Stimulation by tumor necrosis factor of HL‐60 thymidine salvage pathway metabolism dissociated from proliferation

The effect of human tumor necrosis factor (TNF) on early‐passage HL‐60 cells was studied. A transient phase of increased [ 3 H]thymidine (TdR) incorporation was noted at 20‐24 hr of exposure to TNF. This increase was disproportionate to the much slighter stimulation of the percentage of S‐phase cells, which was measured by flow cytometry. Evidence for increased metabolic trapping of [ 3 H] TdR following TNF treatment was apparent from whole cell uptake experiments. The salvage pathway enzyme TdR kinase was therefore measured and was found to be elevated comparably to [ 3 H]TdR uptake. The mechanism of TNF regulation of TdR kinase was further investigated by a series of combination treatment experiments using other biologic factors and pharmacologic inhibitors of various intracellular steps. The response to TNF was not potentiated or reproduced by IL‐1, IL‐2, IL‐3, IL‐4, G‐CSF, M‐CSF, GM‐CSF or α‐ or γ‐interferon. Blockers of early signal transduction steps, including H7, W7, sphingosine, and pertussis toxin, failed to inhibit TNF stimulation of [ 3 H]TdR incorporation. mRNA synthesis inhibition with α‐amanitin blocked this TNF effect, as did cAMP but not cGMP analogues. A sensitizing effect was noted with amiloride or cytochalasin B, characterized by greater relative increases of [ 3 H]TdR incorporation and TdR kinase activity in response to TNF. In the presence of cytochalasin B, TNF treatment resulted in no change or slight decreases in the percentage of S‐phase cells. Regulation of TdR kinase could thereby be dissociated from the usual cell cycle control. This study thus documents a unique example of stimulation of thymidine salvage pathway metabolism by a biologic factor, dissociable from overall cell cycle regulation.