title: Ischemic time impacts biological integrity of phospho-proteins in PI3K/Akt, Erk/MAPK, and p38 MAPK signaling networks.
abstract: Post-translational modifications of proteins, such as phosphorylation, are labile events dynamically regulated by opposing kinase and phosphatase activities. Preanalytical factors, such as ischemic time before fixation, affect these activities and can have a significant impact on the ability to elucidate signaling pathways in tissue. Immunohistochemical analysis of phosphorylated proteins involved in PI3K/Akt, Erk/MAPK, and p38 MAPK signaling networks was performed in human cell line xenografts from lung, brain, ovary, and prostate tumors. In order to replicate real-world practices, the tissues were subjected to ischemic times of 0 (baseline), 1, 4, and 24 hours before fixation in formalin. Two key concepts emerge from this analysis: (1) the stability of different phospho-epitopes within a given tumor type is variable (e.g. phospho-PRAS40 is more labile than phospho-S6 ribosomal protein) and (2) the stability of a given phospho-epitope (e.g. phospho-MAPKAPK2) varies significantly across different tumor types. These results highlight the importance of proper tissue acquisition and rapid fixation to preserve the biological integrity of signal transduction pathways that may guide therapeutic decision making.
authors: Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, DC0424, Indianapolis, IN 46285, USA. holzertr@lilly.com
authors: Timothy R
authors: TR
authors: Holzer
authors: Angie D
authors: AD
authors: Fulford
authors: Austin M
authors: AM
authors: Arkins
authors: Janet M
authors: JM
authors: Grondin
authors: Christopher W
authors: CW
authors: Mundy
authors: Aejaz
authors: A
authors: Nasir
authors: Andrew E
authors: AE
authors: Schade
mesh_terms: D000818:Animals; D001932:Brain Neoplasms / Q000098:blood supply / Q000201:enzymology; D005260:Female; D005909:Glioblastoma / Q000098:blood supply / Q000201:enzymology; D006801:Humans; D007150:Immunohistochemistry; D007511:Ischemia / Q000201:enzymology*; D008175:Lung Neoplasms / Q000098:blood supply / Q000201:enzymology; D020935:MAP Kinase Signaling System; D008297:Male; D051379:Mice; D008819:Mice, Nude; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D009369:Neoplasms / Q000098:blood supply* / Q000201:enzymology*; D010051:Ovarian Neoplasms / Q000098:blood supply / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D011471:Prostatic Neoplasms / Q000098:blood supply / Q000201:enzymology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D048051:p38 Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases