Flow cytometric detection of progastrin interaction with gastrointestinal cells.
The unprocessed gastrin precursor, progastrin (PG), is often overexpressed in colon cancer and other malignancies where it appears to stimulate colonic growth. Overexpression of progastrin also stimulates proliferation of normal colonic mucosa, but the receptors mediating these effects have not been identified. Here we report the development of a non-radioactive assay for assessment of PG binding to normal and transformed cells. Progastrin was labeled using biotinylation, and binding of biotinylated PG to cells was assessed using flow cytometry. Using this approach, we show strong and specific binding of PG to some cell lines (IEC-6, IEC-18, HT-29, COLO320) and minimal binding to others (HeLa, DC2.4, Jurkat). We also found PG binding to several non-gut epithelial lines, such as CHO-K1, COS-6 and HEK293 cells. The specificity of binding was confirmed by competition with cold, unlabeled PG but not with glycine-extended gastrin or amidated gastrin-17. Binding was not influenced by the presence of the classical CCK-2 receptor, but was partially dependent on the charged glycosaminoglycans (GAG). The analysis of primary colonic tissues isolated from wild type C57BL/6 mouse, revealed a small epithelial subpopulation of non-hematopoietic (CD45-negative) cells that strongly interacted with PG. Surprisingly, this population was greatly expanded in gastrin knockout mice. This non-radioactive, FACS-based assay should prove useful for further characterization of cells expressing the progastrin receptor.
Dubeykovskiy
Alexander
A
Division of Digestive and Liver Diseases, Columbia University Medical Center, 1130 St Nicholas Avenue, Room #901, New York, NY 10032, USA.
Nguyen
Thomas
T
Dubeykovskaya
Zinaida
Z
Lei
Shi
S
Wang
Timothy C
TC
D000818:Animals; D017306:Annexin A2 / Q000378:metabolism; D016466:CHO Cells; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D003106:Colon / Q000166:cytology / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004847:Epithelial Cells / Q000378:metabolism; D005434:Flow Cytometry; D005755:Gastrins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D041981:Gastrointestinal Tract / Q000166:cytology / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D019169:Jurkat Cells; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D011498:Protein Precursors / Q000378:metabolism*; D043887:Receptor, Cholecystokinin B / Q000378:metabolism
D017306:Annexin A2; D005755:Gastrins; D006025:Glycosaminoglycans; D011498:Protein Precursors; D043887:Receptor, Cholecystokinin B; C013706:big gastrin