Figure 2.

Gln deprivation increased oxidative stress and PCa cell sensitivity to X-ray radiation in Gln-dependent PCa cells. (A) Gene expression analysis showed that Gln deprivation results in consistent deregulation patterns of genes that control oxidative stress response and DNA damage response (DDR), although this deregulation is more pronounced for radiosensitive (RS) parental cells in each pair as compared to radioresistant (RR) cells, as well as for DU145 in comparison to LNCaP cells. (B) Absolute log2 fold change of genes involved in oxidative stress response and DNA damage response signatures. (C) Western blot analysis of key DDR regulators in response to Gln depletion for 72 h and quantification of the Western blot data. Cells grown in presence of glutamine were used as control. Data are mean ± s.d. (D) Gln starvation increased the intracellular level of ROS in DU145 and DU145 RR cells. Data are mean ± s.d. (E) Radiobiological colony-forming assay of PCa cells grown with or without Gln supplementation. Data are mean ± s.d. (F) Analysis of residual γH2A.X foci number after 24 h of glutamine deprivation followed by 4 Gy of X-ray irradiation. Cells were analyzed 24 h after irradiation. Control cells were cultured in presence of glutamine. Data are mean ± s.d. Scale bars = 10 µm. (G) Comparative analysis of gene expression data from patient-derived primary cultures (PDPC) and Gln-deprived PCa cell lines. PDPC cultures were analyzed for their relative radioresistance using a 3-D radiobiological clonogenic assay. Data are mean ± s.d. Lists of genes significantly deregulated (p<0.05) in DU145 and LNCaP in response to glutamine starvation were compared with the list of genes differentially expressed (p < 0.05) between radioresistant (RR) and radiosensitive (RS) PDPC samples. For the genes present in both lists, Gln-/Gln+ and RR/RS fold changes were calculated. Finally, the Pearson correlation for those fold change values was determined. *p < 0.05; **p < 0.01; ***p < 0.001.