Related Experiment Videos
Characterization of p53 and p21 functional interactions in glioma cells en route to apoptosis
C Gomez-Manzano1, J Fueyo, A P Kyritsis
1Department of Neuro-Oncology, University of Texas M. D. Anderson Cancer Center, Houston 77030, USA.
Background:
Alterations of the p53 (also called TP53) gene are one of the most common abnormalities in gliomas. We have previously reported that restoration of wild-type p53 protein function in glioma cells results in programmed cell death (apoptosis). Since p53 functions are mediated by genes that directly control the tumor suppressor effect of the p53 protein, understanding the relationship between p53 and p53-related genes in glioma cells will aid in the design of more rational treatment strategies for brain tumors.
Purpose:
We conducted this study to examine the timing of the p53-mediated events preceding apoptosis. More specifically, we undertook this work to characterize the genetic and cell cycle-related factors that may increase the resistance of glioma cells to p53-induced apoptosis.
Methods:
Two human glioma cell lines (U-251 MG and U-373 MG) that express mutant p53 protein and two (U-87 MG and EFC-2) that express wild-type p53 protein were used. Replication-deficient adenovirus was utilized as an expression vector to transfer exogenous p53 and p21 complementary DNAs into the glioma cells; control cells were infected with the viral expression vector alone. To monitor gene transfer and the expression of exogenous genes (as well as the expression of endogenous genes), we used western blot analyses and immunohistochemistry analyses. Flow cytometry studies of cellular DNA content were performed to determine the cell cycle phenotype of the glioma cells before and after treatment.
Results:
p53-mediated apoptosis was preceded by elevation in the levels of the p21 (cell cycle-related) and Bax (apoptosis-related) proteins. In addition, cell cycle analyses showed that glioma cells were arrested in the G2 phase before undergoing cell death. Transfer of p21 induced a G2 block but did not induce apoptosis. Moreover, coexpression of p21 and p53 prevented glioma cells from undergoing apoptosis. Expression of exogenous p53 in wild-type p53 cells did not induce elevation of Bax levels, arrest in G2 phase, or apoptosis.
Conclusions And Implications:
Our data confirmed the ability of wild-type p53 to induce apoptosis in p53 mutant glioma cells. In addition, our results document that p21 plays a role in protecting cells from p53-mediated programmed cell death and suggest that p53-mediated apoptosis and p21 induction may represent, at least in certain cases, opposite signals. Finally, our data suggest that over expression of p21 in gliomas may be related to resistance to treatments that induce apoptosis.
Insights
Wild-type p53 induces apoptosis in glioma cells, but p21 expression blocks this effect. Overexpression of p21 may lead to resistance to apoptosis-inducing treatments for gliomas.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- TP53 gene alterations are common in gliomas.
- Wild-type p53 restoration induces apoptosis in glioma cells.
- Understanding p53-related genes is crucial for brain tumor treatment strategies.
Purpose of the Study:
- Examine the timing of p53-mediated events before apoptosis.
- Characterize genetic and cell cycle factors influencing glioma cell resistance to p53-induced apoptosis.
Main Methods:
- Used human glioma cell lines with mutant and wild-type p53.
- Employed adenovirus vectors for exogenous p53 and p21 cDNA transfer.
- Analyzed protein expression via western blot and immunohistochemistry; assessed cell cycle via flow cytometry.
Main Results:
- p53-mediated apoptosis correlated with increased p21 and Bax protein levels.
- Glioma cells exhibited G2 phase arrest prior to apoptosis.
- p21 transfer caused G2 block but not apoptosis; coexpression of p21 and p53 inhibited apoptosis.
- Exogenous p53 in wild-type cells did not elevate Bax, induce G2 arrest, or cause apoptosis.
Conclusions:
- Confirmed wild-type p53 induces apoptosis in p53 mutant glioma cells.
- p21 protects cells from p53-mediated apoptosis, suggesting opposing signals.
- p21 overexpression in gliomas may correlate with resistance to apoptosis-inducing therapies.