Background/Significance The deregulation of molecular checkpoints in the cell cycle is associated with a number of cancers. Cyclin E, controlling the transition from G1 to S phase, is overexpressed in many tumors and thus a target of future cancer therapy. Because molecular determinants of radiation sensitivity are differentially regulated via cell cycle phase, cyclin E activity may be an important determinant of radiation sensitivity. Since deregulation of cyclin E is reported to induce chromosome instability, cyclin E overexpression may increase radiosensitivity by interfering with DNA repair mechanisms.
Methods A glioblastoma multiforme (GBM) line was transduced with a recombinant adenovirus containing the human cyclin E cDNA. Cyclin E upregulation was confirmed with Western Blot analysis. Cells lines underwent standard clonogenic radiobiologic assays after Cs-137 irradiation. To quantify radiation-induced double-stranded DNA breaks, agarose-embedded cells were irradiated with a 90 Gy dose and subsequently subjected to pulsed field gel electrophoresis after one hour. To investigate cell kinetics as a modulator of radiation toxicity, parallel flasks cultured in identical conditions underwent FACS analysis.
Results GBM cells were significantly affected by cyclin E overexpression for all parameters measured. FACS analysis demonstrated an almost two-fold increase in cells in G2 or M phase versus G0 or G1 phase in the cyclin E-positive population, compared to control populations. Additionally, cyclin E-transduced GBM cells had higher rates of radiation-induced cell kill at doses over 10 Gy and showed a 60% increase in DNA double-strand breaks compared to control cells, one hour after radiation with a 90 Gy dose.
Conclusions In this study, we show that cyclin E transduction, resulting in cyclin E overexpression, sensitizes GBM cells to ionizing radiation. High levels of cyclin E push cells through S phase and result in a greater percentage of cells in G1 or M. The chromosomally unstable cells exposed to ionizing radiation at later cell cycle phases continue to divide, thus abrogating cell cycle checkpoint control and altering clonogenicity. The data suggest that cyclin E overexpression interferes with DNA double-strand break repair mechanisms regulating clonogenic radiation survival. Similarly, the data support the hypothesis that cyclin E overexpression alters the radiation sensitivity phenotype by both DNA repair and checkpoint control mechanisms.
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