Generation of phosphorylcholine as an essential event in the activation of Raf‐1 and MAP‐kinases in growth factors‐induced mitogenic stimulation

Abstract

Cell proliferation is regulated by an appropriate combination of intracellular signals involving activation of kinases and the generation of phospholipid metabolites. We report here that growth factors induce a biphasic generation of phosphorylcholine (PCho) in quiescent NIH 3T3 cells, resulting in an early and transient increase at 100 s and a larger and sustained increase after 3 h of stimulation. Generation of PCho at both early and late times of growth factors stimulation results from the consecutive activation of phospholipase D (PLD) and choline kinase (ChoK). Production of PCho by specific growth factors seems an essential requirement for the early signals associated to activation of Raf-1 and MAP kinases, since blockage of choline kinase completely inhibited activation of Raf-1 and MAP kinases by PDGF or FGF. Both the transient early increase and the late sustained increase in PCho are required for the induction of DNA-synthesis, besides completion of the activation of the serine/threonine kinases cascade. Thus, our results strongly suggest that generation of PCho by the PLD/choline kinase pathway is one of the critical steps in regulating cell growth in NIH 3T3 stimulated by growth factors.

Choline kinase inhibitors as a novel approach for antiproliferative drug design

“…Neither does this e ect result from interference with other known enzymes involved in phospholipid metabolism (Cuadrado et al, 1993). However, it is possible that the observed antiproliferative e ect of ChoK inhhibition by HC-3 is a consequence of non-speci®c interference with intracellular signaling cascades, since we have found that inhibition of PCho production by HC-3 interferes with the activation of the Raf/MAPK pathway induced by growth factors (Jime nez et al, 1995). Thus, the availability of more selective compounds capable of inhibiting ChoK would be essential to further establish the correlation of inhibition of PCho and blockade of the RAf/MAPK pathway.…”

“…Previous studies have demonstrated that in cells transformed by oncogenes with high incidence in human tumors such as ras oncogenes (found in about 30% of all human tumors), there is a constitutive activation of choline kinase (ChoK) (Lacal et al, 1987;Macara, 1989;Lacal, 1990;Teegarden et al, 1990;Cuadrado et al, 1993;Carnero et al, 1994a;Jime nez et al, 1995;Ratnam and Kent, 1995), an enzyme that is responsible for the conversion of choline into phosphorylcholine (PCho) (Pelech and Vance, 1984). The increase in ChoK activity results in elevated levels of PCho, a putative novel second messenger involved in proliferation (Cuadrado et al, 1993).…”

“…Furthermore, there is evidence that hemicholinium-3 (HC-3), the most potent drug known as a ChoK inhibitor, blocks DNA synthesis stimulation by growth factors such as PDGF, bFGF, EGF, or phorbol esters but not that of serum or insulin (Cuadrado et al, 1993). Furthermore, HC-3 blockage is e ective only when added up to 4 ± 6 h of addition of growth factors (Jime nez et al, 1995), suggesting that it is highly speci®c for a particular step in the cell cycle. All together, these results imply that PCho may be an essential element in some mitogenic routes and most likely those where Ras proteins are involved.…”

“…Thus, blockade of PCho production may constitute a strategy for the speci®c inhibition of tumor cells where ras oncogenes are key players. On the other hand, the speci®city of the inhibitory e ect observed in HC-3 treated cells (Cuadrado et al, 1993;Jime nez et al, 1995) suggests that its e ect is not due to non-speci®c toxicity and makes this compound an interesting model for the design of new antitumor agents. However, the antimitogenic action of HC-3 is achieved at high concentrations (Cuadrado et al, 1993;Jime nez et al, 1995) and it is possible that it leads to its inhibitory e ect on cells stimulated with growth factors as a consequence of some action on other pathways.…”

“…On the other hand, the speci®city of the inhibitory e ect observed in HC-3 treated cells (Cuadrado et al, 1993;Jime nez et al, 1995) suggests that its e ect is not due to non-speci®c toxicity and makes this compound an interesting model for the design of new antitumor agents. However, the antimitogenic action of HC-3 is achieved at high concentrations (Cuadrado et al, 1993;Jime nez et al, 1995) and it is possible that it leads to its inhibitory e ect on cells stimulated with growth factors as a consequence of some action on other pathways. Furthermore, HC-3 is a competitive inhibitor of the high a nity choline transport system and has highly toxic respiratory paralyzant activity (Cannon, 1994), making the use of HC-3 as an in vivo antiproliferative agent impossible.…”