Interference with c-myc expression and RB phosphorylation during TNF-mediated growth arrest in human endothelial cells

Incorporation of [H-3]-thymidine into DNA in nonsynchronized cultures of human endothelial cells was blocked by a 24 h exposure to TNF in a dose dependent manner that resulted in accumulation of cells in G1, as assayed by flow cytometry analysis of DNA content. Proliferation restarted when cells were replated in the absence of TNF. Northern analysis of c-myc mRNA in synchronized untreated cultures showed a transient increase previous to DNA synthesis that was decreased with TNF treatment. Western analysis of the retinoblastoma gene product RE in untreated synchronized cultures showed reduced electrophoretic mobility during the transition from G1 to S, congruent with RE inactivation by phosphorylation. TNF treatment prevented RE retardation and reduced total levels of RE protein. Taken together our results show that the TNF-mediated block of endothelial proliferation correlates with deficient activation of the G1 events necessary for entry into S, despite the presence of serum and endothelial mitogens. (C) 1997 Academic Press.