Delayed cell death associated with mitotic catastrophe in γ-irradiated stem-like glioma cells

Abstract

Background and PurposeStem-like tumor cells are regarded as highly resistant to ionizing radiation (IR). Previous studies have focused on apoptosis early after irradiation, and the apoptosis resistance observed has been attributed to reduced DNA damage or enhanced DNA repair compared to non-stem tumor cells. Here, early and late radioresponse of patient-derived stem-like glioma cells (SLGCs) and differentiated cells directly derived from them were examined for cell death mode and the influence of stem cell-specific growth factors.Materials and methodsPrimary SLGCs were propagated in serum-free medium with the stem-cell mitogens epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2). Differentiation was induced by serum-containing medium without EGF and FGF. Radiation sensitivity was evaluated by assessing proliferation, clonogenic survival, apoptosis, and mitotic catastrophe. DNA damage-associated γH2AX as well as p53 and p21 expression were determined by Western blots.ResultsSLGCs failed to apoptose in the first 4 days after irradiation even at high single doses up to 10 Gy, but we observed substantial cell death later than 4 days postirradiation in 3 of 6 SLGC lines treated with 5 or 10 Gy. This delayed cell death was observed in 3 of the 4 SLGC lines with nonfunctional p53, was associated with mitotic catastrophe and occurred via apoptosis. The early apoptosis resistance of the SLGCs was associated with lower γH2AX compared to differentiated cells, but we found that the stem-cell culture cytokines EGF plus FGF-2 strongly reduce γH2AX levels. Nonetheless, in two p53-deficient SLGC lines examined γIR-induced apoptosis even correlated with EGF/FGF-induced proliferation and mitotic catastrophe. In a line containing CD133-positive and -negative stem-like cells, the CD133-positive cells proliferated faster and underwent more γIR-induced mitotic catastrophe.ConclusionsOur results suggest the importance of delayed apoptosis, associated mitotic catastrophe, and cellular proliferation for γIR-induced death of p53-deficient SLGCs. This may have therapeutic implications. We further show that the stem-cell culture cytokines EGF plus FGF-2 activate DNA repair and thus confound in vitro comparisons of DNA damage repair between stem-like and more differentiated tumor cells.

Cell Death Pathways, with Special Regard to Ionizing Radiation and Temozolomide

“…Much of the information about this type of PCD is derived from in vitro studies on GB cell lines. Moreover, mitotic catastrophe associated with delayed apoptosis has also been observed in patient-derived p53-defi cient stem-like glioma cells and differentiated cells treated with IR (5-10 Gy) [ 21 ]. Recently, transcriptome studies in vitro, through total RNA sequencing analysis, identifi ed, after irradiation, an altered expression pattern of genes involved in the mitotic process, including G2-, spindle assembly checkpoint-, and centrosomeassociated genes [ 1 , 22 ].…”

The nitric oxide donor JS-K sensitizes U87 glioma cells to repetitive irradiation

“…17 Repetitive irradiation (3 × 2 Gy) showed no significant increase in MC features compared to the untreated controls at any point in time (Figure 3(g)). JS-K monotherapy resulted in a timeand dose-dependent increase in MC features reaching its maximum when using 7.5 µM JS-K 24 h after the end of the treatment: 15% of the treated cells showed signs of MC compared to cells with spontaneous MC in the untreated control group (<0.5%).…”

“…Cells with nuclei that showed multiple fragmentations and a nuclear diameter >20 µm were considered positive for MC according to the criteria described by Firat et al 17 Evaluation of cell nuclei was performed using fluorescence microscopy (Zeiss Axiovert 135). For each point in time and each treatment group, three culture coverslips were evaluated.…”

Knockdown of Checkpoint Kinase 1 Is Associated with the Increased Radiosensitivity of Glioblastoma Stem-Like Cells

“…GSCs play a pivotal role in tumor development and maintenance Wan et al 2010). Because radiation can kill differentiated tumor cells while sparing the rare cancer stem cell population, GSCs are regarded as a "culprit" of tumor recurrence (Bao et al 2006;Firat et al 2011). Since the cell cycle checkpoint kinase 1 and 2 (Chk1, Chk2) are the key regulators in cell checkpoint signaling pathway (Smith et al 2010;Dai and Grant 2010), we hypothesize that restraining Chk1 or Chk2 of GSCs may abrogate cell cycle checkpoint and improve the radiosensitivity.…”