Purpose Glioblastoma multiforme (GBM) is the most lethal form of brain cancer with a median survival of only 12–15 months. Current standard treatment consists of surgery followed by chemoradiation. The poor survival of GBM patients is due to aggressive tumor invasiveness, an inability to remove all tumor tissue, and an innate tumor chemo- and radioresistance. ATM, ataxia telangiectasia (A-T) mutated, is an excellent target for radiosensitizing GBM because of its critical role in regulating the DNA damage response and p53, among other cellular processes. As a first step toward this goal, we recently showed that the novel ATM kinase inhibitor KU-60019 reduced migration, invasion, growth, and potently radiosensitized human glioma cells in vitro. Experimental Design Using orthotopic xenograft models of GBM, we now show that KU-60019 is also an effective radiosensitizer in vivo. Human glioma cells expressing reporter genes for monitoring tumor growth and dispersal were grown intra-cranially, and KU-60019 was administered intra-tumorally by convection-enhanced delivery or osmotic pump. Results Our results demonstrate that the combined effect of KU-60019 and radiation significantly increased survival of mice 2–3 fold over controls. Importantly, we show that glioma with mutant p53 is much more sensitive to KU-60019 radiosensitization than genetically matched wild-type glioma. Conclusions Taken together, our results suggest that an ATM kinase inhibitor may be an effective radiosensitizer and adjuvant therapy for patients with mutant p53 brain cancers.

Previously, we have demonstrated that deoxycholic acid (DCA)-induced signaling of extracellular signalregulated kinases 1 and 2 (ERK1/2) in primary hepatocytes is a protective response. In the present study, we examined the roles of the ERK and c-Jun

In primary hepatocytes and HepG2 hepatoma cells, prolonged activation of the p42/44 mitogen-activated protein kinase (MAPK) pathway is associated with a reduction in DNA synthesis, mediated by increased expression of the cyclin-dependent kinase inhibitor protein p21 Cip-1/WAF1/mda6 (p21). This study was performed to evaluate the contribution of transcriptional and post-transcriptional regulation in this response. Prolonged activation of the MAPK pathway in wild-type or p21 null hepatocytes caused a large decrease and increase, respectively, in DNA synthesis. Prolonged activation of the MAPK pathway in either wild-type or p21 antisense HepG2 cells also caused large decreases and increases, respectively, in DNA synthesis. MAPK signaling increased the phosphorylation of the transcription factors Ets2, C/EBPα, and C/EBPβ, and rapidly increased transcription from the p21 promoter via multiple Ets- and C/EBP-elements within the enhancer region. Eight hours after MAPK activation, loss of C/EBPβ or Ets2 function significantly reduced MAPK-stimulated transcription from the p21 promoter and abolished increased p21 protein expression. At this time, MAPK signaling increased both p21 mRNA and p21 protein stabilities that were also demonstrated to be essential for a profound increase in p21 protein levels. Thirty-six hours after MAPK activation, transcription from the p21 promoter was still significantly reduced in cells without either C/EBPβ or Ets2 function; however, these cells were now capable of exhibiting a partial increase in p21 protein expression. In contrast, loss of C/EBPα function modestly reduced MAPK-stimulated transcription from the p21 promoter but strongly inhibited the ability of prolonged MAPK activation to increase protein levels of p21. This data suggested that prolonged enhancement of p21 protein levels may be under posttranscriptional control. In agreement with this hypothesis, prolonged MAPK signaling further increased p21 mRNA stability at 36 h, compared with the 8-h time point. Our data argue that MAPK signaling increased p21 promoter activity via multiple transcription factors, which alone were insufficient for a robust prolonged increase in p21 protein levels in primary hepatocytes, and that to increase p21 protein levels also required enhanced stabilization of p21 mRNA and p21 protein. Collectively, these data suggest that loss of transcription factor and mRNA/protein stabilization functions correlates with an inability of MAPK signaling to cause growth arrest versus proliferation in primary hepatocytes.

Prolonged activation of the mitogen-activated protein kinase (MAPK) pathway enhances expression of the cyclin kinase inhibitor p21 that can promote growth arrest and cell survival in response to cytotoxic insults. Bile acids can also cause prolonged MAPK activation that is cytoprotective against bile acid-induced cell death. Here, we examined the impact of bile acid-induced MAPK signaling and p21 expression on the survival of primary mouse hepatocytes. Deoxycholic acid (DCA) caused prolonged activation of the MAPK pathway that weakly enhanced p21 protein expression. When DCA-induced MAPK activation was blocked using MEK1/2 inhibitors, both hepatocyte viability and expression of p21 were reduced. Surprisingly, constitutive overexpression of p21 in p21؉/؉ hepatocytes enhanced DCA-induced cell killing. In agreement with these findings, treatment of p21؊/؊ hepatocytes with DCA and MEK1/2 inhibitors also caused less apoptosis than observed in wildtype p21؉/؉ cells. Expression of p21 in p21؊/؊ hepatocytes did not modify basal levels of apoptosis but restored the apoptotic response of p21؊/؊ cells to those of p21؉/؉ cells overexpressing p21. These findings suggest that basal expression of p21 plays a facilitating, proapoptotic role in DCA-induced apoptosis. Overexpression of p21 enhanced p53 protein levels. In agreement with a role for p53 in the enhanced apoptotic response, overexpression of p21 did not potentiate apoptosis in p53؊/؊ hepatocytes but, instead, attenuated the death response in these cells. In conclusion, our data suggest that overexpression of p21 can promote apoptosis, leading to elevated sensitivity to proapoptotic stimuli. (HEPATOLOGY 2002; 36:39-48.)

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