TI - Activation of downstream signaling in KRAS-amplified gastric cancer cells . AB - To investigate KRAS activity in gastric cancer cells , we analyzed the amount of GTP-KRAS in cells using an in vitro pull-down assay . There was a higher amount of GTP-KRAS in HSC45 and SH101P4 cells , which carried amplified mutant KRAS , than in MKN1 cells , in which the level of GTP-KRAS was comparable to AGS cells , which carried non-amplified mutant KRAS ( Figure 4b ) . Serum stimulation had little effect on the level of GTP-KRAS in HSC45 cells , but resulted in a dramatic elevation of GTP-KRAS in MKN1 cells ( Figure 4c ) . As expected , this data was consistent with constitutively active mutant KRAS overexpression due to amplification , and it suggested that overexpression of wild-type KRAS may also promote oncogenic properties when cells are exposed to external stimuli . To gain further insight into the role of overexpressed KRAS in cancer cell growth , we analyzed the activation of p44/42 MAP kinase and AKT ( Figure 4d ) , which are pivotal molecules in the MAP kinase cascade and PI3K signaling pathways that are downstream of KRAS [3,30] . Under normal culture conditions ( Figure 4d , lanes indicated as "N" ) , basal PHOSphorylation of p44/42 was increased in KRAS-amplified cells ( HSC45 , MKN1 and SH101P4 ) as compared to NUGC4 gastric cancer cells , in which there is neither amplification nor mutation of KRAS . While the PHOSphorylation of p44/42 was modestly increased in MKN1 cells after serum stimulation , the effect of serum stimulation on HSC45 and SH101P4 cells was minimal , which indicated that p44/42 is constitutively active in the latter two cell lines . To investigate the biological significance of KRAS amplification in gastric cancer , we used small interfering RNA ( siRNA ) to knock-down the expression of KRAS or KRAS neighboring genes in four gastric cancer cell lines , HSC45 ( carrying amplification and mutation of KRAS ) , MKN1 ( amplification but no mutation of KRAS ) , AGS ( mutation but no amplification of KRAS ) and NUGC4 ( no amplification or mutation of KRAS ) . Knock-down of KRAS and three neighboring genes was verified by qRT-PCR ( Additional file 13 ) and KRAS immunoblot analysis ( Figure 5a ) . While the knock-down of KRAS in HSC45 and MKN1 cells caused a marked reduction in PHOSphorylation of p44/42 , knock-down of neighboring genes had no effect ( Figure 5a , b ) . PHOSphorylation of p44/42 was reduced in KRAS knock-down AGS cells , but not in NUGC4 cells . These results indicated that KRAS amplification is associated with both transient and constitutive activation of p44/42 MAP kinase . Basal PHOSphorylation of AKT was detected under normal culture conditions , and was increased after serum stimulation of MKN1 and AGS cells ( Figure 4d ) . Nucleotide sequence analysis revealed a single nucleotide mutation at codon 545 of PIK3CA in MKN1 and AGS cells ( E545K and E545A , respectively , Additional file 11c ) , which suggested that AKT is potentially activated in these cells through mutational activation of PIK3CA . However , in MKN1 cells , PHOSphorylation of AKT was reduced by KRAS knock-down under both the normal culture condition as well as after serum stimulation ( Figure 5b ) , which suggested that the overexpression of wild-type KRAS might also be involved in enhancing the activation of AKT .