Effects of Tumor Necrosis Factor-alpha on Antimitogenicity and Cell Cycle-related Proteins in MCF-7 Cells

Tumor necrosis factor-alpha (TNF-alpha) demonstrated antimitogenic activity in MCF-7 cells (estrogen receptor-positive human breast cancer cells) in a dose- and time-dependent manner (EC-50 of 2.5 ng/ml). This antimitogenic effect of TNF-alpha was accompanied by a decreased number of cells in S phase in a dose- and time-dependent manner. Based on growth arrest experiments using aphidicolin, it is apparent that TNF-alpha acted in early G1 phase. It did not show antimitogenic effects once cells reentered the S phase based on [3H]thymidine incorporation into DNA and cell cycle analysis. Specificity of TNF-alpha was established by using monoclonal anti-human TNF-alpha antibody. On the basis of Western immunoblot analysis of Rb, p53 and cell cycle inhibitory protein (Cip1) (p21) proteins, TNF-alpha decreased Rb protein expression in a dose- and time-dependent manner whereas it increased the expression level of tumor suppressor p53 protein. TNF-alpha also increased the expression level of Cip1 (p21) protein in a dose-dependent manner. This induction of Cip1 (p21) protein was preceded by the induction of p53 protein in MCF-7 cells. Cip1 (p21) protein associated with cyclin D was also increased. Tumor suppressor Rb protein expression was increased during G1 to S phase progression. Cyclin D protein expression levels were not changed in response to TNF-alpha treatment, although serine/threonine kinase inhibitors such as H7 and the protein kinase C inhibitor staurosporine decreased cyclin D expression levels in MCF-7 cells. Based on experiments with staurosporine, it appears that TNF-alpha does not utilize a protein kinase C pathway in MCF-7 cells. Other cell cycle-related proteins such as Cdk2, Cdc2, and Cdk4 did not show any change in response to TNF-alpha. TNF-alpha did not affect complexes between cyclin D and Cdk2, Cdk4, and Rb proteins in MCF-7 cells. Taken together these results suggest that Rb, p53, and Cip1 (p21) proteins mediate TNF-alpha antimitogenic activity, and TNF-alpha induces growth arrest in the G1 phase in MCF-7 cells.