The Hydroxyurea-induced Loss of Double-Minute Chromosomes Containing Amplified Epidermal Growth Factor Receptor Genes Reduces the Tumorigenicity and Growth of Human Glioblastoma Multiforme

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Abstract
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. Glioblastoma multiforme cells were treated in vitro with 0 (HUO) or 100 jumol/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. Prior to injection, HU100 cells lost 95% of their amplified EGFR genes and developed into tumors 6 weeks after injection versus 3 weeks for HUO cells. Mice with HU100 tumors had a median survival of 62 days versus 43 days for control mice with HUO 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 HUO control cells in culture and showed a decreased number of cells entering the cell cycle. Immunofluorescent staining of HU150 (150 μmol/L) cells showed decreased EGFR protein expression.. 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.