Partial degradation of T-cell signal transduction molecules by contaminating granulocytes during protein extraction of splenic T cells from tumor-bearing mice.

Flavone-8-acetic acid plus recombinant human interleukin 2 is a successful antitumor therapy in mice bearing the Renca murine renal cell carcinoma. This report demonstrates that T cells, particularly CD8+ T cells, are critical for the generation of this response. Initial experiments examining T-cell signal transduction proteins demonstrated that T cells from Renca-bearing mice had undetectable levels of p56lck and zeta-chain of the T-cell receptor and that flavone-8-acetic acid and recombinant human interleukin 2 therapy could be used as a model for reversal of these alterations. However, further experimentation showed that the majority of the reduction in zeta-chain and part of the reduction in p56lck was due to degradation of these molecules during protein extraction caused by mature granulocytes contaminating the enriched T-cell population. This was not the case for nuclear c-Rel or NF kappa B p65, which remained at undetectable/reduced levels in the absence of granulocytes, confirming our previous data that transcription factor alterations exist in tumor-bearing mice. Thus, most of the reduction in zeta-chain in T cells from Renca-bearing mice is due to granulocyte contamination and emphasizes the need to use pure T-cell populations and/or sufficient amounts and types of protease inhibitors when quantitating proteins in T cells from tumor-bearing mice.