PARTIAL DEGRADATION OF T-CELL SIGNAL-TRANSDUCTION MOLECULES BY CONTAMINATING GRANULOCYTES DURING PROTEIN EXTRACTION OF SPLENIC T-CELLS FROMTUMOR-BEARING MICE

Flavone-8-acetic acid plus recombinant human interleukin 2 is a succes sful antitumor therapy in mice bearing the Renca murine renal cell car cinoma, This report demonstrates that T cells, particularly CD8(+) T c ells, are critical for the generation of this response, Initial experi ments examining T-cell signal transduction proteins demonstrated that T cells from Renca-bearing mice had undetectable levels of p56(lck) an d 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 redu ction in p56(lck) was due to degradation of these molecules during pro tein extraction caused by mature granulocytes contaminating the enrich ed T-cell population, This was not the case for nuclear c-Rel or NF ka ppa B p65, which remained at undetectable/reduced levels in the absenc e of granulocytes, confirming our previous data that transcription fac tor alterations exist in tumor-bearing mice. Thus, most of the reducti on in zeta-chain in T cells from Renca-bearing mice is due to granuloc yte contamination and emphasizes the need to use pure T-cell populatio ns and/or sufficient amounts and types of protease inhibitors when qua ntitating proteins in T cells from tumor-bearing mice.