[1914] Ischemic Time Impacts Biological Integrity of Phosphoproteins in PI3K/Akt, Erk/MAPK, and p38 MAPK Signaling Networks.

Timothy R Holzer, Angie D Fulford, Austin M Arkins, Janet M Grondin, Christopher W Mundy, Aejaz Nasir, Andrew E Schade. Eli Lilly and Co., Indianapolis, IN

Background: It is well-established that protein post-translational modifications, such as phosphorylation, are labile events dynamically regulated by opposing kinase and phosphatase activities. Therefore, preanalytical variables such as ischemic time before fixation can have a significant impact on the ability to interrogate signaling pathways in tissue.
Design: We performed immunohistochemical analysis of p-PRAS40 (Thr246), p-p70S6 kinase (Thr389), p-ribosomal protein S6 (Ser240/Ser244), p-Erk (Thr202/Tyr204), p-BAD (Ser112), p-p38 MAP kinase (Thr180/Tyr182), and p-MAPKAP kinase 2 (Thr334) in human cell line xenografts from lung (A549), brain (U87MG), ovary (A2780), and prostate (PC3) tumors. In order to replicate real-world practices, the tissues were subjected to ischemic times ranging from 0 (baseline) to 24 hours before fixation in formalin. Staining was quantified by a positive pixel counting algorithm, and stability profiles were generated for each phosphoprotein in each tissue.
Results: Phosphoprotein stability profiles show either an increase (n=1), an initial increase followed by a decrease (n=7), a decrease (n=10), or a relatively stable level of staining (n=6). Figure 1 shows representative stability profiles for p-MAPKAP kinase 2 in all four tissues.

In Figure 2, representative stability profiles for all phosphoproteins in U87MG are shown.

Conclusions: Two key concepts emerge from this analysis: (a) the stability of a given phosphoepitope varies significantly across multiple tumor types, and (b) the stability of different phosphoepitopes within a given tumor type is variable. These results highlight the importance of proper tissue acquisition and rapid fixation to preserve the biological integrity of signal transduction pathways that will guide therapeutic decision making.
Category: Techniques

Monday, February 28, 2011 1:00 PM

Poster Session II # 269, Monday Afternoon


Close Window