The hydroxyurea-induced loss of double-minute chromosomes containing amplified epidermal growth factor receptor genes reduces the tumorigenicity and growth of human glioblastoma multiforme.

OBJECTIVE: We investigated whether the hydroxyurea-induced loss of double-minute chromosomes containing amplified epidermal growth factor receptor (EGFR) genes would lead to a loss of tumorigenicity of a glioblastoma multiforme cell line. METHODS: Glioblastoma multiforme cells were treated in vitro with 0 (HU0) or 100 micromol/L (HU100) hydroxyurea and then injected into the flanks of nude mice. Survival and tumor volumes were evaluated. Pulsed-field gel electrophoresis, Southern blot hybridization, and slot-blot analysis were used to determine EGFR amplification levels. Flow cytometry and immunofluorescent staining were used for cell-cycle analysis and EGFR protein expression. RESULTS: Prior to injection, HU100 cells lost 95% of their amplified EGFR genes and developed into tumors 6 weeks after injection versus 3 weeks for HU0 cells. Mice with HU100 tumors had a median survival of 62 days versus 43 days for control mice with HU0 tumors. Pulse-field gel electrophoresis analysis showed that HU100 tumors had reamplified the EGFR gene as double-minute chromosomes of the same size as those originally present before hydroxyurea treatment. When HU100 cells were cultured in the absence of hydroxyurea, the EGFR gene also reamplified. HU100 cells grew at less than half the rate of untreated HU0 control cells in culture and showed a decreased number of cells entering the cell cycle. Immunofluorescent staining of HU150 (150 micromol/L) cells showed decreased EGFR protein expression. CONCLUSION: The EGFR gene is important for tumorigenicity in mice and growth in culture. Hydroxyurea induces the loss of double-minute chromosome-amplified EGFR genes against a selection gradient and significantly delays the onset of tumors. These results support the potential use of low-dose hydroxyurea for the treatment of human glioblastoma multiforme.

In vitro and in vivo cytotoxicity of gossypol against central nervous system tumor cell lines.

Gossypol is a lipid soluble polyphenolic compound isolated from cotton seed oil which has been previously shown to have antiproliferative activity in vitro against a variety of human solid tumor cell lines. It has been extensively tested in clinical trials as a male contraceptive agent and found to be well tolerated. Its mechanism of action is thought to be inhibition of cellular energy metabolism. It inhibits glycolysis through inhibition of LDH isoenzyme type 5, and it inhibits mitochondrial oxidative phosphorylation and electron transport. We tested the in vitro antiproliferative effect of gossypol against four well characterized human glioma cell lines, HS 683, U373, U87 and U138, and one rat glioma cell line, C6, using the colorimetric Microculture Tetrazolium Assay (MTT). Cytotoxicity was found to be concentration and time dependent and increased with incubation times up to 8 days. The relative sensitivity of the glioma cell lines to gossypol at 48 hour incubation correlated with their respective LDH isoenzyme profiles, with the more sensitive cell lines expressing increased cathodal LDH isoenzymes (LDH5). The in vitro cytotoxicity of gossypol to these CNS tumor lines was compared to the other non central nervous system solid tumor cell lines which had been previously reported as being sensitive to gossypol, including SW-13 (adrenal), MCF-7 (breast), T47-D (breast), and HeLa (cervical). Additional lines tested included SK-MEL-3 (melanoma), Colo 201 (colon) and BRW, a line established in our laboratory from a patient with a Primitive Neuroectodermal tumor. C6, HS 683, and BRW had similar IC50s as the sensitive solid tumor cell lines. U373, U87 and U138 had significantly less sensitivity at 48 hours. There was greater cytotoxicity and no significant differences in the IC50s between any of cell lines at 8 day incubations. Additionally, we tested the cytotoxicity of gossypol against BRW in vivo, using the nude mouse xenograft model. Gossypol, given at a dose of 30 mg/kg per day five days a week for four weeks orally via gavage, was found to decrease the mean tumor weight of treated xenografts by more than 50% as compared to untreated xenografts. These findings suggest that gossypol has potential for further study as an agent for the treatment of primary CNS malignancies.

Highly specific micromethod for the enzymatic determination of radioactive [14C]lactate.

By collecting released 14CO2 following the enzymatic decarboxylation of radiolabeled lactate, picomoles of the latter can be precisely, easily, and reproducibly measured in small biological fluids. This radioactive [14C]lactate microassay does not require a neutralization step, nor does it require chemical extractions and partioning procedures, ion exchange, or pyruvate derivatization. Under our specified conditions this simple reaction goes to completion in 90 min. Using this assay in porous adipose cells, with the cell number logarithmically less than that found in other literature methods, the measured glycolytic flux rates were consistent with those previously reported. In these studies, glycolysis was initiated with [U-14C]glucose 6-phosphate. This microradioactive lactate assay is useful when dealing with minute tissue samples and/or microliter aliquots of biological fluids.