Valproic acid (VA) is a well-tolerated drug used to treat seizure disorders and has recently been shown to inhibit histone deacetylase (HDAC). Because HDAC modulates chromatin structure and gene expression, parameters considered to influence radioresponse, we investigated the effects of VA on the radiosensitivity of human brain tumor cells grown in vitro and in vivo. The human brain tumor cell lines SF539 and U251 were used in our study. Histone hyperacetylation served as an indicator of HDAC inhibition. The effects of VA on tumor cell radiosensitivity in vitro were assessed using a clonogenic survival assay and ␥H2AX expression was determined as a measure of radiation-induced DNA double strand breaks. The effect of VA on the in vivo radioresponse of brain tumor cells was evaluated according to tumor growth delay analysis carried out on U251 xenografts. Irradiation at the time of maximum VA-induced histone hyperacetylation resulted in significant increases in the radiosensitivity of both SF539 and U251 cells. Key words: valproic acid; radiosensitization; histone deacetylase; in vivo; murine Valproic acid (VA), an 8-carbon branched-chained fatty acid, has well-established efficacy in the treatment of epilepsy and other seizure disorders. 1,2 Its broad-spectrum anticonvulsant activity has been suggested to result from a combination of mechanisms including the increase in ␥-aminobutyric acid, a decrease in ␥-hydroxybutyric acid and a direct interaction with the neuronal membrane blocking voltage dependent sodium channels. 3 Because of its effectiveness, oral bioavailability and generally low toxicity profile VA has been frequently used as a chronic anti-epileptic therapy. 4 Whereas generally free of other major side effects, VA is teratogenic in humans and rodents. In each case administration of VA during pregnancy can result in major malformations, dysmorphic syndromes and an estimated risk of neural tube defects of 1-3%. 5 Structure activity studies using VA and VA-related compounds, however, showed that the teratogenic effects could be separated from the anticonvulsant actions. 6,7 Moreover, the teratogenic activity of VA was attributed to reduced rate of cell proliferation resulting in a disruption in neuroepithelial-mesenchymal interactions. 8 Attempts to define the mechanism responsible for the neural tube defects led to in vitro studies using neuroblastoma cell lines, which showed that VA inhibited cell proliferation and induced morphological differentiation. 9 Subsequent investigations supported a potential anti-cancer action with VA reported to inhibit the proliferation of a variety of human tumor cells grown in vitro and as in vivo xenografts. 10 -12 Valproic acid was shown recently to be an effective inhibitor of histone deacetylase (HDAC). 6,11 Using VA and VA analogs, Phiel et al. 6 showed that the teratogenic but not the anti-epileptic activity of VA is likely due to HDAC inhibition. Moreover, their results along with previous reports 11 indicate that the anti-tumor effects of VA are likely the resu...

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Defining the molecules that regulate tumor cell survival is an essential prerequisite for the development of targeted approaches to cancer treatment. Whereas many studies aimed at identifying such targets use human tumor cells grown in vitro or as s.c. xenografts, it is unclear whether such experimental models replicate the phenotype of the in situ tumor cell. To begin addressing this issue, we have used microarray analysis to define the gene expression profile of two human glioma cell lines (U251 and U87) when grown in vitro and in vivo as s.c. or as intracerebral (i.c.) xenografts. For each cell line, the gene expression profile generated from tissue culture was significantly different from that generated from the s.c. tumor, which was significantly different from those grown i.c. The disparity between the i.c gene expression profiles and those generated from s.c. xenografts suggests that whereas an in vivo growth environment modulates gene expression, orthotopic growth conditions induce a different set of modifications. In this study the U251 and U87 gene expression profiles generated under the three growth conditions were also compared. As expected, the profiles of the two glioma cell lines were significantly different when grown as monolayer cultures. However, the glioma cell lines had similar gene expression profiles when grown i.c. These results suggest that tumor cell gene expression, and thus phenotype, as defined in vitro is affected not only by in vivo growth but also by orthotopic growth, which may have implications regarding the identification of relevant targets for cancer therapy.microarray ͉ intracerebral ͉ brain tumor

Purpose: Histone deacetylase (HDAC) inhibitors are undergoing clinical evaluation in cancer therapy. Because HDAC modulation has been shown to enhance the radiosensitivity of tumor cells in vitro, we investigated the effects of the HDAC inhibitor MS-275 on the radioresponse of DU145 prostate carcinoma xenografts.Experimental Design: As an indicator of HDAC inhibition in vivo, the histone acetylation status in tumor lysates was determined after two, four, and six injections of MS-275 delivered at 12-hour intervals, as well as 24 and 48 hours after the last injection. Tumor growth delay studies were then performed using this DU-145 xenograft model with radiation administered to leg tumors after the fourth dose of MS-275, which corresponded to the time of maximum histone hyperacetylation.Results: An increase in histone hyperacetylation was detected in each tumor after two injections of MS-275 with a maximum hyperacetylation occurring after four to six injections. In tumor growth delay studies, the combination of MS-275 and radiation resulted in a greater than additive inhibition of tumor growth as compared with the individual modalities. As alternative sources for an indicator of drug radiosensitizing activity, histone hyperacetylation was determined in a series of normal tissues, including lymphocytes. Each of the normal tissues also had a maximal histone hyperacetylation after four to six injections of MS-275.Conclusions: These studies show that MS-275 enhances the radiosensitivity of DU145 xenografts and suggest that histone hyperacetylation status can serve as a useful marker for drug radiosensitizing activity.