Gilles L'Allemain

Abrogation of p27Kip1 by cDNA Antisense Suppresses Quiescence (G0 State) in Fibroblasts

Progression of eukaryotic cells through the cell cycle is governed by the sequential formation, activation, and subsequent inactivation of a series of cyclin-dependent kinase (Cdk) complexes. p27Kip1 (p27) is a Cdk inhibitor that blocks, in vitro, the activity of cyclin D-Cdk4, cyclin D-Cdk6, cyclin E-Cdk2 as well as cyclin A-Cdk2, a complex active during S phase. The level of p27 protein expression, usually high in G0/G1 resting cells, declines as cells progress toward S phase and enforced expression of p27 in fibroblasts causes G1 arrest. This situation prevails in CCL39, a Chinese hamster lung fibroblast cell line (this report). However, in addition to p27, several other Cdk inhibitors known to alter G1 progression coexist in most mammalian cells. To investigate the specific contribution of p27 in the control of the mitogen-sensitive G0/G1 arrest, we specifically reduced its synthesis by expressing a full-length p27 antisense cDNA in CCL39 cells. Interestingly, reduction of up to 90% of p27 protein expression increased both basal and serum-stimulated gene transcription of cyclin D1, cyclin A, dihydrofolate reductase, and DNA synthesis reinitiation. Moreover, overexpression of this antisense allows cells to grow for several generations in a serum-free medium supplemented with insulin and transferrin only, thus suggesting that p27-depleted cells cannot exit the cell cycle. These effects were fully reversed by coexpression of a plasmid encoding p27 sense. We conclude that p27, by setting the level of growth factor requirement, plays a pivotal role in controlling cell cycle exit, a fundamental step in growth control.

Cytoplasmic pH, a key determinant of growth factor-induced DNA synthesis in quiescent fibroblasts

In response to growth factors, quiescent fibroblast mutants lacking Na+/H+ exchange activity fail to elevate their cytoplasmic pH (pHi) and to reinitiate DNA synthesis at neutral and acidic pHo. A pHi threshold of ~7.2 exists, below which growth factors cannot set in motion the G0 to S phase transition. Restoration of the pHi defect in mutant cells restores the wild‐type phenotype. These findings, combined with the properties of another class of mutants able to grow at very low pHo, demonstrate that pHi modulated by growth factor activation of the Na+/H+ antiporter, plays a determinant role in growth control.

Deciphering the MAP kinase pathway.

MAP kinases (MAPK) are serine/threonine kinases which are activated by a dual phosphorylation on threonine and tyrosine residues. Their specific upstream activators, called MAP kinase kinases (MAPKK), constitute a new family of dual-specific threonine/tyrosine kinases, which in turn are activated by upstream MAP kinase kinase kinases (MAPKKK). These three kinase families are successively stimulated in a cascade of activation described in various species such as mammals, frog, fly, worm or yeast. In mammals, the MAP kinase module lies on the signaling pathway triggered by numerous agonists such as growth factors, hormones, lymphokines, tumor promoters, stress factors, etc. Targets of MAP kinase have been characterized in all subcellular compartments. In yeast, genetic epistasis helped to characterize the presence of several MAP kinase modules in the same system. By complementation tests, the relationships existing between phylogenetically distant members of each kinase family have been described. The roles of the MAP kinase cascade have been analyzed by engineering various mutations in the kinases of the module. The MAP kinase cascade has thus been implicated in higher eukaryotes in cell growth, cell fate and differentiation, and in low eukaryotes, in conjugation, osmotic stress, cell wall construct and mitosis.

Early and late mitogenic events induced by FGF on Bovine Epithelial Lens cells are not triggered by hydrolysis of polyphosphoinositides

Basic or acidic forms of FGF, a potent mitogen for Bovine Epithelial Lens cells caused a rapid and transient rise in cytoplasmic Ca2+ followed by an increase in intracellular pH of 0.4 units. When cells were labeled at equilibrium with [3H]-inositol, no significant breakdown of polyphosphoinositides (in the presence of 20 mM LiCl) could be detected in response to 10-100 ng/ml of FGF. Similarly, fetal calf serum efficiently reinitiated DNA synthesis in these cells with little stimulation of polyphosphoinositide hydrolysis. In contrast, prostaglandin F2 alpha and angiotensin II, two weak mitogens for BEL cells, were found potent agonists of polyphosphoinositide breakdown. These results strongly indicate that the mitogenic action of FGF is not coupled to phospholipase C activation, a conclusion consistent with the fact that the FGF-induced [Ca2+]i rise is strictly dependent upon external Ca2+.

Chapter 12 Na+-H+ Exchange and Growth Control in Fibroblasts: A Genetic Approach

Publisher Summary This chapter presents genetic and biochemical evidence establishing that the Na + –H + antiporter is a major pH i -regulating system in fibroblasts, that growth factors activate the antiporter by increasing its pH i sensitivity, and that growth factor-induced cytoplasmic alkalinization is essential for reinitiation of DNA synthesis and growth at neutral and acidic pH o . In addition, the chapter presents specific selection procedures that have led to the isolation of three classes of mutants of Na + –H + antiport system: (1) mutants partially or totally defective, (2) mutants with altered Na + or amiloride binding sites, and (3) mutants overproducing the antiporter. The first advances in genetics of Na + –H + antiport system offered new approach to analyze the physiology of pH i -regulating system in fibroblasts, the role of pH i in growth control, and the identification of Na + –H + antiport at a molecular level. Na + –H + antiporter is a major pH i -regulating system in fibroblasts. In the presence of HCO − 3 , the operation of a 4-acetamide-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS)-sensitive and Na + -dependent Cl −- -HCO − 3 antiporter is a good substitute for Na + -H + exchange in pH, regulation. Growth factor-induced cytoplasmic alkalinization is triggered by activation of the Na + –H + antiporter. Growth factor activation results from an increased affinity of the system for internal H + . pH i exerts a control on the rate of cell entry into S phase. α-thrombin, a potent activator of polyphosphoinositide breakdown in CCL39 cells, activates as serum growth factors and phorbol esters, the Na + –H + antiporter by increasing its affinity for internal H + . The molecular identification of the antiporter molecule(s) should demonstrate if the change in pH i sensitivity reflects different states of kinase C-dependent phosphorylation.