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Basic and Translational Investigations |
1 Department of Neurosurgery, Brain Tumor Center, The University of Texas M. D. Anderson Cancer Center, Houston, TX , USA; Department of Neurosurgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
2 Department of Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, TX , USA
3 Department of Genitourinary Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX , USA
4 Department of Experimental Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX , USA
5 Department of Neuro-Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX , USA
6 Department of Biostatistics, The University of Texas M. D. Anderson Cancer Center, Houston, TX , USA
7 Department of Neurosurgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
8 Department of Neurosurgery, Brain Tumor Center, The University of Texas M. D. Anderson Cancer Center, Houston, TX , USA
* To whom correspondence should be addressed. E-mail: flang{at}mdanderson.org.
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Abstract |
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The p53 protein plays a critical role in inducing cell cycle arrest or apoptosis. Because p53 is inactivated in human gliomas, restoring p53 function is a major focus of glioma therapy. The most clinically tested strategy for replacing p53 has been adenoviral-mediated p53 gene therapy (Ad-p53). In addition to their therapeutic implications, investigations into Ad-p53 provide model systems for understanding p53's ability to induce cell cycle arrest versus apoptosis, particularly because wild-type p53 cells are resistant to Ad-p53-induced apoptosis. Here we use Ad-p53 constructs to test the hypothesis that simultaneous phosphorylation of p53 at threonine 18 (Thr18) and serine 20 (Ser20) is causally associated with p53-mediated apoptosis. Studies using phosphorylation-specific antibodies demonstrated that p53-induced apoptosis correlates with phosphorylation of p53 at Thr18 and Ser20 but not with carboxy-terminal phosphorylation (Ser392). To prove a causal relationship between apoptosis and Thr18 and Ser20 phosphorylation of p53, the effects of an adenoviral p53 construct that was not phosphorylated (Ad-p53) was compared with a Thr18/Ser20 phosphomimetic construct (Ad-p53-18D20D) in wild-type p53 gliomas. Whereas treatment with Ad-p53 resulted only in cell cycle arrest, treatment with Ad-p53-18D20D induced dramatic apoptosis. Microarray and Western blot analyses showed that only Ad-p53-18D20D was capable of inducing expression of apoptosis-inducing proteins. Chromatin immunoprecipitation assays indicated that the protein product of Ad-p53-18D20D, but not Ad-p53, was capable of binding to apoptosis-related genes. We thus conclude that phosphorylation of Thr18 and Ser20 is sufficient for inducing p53-mediated apoptosis in glioma cells. These results have implications for p53 gene therapy and inform other strategies that aim to restore p53 function.
Key Words: apoptosis, cell cycle arrest, glioma, p53, phosphorylation
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