br Methods br Results and discussion Protein analysis of

Methods
Results and discussion Protein analysis of commonly used cell lines (C6, D54-MG, U87, U251 and STTG1) reveals high j 14 of SXC (Cho and Bannai, 1990, Ye et al., 1999, de Groot et al., 2005, Lyons et al., 2007, Savaskan et al., 2008). Since protein expression can be altered over time in cell lines, we have adopted a xenograft model of glioma in which patient tissue is propagated in the flank of nude mice (Giannini et al., 2005). Using this model, we found that SXC expression in patient tumors is heterogeneous, and, as a result we chose to functionally study tumors that express high and low levels of SXC. Fig. 3A shows the xCT and CD98 expression of four of these ‘xenolines’, with two expressing high levels (GBM22 and GBM59) and two expressing low levels (GBM14 and GBM39) of the catalytic xCT subunit. We first tested functionality of SXC by performing radiolabeled 3[H]-glutamate uptake assays, a commonly used method of testing SXC function (Ye et al., 1999). High SXC expressing GBM22 xenolines showed Na+-independent, Cl--dependent uptake, which was inhibited with the SXC inhibitors Sulfasalazine (SAS) and (S)-4-Carboxyphenylglycine (S4CPG), confirming SXC-mediated uptake in high SXC expressing GBM22, but not in low SXC expressing GBM14 (Fig. 3B).
Conclusions High SXC expressing tumors exhibit a growth advantage over low SXC expressing tumors, as seen by the growth differences between the GBM22 and GBM14 xenolines. These findings are also replicated in vivo, as GBM22 xenolines that are implanted intracranially into mice kill the animals ∼2× faster than implanted GBM14 tumors (data not shown). This enhanced growth of high SXC expressing tumors is a result of their ability to utilize extracellular cystine for intracellular GSH synthesis. Our data suggests that high SXC expressing tumors are more radiation resistant, at least at lower radiation doses, due to their ability to increase synthesis of GSH to neutralize the free radicals formed as a consequence of radiation exposure. SXC inhibition reduced the resistance of GBM22 cells to 1Gy radiation, bringing their radiation sensitivity closer to that seen in the GBM14 cells. We found that the GBM14 cells grew much slower in culture than the GBM22 cells, which may be masking a difference in radiation sensitivity at higher doses. Since radiation more efficiently damages cells that are actively dividing, one could argue that the GBM14 cells are not being exposed to equivalent doses of radiation due to their slower growth. Treating these cells with a fractionated radiation dose schedule (ex. spreading a dose of 5Gy over 5days, administering 1Gy/day), may uncover a greater separation of radiation sensitivity in these xenolines. Additionally, we hypothesize that at least under in vitro conditions, higher levels of radiation may cause cellular damage too quickly for GSH to neutralize and therefore these findings should be investigated using an in vivo model to more accurately determine radiation dose and tumor resistance with and without SAS at higher doses.
Acknowledgements
Introduction Glutamate is the primary excitatory amino acid in the central nervous system, and critically participates in various physiological and pathological processes. Disruption of glutamate homeostasis has been indicated to contribute to a number of major neurological diseases [[1], [2], [3], [4]]. Excitatory amino acid transporters (EAATs) play a critical role in the synaptic reuptake of glutamate. The EAAT family includes five subtypes, namely EAAT1–5 in the human, and corresponding GLAST, GLT-1, EAAC1, EAAT4 and EAAT5 in rodents [5]. GLAST (EAAT1) and GLT-1 (EAAT2) are predominantly expressed in astrocytes while EAAC1 (EAAT3), EAAT4 and EAAT5 are neuronal-specific transporters [6]. Among of them, GLT-1 is the major glutamate transporter that accounts for >90% of total glutamate uptake in the brain [5,7].