Hypoxic Activation of K-Ras normoxic conditions (Fig. 1A). All of these lines have a wild-type KRAS gene. However, when these cells were incubated in hypoxic conditions (1% O2), there was a dramatic increase (from 3 to 6.8 fold) in the levels of activated Ras (Fig. 1A). In contrast, the basal activities of Ras in DLD1, HCT116 (both heterozygous for the KRASD13 mutation), and SW480 (homozygous for the KRASV12 mutation) cells in normoxia were high, and there was no subsequent increase in hypoxia (Fig. 1A, right panel). SW480 cells had the strongest levels of Ras activation in normoxic conditions. Hypoxia is thus a strong activator of Ras in colon cancer cells, but this induction was observed only in those cells with a wild-type KRAS gene. To verify that the K-ras isoform was activated by hypoxia, a K- ras specific antibody was utilized. As illustrated in Figure 1B, levels of GTP bound-K-ras were induced by hypoxia in colon cancer cells with wild-type KRAS (Caco2, HT29) whereas there were no changes in cell lines with a mutant KRAS oncogene (DLD1, HCT116, SW480). A role for N-ras in the regulation of apoptosis has been suggested in colon cancer, but an N-ras specific antibody failed to detect GTP-bound-N-ras in Caco2, DLD1, and HCT116 cell lines in either normoxic or hypoxic conditions (Fig. S1) [10]. Tumor hypoxia is often accompanied by a switch to anaerobic glycolysis and a lower pH, so we investigated whether the hypoxic regulation of Ras activity was mediated through changes in pH. Incubation of Caco2 cells in acidic conditions (pH 6.5) for 4 hours did not increase the activity of Ras, suggesting that changes in pH do not regulate levels of GTP-bound Ras (Fig. 1C). Upregulation of Ras activity by hypoxia requires c-Src c-Src lies upstream of K-ras, and we next sought to explore whether c-Src is also activated by hypoxia. A time-dependent activation of c-Src, as measured by phosphorylation at Tyr416, was observed in both Caco2 and HT29 cells after incubation in hypoxic conditions (Fig. 2A). To determine if c-Src activity Figure 1. Hypoxia activates Ras in colon cancer cell lines with a wild-type KRAS. The levels of GTP-bound Ras (A) and GTP-bound K-ras (B) were evaluated in wild-type (left panel) and mutant (right panel) KRAS cell lines grown either in normoxic (N) or hypoxic (H) conditions for 4 hours. Two milligrams of cell extracts were used for a Ras activation assay, followed by Western blotting with the indicated antibodies. Densitometry values are expressed as fold change compared with control values normalized to 1. C, Caco2 cells were cultured in DMEM with pH adjusted to 7.5 or 6.5 for 4 hours. Cell lysates were used for a Ras activation assay followed by Western blotting with a Ras-GTP specific antibody. Densitometry values are expressed as fold change compared with control values normalized to 1. doi:10.1371/journal.pone.0010966.g001 PLoS ONE | www.plosone.org 2 June 2010 | Volume 5 | Issue 6 | e10966