Suberoylanilide hydroxamic acid affects γH2AX expression in osteosarcoma, atypical teratoid rhabdoid tumor and normal tissue cell lines after irradiation.

PURPOSE: Osteosarcoma and atypical teratoid rhabdoid tumors are tumor entities with varying response to common standard therapy protocols. Histone acetylation affects chromatin structure and gene expression which are considered to influence radiation sensitivity. The aim of this study was to investigate the effect of the combination therapy with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) and irradiation on atypical teratoid rhabdoid tumors and osteosarcoma compared to normal tissue cell lines. METHODS: Clonogenic assay was used to determine cell survival. DNA double-strand breaks (DSB) were examined by pulsed-field electrophoresis (PFGE) as well as by γH2AX immunostaining involving flow cytometry, fluorescence microscopy, and immunoblot analysis. RESULTS: SAHA lead to an increased radiosensitivity in tumor but not in normal tissue cell lines. γH2AX expression as an indicator for DSB was significantly increased when SAHA was applied 24 h before irradiation to the sarcoma cell cultures. In contrast, γH2AX expression in the normal tissue cell lines was significantly reduced when irradiation was combined with SAHA. Analysis of initial DNA fragmentation and fragment rejoining by PFGE, however, did not reveal differences in response to the SAHA pretreatment for either cell type. CONCLUSION: SAHA increases radiosensitivity in tumor but not normal tissue cell lines. The increased H2AX phosphorylation status of the SAHA-treated tumor cells post irradiation likely reflects its delayed dephosphorylation within the DNA damage signal decay rather than chromatin acetylation-dependent differences in the overall efficacy of DSB induction and rejoining. The results support the hypothesis that combining SAHA with irradiation may provide a promising strategy in the treatment of solid tumors.

Mutagenic activity of carcinogens detected in transgenic rodent mutagenicity assays at dose levels used in chronic rodent cancer bioassays.

Data on transgenic rodent mutagenicity of five human carcinogens were summarised and compared with the results from rodent carcinogenicity studies. Four out of five carcinogens showed mutagenic activity already at daily dose levels which induced cancer in long-term rodent bioassays in at least one target tissue of carcinogenesis. In several of these studies, even single dose applications were sufficient to significantly increase the mutation frequency in vivo. Other genotoxic carcinogens required application of multiple dosing at dose-levels used in rodent cancer bioassays to show their in vivo mutagenicity. A rodent respiratory tract carcinogen, 1,2-dibromoethane (DBE), following inhalation exposure, displayed no mutagenic activity, neither in lung nor in nasal mucosa, at a single 2-h exposure to 30 ppm, which is below the highest concentration used in a NTP cancer bioassay. In contrast, after multiple treatment for 10 days at the same daily doses, a significant increase of the mutation frequency in nasal mucosa was apparent. We conclude, that especially when studying new chemicals in these transgenic rodent mutation assays, a multiple dosing protocol should be preferred. For dose selection, the same criteria could be applied as for chronic rodent bioassays.

Antineoplastic efficacy of melphalan and N-(2-chloroethyl)-N-nitrosocarbamoyl-omega-lysine, in combination with diazoxide or insulin in autochthonous mammary carcinoma of the Sprague-Dawley rat.

The anticancer activity of melphalan and N-(2-chloroethyl)-N-nitrosocarbamoyl-omega-lysine (CNC-omega-Lys), was compared in the autochthonous, methylnitrosourea-induced mammary carcinoma of the Sprague-Dawley rat. In addition, the influence on the therapeutic efficacy of the combination with diazoxide, causing a mild, reversible diabetes, and with insulin was investigated. The comparison of melphalan and CNC-omega-Lys clearly showed the superiority of melphalan. Both compounds displayed a significant tumour inhibition in their medium and the highest dosages in comparison to the untreated control. The combination with diazoxide resulted for almost all groups in an increased tumour inhibition. Only the lowest dose of CNC-omega-Lys + diazoxide did not reduce the tumour volume significantly versus the control group. The combination with insulin, however, resulted in a loss of tumour inhibition compared to the effect of the cytotoxic drug alone, although in these groups, too, a significant decrease of tumour volumes versus controls could be observed. Mortality was within tolerable limits (less than 20%) through the treatment period for all experimental groups. Median lifespans were increased in all therapy groups, but no additional benefit could be observed in the combination treatment groups.

Synthesis and characterization of steroid-linked N-(2-chloroethyl)nitrosoureas.

Syntheses of steroid-linked N-[N'-(2-chloroethyl)-N'-nitrosocarbamoyl]-(CNC-) amino acid esters and -amides with potential antineoplastic activity are described. The esters are prepared by reaction of CNC-amino acids with steroids using N,N'-carbonyldiimidazole and N,N'-dicyclohexylcarbodiimide. The corresponding amides are prepared by reaction of 1-(CNC-amino acyloxy)-pyrrolidine-2,5-diones with 17 beta-amino-3-hydroxy-1,3,5(10)-estratriene or 17 beta-O-[4-(6-aminohexylamino)-1,4-dioxo-butyl]-estradiol. Estradiol-17 beta-hemisuccinate is esterified with N-(2-hydroxyethyl)-N'-(2-chloro-ethyl)-N'-nitrosourea (HECNU). Spectroscopic characteristics and relative binding affinities to steroid receptors are given.