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 dou ble-minute chromosomes containing amplified epidermal growth factor re ceptor (EGFR) genes would lead to a loss of tumorigenicity of a gliobl astoma multiforme cell line. METHODS: Glioblastoma multiforme cells we re treated in vitro with 0 (HU0) or 100 mu mol/L (HU100) hydroxyurea a nd then injected into the flanks of nude mice. Survival and tumor volu mes were evaluated. Pulsed-field gel electrophoresis, Southern blot hy bridization, and slot-blot analysis were used to determine EGFR amplif ication levels. Flow cytometry and immunofluorescent staining were use d for cell-cycle analysis and EGFR protein expression. RESULTS: Prior to injection, HU100 cells lost 95% of their amplified EGFR genes and d eveloped into tumors 6 weeks after injection versus 3 weeks for HU0 ce lls. Mice with HU100 tumors had a median survival of 62 days versus 43 days for control mice with HU0 tumors. Pulse-field gel electrophoresi s analysis showed that HU100 tumors had reamplified the EGFR gene as d ouble-minute chromosomes of the same size as those originally present before hydroxyurea treatment. When HU100 cells were cultured in the ab sence 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. Immuno fluorescent staining of HU150 (150 mu mol/L) cells showed decreased EG FR protein expression. CONCLUSION: The EGFR gene is important for tumo rigenicity 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 s upport the potential use of low-dose hydroxyurea for the treatment of human glioblastoma multiforme.