The p42/p44 MAP kinase pathway prevents apoptosis induced by anchorage andserum removal

Anchorage removal like growth factor removal induces apoptosis. In the pres ent study we have characterized signaling pathways that can prevent this ce ll, death using a highly growth factor- and anchorage-dependent line of lun g fibroblasts (CCL39). After anchorage removal from exponentially growing c ells, annexin V-FITC labeling can be detected after 8 h. Apoptosis was conf irmed by analysis of sub-G1 DNA content and Western blot ting of the caspas e substrate poly (ADP-ribose) polymerase. Growth factor withdrawal accelera tes and potentiates suspension-induced cell death. Activation of Raf-1 kina se in suspension cultures of CCL39 or Madin-Darby canine kidney cells stabl y expressing an estrogen-inducible activated-Raf-1 construct (Delta Raf-1:E R) suppresses apoptosis induced by growth factor and/or anchorage removal. This protective effect appears to be mediated by the Raf, mitogen-or extrac ellular signal-regulated kinase kinase (MEK), and mitogen-activated protein kinase module because it is sensitive to pharmacological inhibition of MEK -1 and it can be mimicked by expression of constitutively active MEK-1 in C CL39 cells. Finally, apoptosis induced by disruption of the actin cytoskele ton with the Rho-directed toxin B (Clostridium difficile) is prevented by a ctivation of the Delta Raf-1:ER chimeric construct. These findings highligh t the ability of p42/p44 mitogen-activated protein kinase to generate survi val signals that counteract cell death induced by loss of matrix contact, c ytoskeletal integrity, and extracellular mitogenic factors.