Figure 5. Hypoxic regulation of VEGF by K-ras. A, Relative mRNA levels of VEGF, as evaluated by quantitative RT-PCR, in Caco2 cells transfected with a K-ras-specific siRNA construct or a non targeting control and exposed to normoxia or hypoxia. The data are expressed as fold change as compared to siRNA control cells in normoxia, normalized to 1. Columns, average of at least three experiments; bars, SEM. *, P,0.05 as compared to control cells. B, Caco2 cells were transiently transfected with either siRNA targeting endogenous K-ras or non targeting control siRNA. After 24 hours, a 2.3 kb VEGF-luciferase reporter construct was co- Hypoxic Activation of K-Ras transfected with pRL-CMV and cells were incubated in normoxia or hypoxia for additional 24 hours. The data are expressed as fold change as compared to siRNA control cells in normoxia, normalized to 1. Columns, average of at least three experiments; bars, SEM. *, P,0.05 as compared to control cells. C, Supernatant from cells in (A) was collected, and an ELISA for VEGF was performed. The data are expressed as fold change as compared to siRNA control cells in normoxia, normalized to 1. Columns, average of at least three experiments; bars, SEM. *, P,0.05 as compared to control cells. doi:10.1371/journal.pone.0010966.g005 engaged by K-ras in hypoxic versus normoxic conditions remain to be defined and illustrates the plasticity of K-ras function depending upon specific micro-environmental cues. Hypoxic conditions can therefore create a milieu in which proto-oncogenes that are not mutated can mimic activated oncogenes. Even though wild-type K-ras can be activated by hypoxia, it is likely that its spectrum of activity does not entirely overlap with that of mutant K-ras and there are other properties specific to oncogenic KRAS alleles. Activation of Akt in hypoxia appears to be a common feature of colon tumors with or without KRAS mutations, but it has become clear that mutant K-ras and physiologically activated wild-type K-ras do not function identi- cally [26]. Nevertheless, these findings indicate a critical role for wild-type KRAS alleles in hypoxia and provide a potential explanation for the aggressive behavior of tumor cells that can survive in the hypoxic microenvironment. Materials and Methods Reagents The antibodies used in this study were purchased from the following vendors: p-Akt (Ser473), p-MAPK (Thr202-Tyr204), p-Src (Tyr416), p-EGF Receptor (Tyr1068), total Akt, MAPK, Src, and EGF receptor, were from Cell Signaling Technology (Beverly, MA); v-Src (Ab-1, clone327) was from Calbiochem (San Diego, CA); K-ras and N-ras were from Santa Cruz Biotechnology (Santa Cruz, CA). Cell lines and culture conditions Human colon cancer cell lines were obtained from the American Type Culture Collection. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO-Invitro- gen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; HyClone, Ogden, UT) and antibiotics (Penicillin-Strepto- mycin, Invitrogen). Hypoxic conditions were achieved by culturing cell lines in a sealed hypoxia chamber (Billups-Rothenberg, Del Mar, CA) with a mixture of 1% O2, 5% CO2, and 94% N2. Cells were switched to serum-free UltraCulture (Lonza, Allendale, NJ) before hypoxia. The Src kinase inhibitor PP2 and antioxidant NAC (Calbiochem) were added 1 hour prior to exposure to normoxia or hypoxia. Acidic DMEM was prepared by dissolving DMEM powder without sodium bicarbonate and sodium pyruvate (Invitrogen) in distilled H2O, adjusting the pH to 6.5, filtering and storing at 4uC. Real Time PCR Assay RNA was extracted using the ISOGEN kit (Nippon gene, Tokyo, Japan) and quantitative reverse transcription PCR was performed using the iScriptTM cDNA Synthesis kit (BIO-RAD, Hercules, CA). 18S rRNA served as an endogenous control. We used a Power SYBR Green Master Mix (Applied Biosystems, Foster City, CA) and iQ5 Real-time PCR detection system (BIO- RAD) for real-time quantification. PLoS ONE | www.plosone.org 8 June 2010 | Volume 5 | Issue 6 | e10966