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 rest arted when cells were replated in the absence of TNF. Northern analysi s of c-myc mRNA in synchronized untreated cultures showed a transient increase previous to DNA synthesis that was decreased with TNF treatme nt. Western analysis of the retinoblastoma gene product RE in untreate d synchronized cultures showed reduced electrophoretic mobility during the transition from G1 to S, congruent with RE inactivation by phosph orylation. TNF treatment prevented RE retardation and reduced total le vels of RE protein. Taken together our results show that the TNF-media ted block of endothelial proliferation correlates with deficient activ ation of the G1 events necessary for entry into S, despite the presenc e of serum and endothelial mitogens. (C) 1997 Academic Press.