Protein tyrosine kinase inhibition and cell proliferation: is the [3H]-thymidine uptake assay representative of the T-lymphocyte proliferation rate?
T-cell growth is controlled to a large degree by extracellular signals that bind to specific receptors on the surface of cells. A number of these receptors have intrinsic protein tyrosine kinase (PTK) activity. Their action on second messenger generation, and thus on cell proliferation, has been indirectly demonstrated by the decrease in [3H]-thymidine (TdR) uptake that follows co-stimulation of T-cells with mitogens and PTK inhibitors such as genistein (GEN). In this paper we report that the [3H]-TdR uptake assay is not a valid and reliable tool for investigating the proliferative activity of certain T-cell lines. In fact, a concomitant assessment of both [3H]-TdR uptake and cell cycle progression demonstrated that GEN is able to block G2/M progression of Jurkat T-lymphocytes even at doses (5 micrograms/ml) that do not influence [3H]-TdR uptake. Pretreatment with sodium o-vanadate (100 nM) could not reverse the GEN-related cell cycle perturbation, but was able to restore optimal [3H]-TdR uptake. Finally, GEN treatment was able to induce concentration-dependent apoptotic cell death of Jurkat T-cells. The control of cell activation, proliferation and programmed cell death is undoubtedly influenced by receptor-associated PTKs. The final effect on cell survival is almost entirely dependent on the activation state of the cell. The [3H]-TdR uptake assay seems to be inadequate for a correct interpretation of the expected results.
Spinozzi
F
F
Institute of Internal Medicine and Oncological Science, University of Perugia, Italy.
Pagliacci
M C
MC
Agea
E
E
Migliorati
G
G
Riccardi
C
C
Bertotto
A
A
Nicoletti
I
I
D002453:Cell Cycle / Q000502:physiology*; D002460:Cell Line; D005434:Flow Cytometry; D019833:Genistein; D006131:Growth Inhibitors / Q000494:pharmacology; D006801:Humans; D007529:Isoflavones / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D013601:T-Lymphocytes / Q000378:metabolism / Q000502:physiology*; D013936:Thymidine / Q000378:metabolism
D006131:Growth Inhibitors; D007529:Isoflavones; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D013936:Thymidine