Measurement of protein kinase B activity in single primary human pancreatic cancer cells.
An optimized peptide substrate was used to measure protein kinase B (PKB) activity in single cells. The peptide substrate was introduced into single cells, and capillary electrophoresis was used to separate and quantify nonphosphorylated and phosphorylated peptide. The system was validated in three model pancreatic cancer cell lines before being applied to primary cells from human pancreatic adenocarcinomas propagated in nude mice. As measured by phosphorylation of peptide substrate, each tumor cell line exhibited statistically different median levels of PKB activity (65%, 21%, and 4% phosphorylation in PANC-1 (human pancreatic carcinoma), CFPAC-1 (human metastatic ductal pancreatic adenocarcinoma), and HPAF-II cells (human pancreatic adenocarcinoma), respectively) with CFPAC-1 cells demonstrating two populations of cells or bimodal behavior in PKB activation levels. The primary cells exhibited highly variable PKB activity at the single cell level, with some cells displaying little to no activity and others possessing very high levels of activity. This system also enabled simultaneous characterization of peptidase action in single cells by measuring the amount of cleaved peptide substrate in each cell. The tumor cell lines displayed degradation rates statistically similar to one another (0.02, 0.06, and 0.1 zmol pg(-1) s(-1), for PANC-1, CFPAC-1, and HPAF-II cells, respectively) while the degradation rate in primary cells was 10-fold slower. The peptide cleavage sites also varied between tissue-cultured and primary cells, with 5- and 8-residue fragments formed in tumor cell lines and only the 8-residue fragment formed in primary cells. These results demonstrate the ability of chemical cytometry to identify important differences in enzymatic behavior between primary cells and tissue-cultured cell lines.
Department of Chemistry, University of North Carolina , Chapel Hill, North Carolina 27599, United States.
Angela
A
Proctor
S Gabriela
SG
Herrera-Loeza
Qunzhao
Q
Wang
David S
DS
Lawrence
Jen Jen
JJ
Yeh
Nancy L
NL
Allbritton
D000230:Adenocarcinoma / Q000201:enzymology* / Q000473:pathology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D006801:Humans; D010190:Pancreatic Neoplasms / Q000201:enzymology* / Q000473:pathology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*
D051057:Proto-Oncogene Proteins c-akt