Journal:

Article Title: CCK 2 RECEPTOR EXPRESSION TRANSFORMS NON-TUMORIGENIC HUMAN NCM356 COLONIC EPITHELIAL CELLS INTO TUMOR FORMING CELLS

doi: 10.1002/ijc.24845

Figure Lengend Snippet: Intracellular Ca2+ ([Ca2+]i) imaging of rat intestinal epithelial (RIE) cells expressing recombinant human CCK2 receptor (RIE/CCK2R) loaded with the Ca2+ indicator dye Fura-2 was used as a bioassay to test the sensitivity, efficacy, and specificity of gastrin peptides. Black arrows indicate the time of addition of each treatment. Black bars indicate duration of exposure to each treatment. (A) Treatment of the RIE/CCK2R cells with 10 nM glycine-extended gastrin (Gly-G) failed to induce an increase in [Ca2+]i, whereas 1 nM of gastrin 1-17 (G17) to RIE/CCK2R cells does. (B) A 100-fold increase in concentration of Gly-G (100 nM) results in calcium response less robust than that of 1 nM of G17. (C) Pretreatment with CCK2 receptor antagonist 100 nM of L365,260 abolishes the Ca2+ response by 100 nM of Gly-G and 1 nM of G17 but not 100 nM of bradykinin (BK). (D) Conditioned media (CM) from NCM365 cultures was applied to the RIE/CCK2R cells, inducing a transient increase in [Ca2+]i in the cells of similar amplitude and duration as treating cells with 1 nM G17 (A,B). (E) Pretreatment with selective CCK2R receptor antagonist 1 μM of JB93182 (JB) abrogates CM-induced Ca2+ response. Cells were treated with 10 μM carbachol (Carb) to insure that the cells were loaded with Fura-2. (F) Although NCM356 cells secrete gastrin, addition of exogenous G17 (100 nM) does not induce an increase in [Ca2+]i indicating the absence of functional CCK2 receptor.

Article Snippet: Gastrin 1-17 (G17) was purchased from Biomol (Plymouth Meeting, PA), and dimethyl sulfoxide (DMSO) was purchased from Sigma (St. Louis, MO).

Techniques: Imaging, Expressing, Recombinant, Concentration Assay, Functional Assay