (wild-type vs. mutant) and etoposide-induced apoptosis in different glioma cell lines.
- To explore the therapeutic potential of targeting apoptosis pathways in human glioma.
Main Methods:
- Utilized three human glioma cell lines: U-87MG (wild-type p53), T-98G (mutant p53), and T-98G/p53 (transfected with wild-type p53).
- Quantified cell growth inhibition using a methylthiazol tetrazolium assay.
- Assessed apoptosis induction via Hoechst 33258 staining and DNA fragmentation assays. Protein and mRNA expression were analyzed by Western blotting and PCR. Caspase activity was measured using caspase assays and a specific inhibitor (DEVD-CHO).
Main Results:
- Etoposide significantly inhibited the growth of U-87MG and T-98G/p53 cells in a dose-dependent manner.
- Etoposide treatment increased wild-type p53 expression, activated CPP32, and induced apoptosis in U-87MG cells.
- Apoptosis was induced in T-98G/p53 cells by enhancing wild-type p53 expression, decreasing bcl-2, and activating CPP32; bax expression and ICE activity remained unaltered. Etoposide-induced apoptosis was suppressed by DEVD-CHO.
Conclusions:
- Wild-type p53, CPP32, and bcl-2 are key mediators of etoposide-induced apoptosis in human glioma cells.
- Forced expression of wild-type p53 enhances etoposide-induced cytotoxicity in glioma cells by promoting apoptosis.
- Targeting wild-type p53 and CPP32 activation holds significant therapeutic potential for human glioma treatment.