Aparajita Sarkar

The effects of O6-benzylguanine and hypoxia on the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea in nitrosourea-resistant SF-763 cells.

O6-Alkylguanine-DNA alkyltransferase (AGT) activity is associated with resistance of brain tumor cell lines to the cytotoxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). SF-763 cells exhibit high AGT activity and are resistant to BCNU. In this study, we compared the effects of the AGT inhibitorO6-benzylguanine (BG) on the cytotoxicity of BCNU in oxic and hypoxic SF-763 cells; we also measured AGT activity, ornithine decarboxylase (ODC) activity, and polyamine levels to determine if there was any correlation with cell survival as determined by colony-forming efficiency assay. Exponentially growing monolayer cells were pretreated with 10 μM BG for 2 h under oxic or hypoxic (95% nitrogen/5% CO2) conditions and then exposed to graded concentrations of BCNU for 1 h. BG significantly lowered AGT activity but had no cytotoxic effect in oxic or hypoxic cells; hypoxia alone was not cytotoxic. The cytotoxicity of BCNU was 4 times higher in BG-treated hypoxic cells than in oxic cells treated with BCNU alone; the BCNU doses required for a 1-log cell kill were 75 and 300 μM, respectively. ODC activity was lowered by hypoxia alone but was not significantly affected by BG in either hypoxic or oxic cells. Polyamine levels were not significantly affected by hypoxia or BG. These results indicate that pretreatment with BG dramatically lowers AGT activity and increases the cytotoxicity of BCNU in both oxic and hypoxic SF-763 cells. The mechanism of this enhanced cytotoxicity is apparently unrelated to ODC activity or polyamine levels.

Effects of fractionated radiation therapy on human brain tumor multicellular spheroids

We investigated the cytotoxic effects of fractionated radiation therapy on multicellular spheroids of human malignant glioma cell lines U-87 MG, U-251 MG, and U-373 MG. Graded doses of x-rays were administered in 1, 3, 8, 15, and 30 fractions over 15 days. The isoeffect dose for a 1 log cell kill ranged from 4-4.5 Gy for a single fraction to 7-8 Gy for an 8-fraction protocol; no additional dose-sparing was achieved with more fractions. Therefore, the effects of individual doses (1.56 Gy) of the 8-fraction protocol were studied in U-251 MG spheroids. A cell survival assay showed that the first dose of radiation killed 30-50% of the cells; subsequent doses usually killed fewer cells. The cell kill after all 8 doses was about 1.0 log. No consistent relationship between the intracellular glutathione level and fraction number was observed. The 24-hour labeling index of the spheroids did not decrease until after the second fraction. Thus, the higher cell kill of the first dose does not seem to be related to cell cycle synchrony. Multinuclear and mononuclear giant cells were limited almost entirely to the periphery of the spheroids and increased with the number of radiation fractions. We conclude that multicellular spheroids can be used to study the biological effects of fractionated radiation therapy on human brain tumor cells. Although this model cannot be used to evaluate the effect of radiation on normal tissue, it may be useful in developing more effective radiation therapy protocols for human brain tumors.