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Vol. 11, Issue 3, 1103-1112, March 2000
*Centre National de la Recherche Scientifique, Unite Mixte de
Recherche, 6543, Centre A. Lacassagne, 06189 Nice Cedex 2, France; and Anchorage removal like growth factor removal induces
apoptosis. In the present study we have characterized signaling
pathways that can prevent this cell death using a highly growth
factor- and anchorage-dependent line of lung fibroblasts
(CCL39). After anchorage removal from exponentially growing
cells, annexin V-FITC labeling can be detected after 8 h.
Apoptosis was confirmed by analysis of sub-G1 DNA content and Western
blotting of the caspase substrate poly (ADP-ribose) polymerase.
Growth factor withdrawal accelerates and potentiates suspension-induced
cell death. Activation of Raf-1 kinase in suspension cultures of CCL39
or Madin-Darby canine kidney cells stably expressing an
estrogen-inducible activated-Raf-1 construct (Institut National de la Santé et de la
Recherche Médicale U 343, Hopital de l'Archet, 06202 Nice Cedex
3, France
Raf-1:ER)
suppresses apoptosis induced by growth factor and/or anchorage removal.
This protective effect appears to be mediated by the Raf, mitogen- or
extracellular 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 CCL39 cells. Finally,
apoptosis induced by disruption of the actin cytoskeleton with the
Rho-directed toxin B (Clostridium difficile) is prevented by
activation of the Raf-1:ER chimeric construct. These findings
highlight the ability of p42/p44 mitogen-activated protein kinase to
generate survival signals that counteract cell death induced by loss of
matrix contact, cytoskeletal integrity, and extracellular mitogenic factors.
Corresponding author. E-mail address:
vanobber{at}unice.fr.
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