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.
Franco
J L
JL
Laboratory of Experimental Immunology, Program Resources, Inc/DynCorp, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201, USA.
Ghosh
P
P
Wiltrout
R H
RH
Carter
C R
CR
Zea
A H
AH
Momozaki
N
N
Ochoa
A C
AC
Longo
D L
DL
Sayers
T J
TJ
Komschlies
K L
KL
D000818:Animals; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D015496:CD4-Positive T-Lymphocytes / Q000737:chemistry / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000737:chemistry / Q000276:immunology*; D002292:Carcinoma, Renal Cell / Q000276:immunology / Q000628:therapy*; D005419:Flavonoids / Q000627:therapeutic use*; D006098:Granulocytes / Q000276:immunology*; D007167:Immunotherapy; D007376:Interleukin-2 / Q000627:therapeutic use*; D007680:Kidney Neoplasms / Q000276:immunology / Q000628:therapy*; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051379:Mice; D008807:Mice, Inbred BALB C; D011505:Protein-Tyrosine Kinases / Q000032:analysis*; D011948:Receptors, Antigen, T-Cell / Q000032:analysis*; D015398:Signal Transduction / Q000502:physiology*
D000970:Antineoplastic Agents; D005419:Flavonoids; D007376:Interleukin-2; D011948:Receptors, Antigen, T-Cell; C049328:flavone acetic acid; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)