Stability of phosphoprotein as a biological marker of tumor signaling.
OBJECTIVE
The purpose of the study was to evaluate the stability of phosphoprotein as a marker of signaling activity in human tumors using clinical samples and xenografts.


METHODS
The expression of phospho-Ser473-Akt (p-Akt) was assessed by immunohistochemistry in paraffin-embedded samples from patients enrolled in a Southwest Oncology Group clinical trial of gastroesophageal junction tumors and by immunohistochemistry and Western blotting in human colon tumor xenografts at various times after removal from the animal.


RESULTS
Clinical samples had evaluable p-Akt staining only when obtained as biopsies (9 of 13) and no staining was observed in tumors obtained as surgically resected samples (0 of 15). In HT-29 colon cancer xenografts, p-Akt staining was present in fresh sample but not in tissue that had been allowed to stand for 30 minutes at room temperature. Western blotting of HT-29 tumor xenografts at room temperature showed a slow decrease in total Akt with a half-life of 180 minutes and a rapid decrease in p-Akt with a half-life of 20 minutes.


CONCLUSIONS
Caution should be used when using phosphoprotein levels in human tumor specimens to measure intrinsic signaling activity or drug effects because of the potential for rapid dephosphorylation. Rapid processing of biopsies is essential and postoperative surgical samples may be of limited value because of the time to fixation.
Baker
Amanda F
AF
Arizona Cancer Center, University of Arizona, Tucson, Arizona 85724, USA.
Dragovich
Tomislav
T
Ihle
Nathan T
NT
Williams
Ryan
R
Fenoglio-Preiser
Cecilia
C
Powis
Garth
G
D000818:Animals; D014408:Biomarkers, Tumor / Q000032:analysis*; D015153:Blotting, Western; D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology* / Q000503:physiopathology; D019073:HT29 Cells; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D016513:Mice, SCID; D009368:Neoplasm Transplantation; D010750:Phosphoproteins / Q000032:analysis*; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction; D014183:Transplantation, Heterologous
D014408:Biomarkers, Tumor; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D012694:Serine; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt