Transient transfection Plasmid constructs of mouse xCT and 4F2hc cDNAs cloned into the pcDNA3.1+ vector were transformed into Gemcitabine IC50 the E.coli strain DH5��, and plasmid DNA harvested using a Qiagen Maxiprep kit according to the manufacturer’s recommendations. For xc? transporter overexpression studies, pancreatic cancer cells were plated at a density of 0.5 �� 103 cells per well in 96-well plates. After 24h, wells were aspirated and 125��l of fresh media were added. Cells were transiently transfected with 0.1��g DNA (empty vector pcDNA3.1 control, pcDNA3.1-xCT, or pcDNA3.1-4F2hc) diluted in 12.5��l 150mM NaCl and 0.75��l ExGen 500 in vitro transfection reagent (Fermentas, Burlington, ON, Canada) for 24h. Glutamate uptake assay Glutamate uptake by cultured cells was used to measure xc? transporter activity as described previously (Shih and Murphy, 2001).

Briefly, cells were plated in 24-well plates at 5 �� 105 cells per well and incubated overnight. Cells were washed with and pre-incubated in 1ml per well Na+-free buffer A consisting of 140mM N-methyl-D-glucamine, 5.4mM KCl, 0.4mM KH2PO4, 10mM HEPES, 5mM D-glucose, 1.8mM CaCl2, 0.8mM MgSO4 (pH 7.4) for 20min at 37��C. The medium was then replaced with 300��l Na+-free buffer A containing 33nM L-[3H]-glutamate (49 Cimmol?1) (Amersham Pharmacia/GE Healthcare, Pittsburg, PA, USA) in the presence or absence of 1��M unlabeled amino-acid competitors (L-glutamate, L-cystine) or non-competitors (L-leucine) for 20min at 37��C. Uptake was terminated by washing three times with ice-cold Na+-free buffer A, after which cells were solubilised with 200��l 0.

5% Triton X-100 in 0.1M potassium phosphate buffer (pH 7.0). To determine intracellular L-[3H]-glutamate uptake, 100��l of cell lysate was mixed with 5ml of scintillation cocktail (Fisher, Pittsburg, PA, USA), and radioactivity was measured using an LKB Wallac 1214 Rackbeta (American Instrument Exchange Inc., Haverhill, MA, USA) liquid scintillation counter. A 10��l aliquot of cell lysate was used in a BCA protein assay kit (Pierce Chemical Co, Rockford, IL, USA) to determine protein concentration. Statistical analysis Student’s t-test was used unless otherwise stated to determine statistical significance. Results with a P0.05 were considered significant.

Results Growth requirement for extracellular cystine It is known that cysteine, the reduced form of cystine, is a non-essential amino acid in the human diet as tissues in the body such as the liver can generate cysteine GSK-3 through the transsulphuration pathway (Stabler et al, 1993; Brosnan and Brosnan, 2006). This pathway involves the metabolism of methionine, a nutritionally essential amino acid, to a cystathionine intermediate and its subsequent cleavage by ��-cystathionase to ��-ketobutyrate and cysteine (Stabler et al, 1993; Brosnan and Brosnan, 2006).