Growth arrest of the breast cancer cell line, T47D, by TNF alpha; cell cycle specificity and signal transduction

Abstract

The effects of tumour necrosis factor-alpha (TNF alpha) on the growth and DNA synthesis of the human breast cell line, T47D, were studied. A dose-dependent, reversible inhibition of thymidine incorporation and cell growth was observed in the range of 0.1 ng ml-1 to 100 ng ml-1 of TNF alpha. Cell viability was not impaired in any of the experiments. Flow-cytometric DNA analysis demonstrated that after 24 h exposure to TNF alpha, T47D cells accumulated in the G1 phase of the cell cycle, and were depleted in the G2/M and S phases, suggesting a block in the progression of the G1/S transition. The involvement of protein kinases (PK) and protein phosphatases in TNF alpha-induced signal transduction was also investigated. A transient and rapid 2-fold increase in total cellular protein kinase C (PKC) activity was detected after 10 min exposure to TNF alpha. To study the role of the observed PKC activation in the cytostatic effect of TNF alpha, T47D cells were exposed to the cytokine in the presence of the potent PKC inhibitor, H7. The inhibitory effect of TNF alpha on thymidine incorporation was not affected by exposure to H7 at concentrations sufficient to block the stimulation of thymidine up-take induced by the PKC agonist, phorbol-12-myristate-13-acetate (PMA). The involvement of other signalling pathways was addressed using the cyclic nucleotide-dependent PK inhibitor, H8; the calmodulin-dependent PK inhibitor, W7; and the inhibitor of protein phosphatases PP1 and PP2B, okadaic acid. Exposure of T47D cells to these enzyme inhibitors failed to antagonise the inhibition of thymidine incorporation by TNF alpha. Taken together, these results indicate that the cytostatic effect of TNF alpha on T47D cells occurs at the G1/S transition of the cell cycle, and is mediated by an intracellular pathway which does not involve the activity of protein kinases C and A, nor protein phosphatases PP1, PP2B.