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DAG is highly hydrophobic and remains in the cell membranes where is rapidly metabolised by at least two different routes.

(i) DAG is de-acylated by the sequential action of 1,2-diacylglycerol and monoacylglycerol lipases. Since inositol lipids are enriched in arachidonic acid, DAG de-acylation may serve as source of arachidonic acid for the synthesis of prostaglandins and leukotrienes [Habenicht AJR et al., 1981].

(ii) DAG is rapidly phosphorylated by a DAG kinase to generate phosphatidic acid (PtdAcid) [MacDonald ML et at., 1988]. PtdAacid can be re-utilized for the biosynthesis of inositol lipids or of other acidic phospholipids such as phosphatidylserine [Vance DE,

1985]. In many cell types, this second pathway appears to have a primary importance in the removal of DAG.

Besides second messenger functions, diacylglycerols are also intermediates in phospholipid metabolism. Therefore only a fraction of the DAG detectable in most of the

cell types, derives from Pdtlns(4,5)f’2 hydrolysis [Kennerly DA 1987]. Depending on cell type and status, the ratio of PdtIns(4,5)P2 derived DAG to the total amount of cell diacylglycerol may vary considerably.

DAG can also derive from the breakdown of other phospholipids such as phosphatidylcholine (PtdCho). Production of significant amounts of DAG and phosphocholine (PCho), in absence of phosphoinositide turnover was observed in murine fibroblasts and Hela cells briefly exposed to phorbol esters [Besterman JM et a/., 1986], [Glatz et al., 1987]. Transformation with activated ras oncogenes or the introduction of ras oncogene products into murine fibroblasts or Xenopus laevis oocytes also resulted in the rapid generation of DAG which was paralleled by increased PtdCho turnover and production of PCho [Piice BD gf al., 1989-B] [Lacal JC, et al., 1987]. Importantly, phorbol ester as well as ra^-induced PtdCho breakdown was abolished by PKC down regulation following prolonged pre-treatment with PM A [Price BD et al., 1989-A]. Taken together these observations demonstrated that significant amounts of DAG and PtdAcid are generated by PtdCho hydrolysis. These data also suggested that PKC activation plays an important role in regulating the turnover of PtdCho.

1.5.2. 1.2-DAG and PKC regulation.

PKCs are a family of closely related and widely distributed protein kinases which phosphorylate target proteins on serine and threonine residues. Sevrai PKC isozymes have been identified. They differ from each other in tissue distribution, molecular structure and activation requirements [Ohno S et al., 1987], [Nishizuka Y, 1988]. The release of DAG in the cell membrane following inositol lipid hydrolysis or, phosphatidylcholine hydrolysis, constitutes the primary trigger of PKC activation [Nishizuka Y, 1986]. Most PKC isozymes also require the presence of Ca^^ ions and phosphatidylserine for optimal activation. DAG increases the affinity of PKC for Ca^^ ions, thereby causing enzyme activation at physiological Ca^^ levels [Nishizuka Y 1988]. DAG action on PKC is strictly stereospecific, therefore not all the diacylglycerols are able to activate PKC. The enzyme

requires the l,2-^/;-DAG configuration, while 1,3-DAG and 2,3-DAG isomers are not active. Furthermore DAG needs to be esterified to at least one unsaturated fatty acid y [Nomura H et al., 1986]. Several other compounds, especially tumour promoters such as phorbol esters, can cause PKC activation in absence of inositol lipid breakdown. These compounds compete with each other and with synthetic DAGs for the same binding site on PKC, and, because of their importance in many experimental models, they will be discussed in some detail in the following paragraphs.

1.5.3. Mechanisms of DAG-induced PKC activation.

Ligand-stimulated generation of DAG constitutes, together with the mobilization of Ca^^ ions, represents one of the critical events that cooperate to activate PKC [Nishizuka Y, 1984]. Since DAG is rapidly inactivated, ligand-stimulated PKC activation constitutes a transient and reversible event, during which the enzyme is briefly translocated from the cytosol to the plasma membrane. Membrane association seems critical for PKC activity [Homma Y et a!., 1986], but also exposes the activated PKC to proteolytic cleavage and consequently to inactivation.

Exposure to certain phorbol esters, e.g. phorbol 12-myristate 13-acetate (PMA), results in strong PKC activation These compounds activate PKC by a mechanism similar to that described for the DAGs: they increase PKC affinity for the Ca^^ ions and trigger PKC activation at ‘resting’ Ca‘^ levels. By contrast to DAGs, the phorbol esters are slowly degraded. Therefore PMA-triggered PKC activation and membrane translocation is longer than that generated by DAGs [Nishizuka Y, 1984]. Recent observations have demonstrated that introduction of activated ras oncogene products into murine fibroblasts or Xenopos laevis oocytes also resulted in PKC activation, suggesting a relationship between the ras oncogene signalling pathway and PKC [Price BD et al., 1988-B].

1.5.4. Physiological activity of PKC.

PKC activation positively synergizes with Ca^^ mobilization in triggering cell responses such as secretion, induction of gene expression and notably induction of early

events of mitosis [Nishizuka, 1984]. The role of PKC activation in cell mitosis was stressed by the observation that fibroblasts or lymphocytes treated with PMA, alone or in association with Ca^^ ionofores, initiated DNA synthesis and underwent other early mitotic changes [Geenberg ME & Ziff EB, 1984], [Rabin MS et aL, 1986], [Truneh A et a i,

1985]. Interestingly, PKC can also activate the NF-/:B transcription factor in vitro [Gosh S & Baltimore D, 1990].

PKC activation also provides a negative feed back on ligand-stimulated inositol lipid turnover and on other membrane events [Nishizuka Y, 1986]. In mouse fibroblasts activation of PKC by PMA abolished bombesin or PDGF- stimulated inositol lipid hydrolysis [Brown KD et al., 1987] [Price BD et al., 1989-A]. The mechanisms of PKC- induced down regulation of inositol lipid turnover have only been partially elucidated. PKC may inhibit PI-PLC directly [Ryu SH al., 1990]. Following prolonged PMA treatment, ligand-stimulated activation of inositol lipid breakdown was restored and, in some case enhanced. Since membrane translocation of PKC exposes the enzyme to inactivation by proteolysis, it is plausible that prolonged PKC activation results in PKC down-regulation. The enhanced inositol lipid hydrolysis following PKC down regulation, would then be the result of the loss of negative feed back on inositol lipid breakdown [Helper JR et al.,

1988].

In several cell types, activation of PKC, triggered by ligand stimulation, ras oncogene transformation or by exposure to PMA, resulted in PLC or PLD-mediated hydrolysis of PtdCho or of other phospholipids, eg phosphatidylethanolamine (PdtEt) [Price BD et al„ 1989-B], [Billah MM et al., 1989], [Kiss Z & Anderson WB, 1989]. Although the regulatory role of these events has not been entirely clarified, the generation of DAG and PtdAcid was involved in the transduction of chemotactic signals [Pai JK et al., 1988] [Bonser RW et al., 1989] and in the activation of early mitotic events in murine fibroblasts and epidermal carcinoma cell [Moolenar WH et al., 1986], [Yu CL et al., 1988].

The role of inositol lipid breakdown and the regulation of proliferation of normal and neoplastic cells

1.6, Inositol lipid turnover and the regulation o f proliferation o f normal cells.

The proliferation of eucaryotic cells is regulated by growth factors and mitogens. The binding of a growth factor to its cell surface receptor triggers cascades of intracellular events or signals that will secure commitment to mitosis. Binding of certain growth factors and mitogens to their receptors triggers inositol lipid breakdown, suggesting that this pathway constitutes a critical step in the regulation of cell proliferation [Whitman M & Cantley L, 1988].

Growth factors and mitogens which trigger the activation of the inositol lipid signalling pathway can be divided in two broad families:

i. Growth factors and mitogens acting through receptors which are coupled to PI-PLC activation by G proteins.

ii. Growth factor and mitogens acting through receptors containing ligand activated tyrosine kinase activity.

1.6.1. Growth factor and mitogen receptors coupled to PI-PLC via G proteins.

Several mitogens and vasoactive polypeptides, including thrombin, bombesin, serotonin, bradykinin, vasopressin, epinephrine, substance P, fmet-leu phe, etc, function by binding to receptors which are coupled to PI-PLC by specific G proteins [Heslop JP et a i, 1986] [Martin TFJ, 1989]. In hamster fibroblasts, pertussis toxin treatment abolished thrombin-induced inositol lipid hydrolysis [Paris S et al. 1987] and caused significant reduction of thrombin-induced DNA synthesis [Chambard JC et al. 1987]. Similar observations have been made using fibroblasts, macrophages, neuroblastoma-glioma cells.

hépatocytes and haemopoietic cells. In these cell types the coupling of receptors to PI-PLC activation was pertussis toxin- or cholera toxin-sensitive [Martin TFJ, 1989].

Activation of resting T lymphocytes, following T cell receptor (TCR/CD3) stimulation by PHA or other ligands, resulted in PI-PLC mediated inositol lipid hydrolysis. Treatment of intact or permeabilized T lymphocytes with the AIF/ ion or with non hydrolysable GTP analogs, like GTP-y-S, also resulted in PI-PLC activation suggesting the existence of G proteins that couple the (TCR/CD3) complex to a PI-PLC [Mire-Sluis AR et al., 1987]

1.6.2. Growth factors acting through receptors with ligand activated tvrosine kinase activitv.

This group includes PDGF, EGF, fibroblast growth factor (FGF), insulin like growth factors. The receptors for these growth factors possess intrinsic ligand-activated tyrosine kinase activity. The binding of a growth factor results in receptor autophosphorylation. PI-PLCy and other intracellular enzymes, including Pdtlns-3-kinase, ra^GAP and c-raf serine kinase, recognize and bind to the cytosolic domain of the activated receptor on specific phosphotyrosine residues [Cantley LC et al., 1991]. Following binding, the activity of PI-PLCy and of the other enzymes is modulated by tyrosine phosphorylation. These observations have provided direct evidence that concomitant activation of various signalling pathways occurs following growth factor stimulation, and that these signals cooperate to trigger mitosis [Cantley LC et al.,1991].

Both PDGF and EGF treatment resulted in tyrosine phosphorylation of PI-PLCy in intact 3T3 fibroblasts and A431 epidermoid cells as well as in vitro [Meisenhelder J et al.

1989]. Exposure to PDGF of Rat-2 cells line, overexpressing the isozymes PI-PLCy^ and PI-PLCy^, caused tyrosine phosphorylation of PI-PLCy isozymes and generation of inositol phosphates which was higher than in the parental cells, and which correlated with the levels of tyrosine-phosphorylated PLCy. Furthermore, in the same cells, G protein activators like AIF/, failed to cause inositol phosphate accumulation [Sultzman L et al.

1991]. Direct evidence that tyrosine phosphorylation caused an increase of PI-PLCy catalytic activity in vitro, was obtained in PI-PLCy immunoprecipitates from EGF treated and untreated A-431 cells. PI-PLCy activity was significantly higher in the immunoprecipitates from EGF treated cells, but importantly, PI-PLCy activity was inhibited following the addition of a phosphotyrosine phosphatase to the assays [Nishibe S et al,

1990].

PDGF triggers inositol lipid hydrolysis, and inositol lipid re-synthesis in several types of cells and cell lines of epithelial or mesenchymal origin. Following exposure to PDGF, generation of Ins(l,4,5)P3 and DAG showed a lag-time that varied with the cell type. Nevertheless, transient rise of cytoplasmic Ca^"^ levels and PKC activation have been documented [Habenicht AJR et al„ 1981] [Habenicht AJR et at., 1985].

Although PI-PLCy isozymes have been shown to be readily tyrosine phosphorylated in response to EGF stimulation, activation of inositol lipid hydrolysis was detected only in certain instances of EGF-induced mitogenesis. Following EGF stimulation, increased synthesis of polyphosphoinositide species and pertussis toxin-insensitive inositol lipid hydrolysis, was detected in the A431 epidermoid cell line. [Pike LJ & Eakes AT, 1987]. Stimulation with EGF of mouse fibroblasts or of epidermoid cells, also triggered the activity of Ptdlns and PtdIns(4)P 3'-kinases and the generation of 3’-phosphorylated phosphoinositide [Bjorge JD et al. 1990]. Although the functions of these inositol lipid species are not yet understood, they are thought to have a signalling role in cell mitosis [Carpenter IC & Cantley LC, 1990].

1.6.3. Relationship between generation of inositol-lipid derived second messengers and the control of cell proliferation.

Several strategies have been devised to study the relationship between growth factor-stimulated inositol lipid turnover and cell proliferation. On the one hand, inositol lipid turnover has been mimicked by pharmacological agents, whereas on the other hand inositol lipid turnover has been selectively blocked by immunological or pharmacological

means.

1.6.3.a. Experiments in which the operation of inositol lipid turnover was artificially reproduced or induced.

Calcium ionophores (A 23187 and ionomycin), and phorbol esters (PMA in particular), have been used to mimic the intracellular signals generated by Pdtlns(4,5)?2 hydrolysis, namely Ca^^ mobilization and PKC activation. The goal of these experiments was to investigate the effects of these signals on cell proliferation in the absence of growth factor stimulation.

Treatment of several epithelial or mesenchymal cell lines, resting lymphocytes or of chronic lymphocytic leukaemia cells, with calcium ionophores or with PKC activators including DAG analogs such as 1-oleoyl 2-acetyl glycerol (OAG) or PMA caused induction of some early mitotic events such as weak stimulation of DNA synthesis, transcription of the immediate-early response genes c-myc and c-fos, activation of Na^/H^ exchange and cytosol alkalinization. Simultaneous exposure to calcium ionophores and phorbol esters caused stronger induction of early mitotic events an, in some instances, S- phase entry [Moore JP et al., 1986], [Truneh A et at., 1985], [Rabin MS et al., 1986], [Drexler HG et al., 1989], [Greenberg ME & Ziff EB, 1984]. Nevertheless treatment with these compounds alone was not able to induce cell mitosis, observations that could be partially explained by their toxic nature [Whitman M & Cantley L. 1988].

The artificial expression of PI-PLC coupled neuroreceptors in non-neuronal cells, such as fibroblasts or lymphoid cells, has recently provided the possibility of ‘physiologically’ activating inositol lipid hydrolysis. The stimulation of the PI-PLC - coupled 5HTlc serotonin neuroreceptor, expressed in NIH 3T3 fibroblasts, caused Ca^^ mobilization and formation of neoplastic foci [Julius D et al. 1989]. Stimulation with carbachol of the muscarinic receptor Hm l, expressed in the CCL39 hamster lung fibroblast cell line, triggered inositol lipid hydrolysis followed by Ca^^ mobilization, PKC activation and initiation of early mitotic events. Nevertheless, carbachol alone did not induce cell

mitosis, suggesting that activation of additional signalling pathways was required [Seuwen K et al. 1990]. Similar experiments have been carried out by expressing the HM l muscarinic receptor in a Jurkat-derived T-cell line. Stimulation with carbachol, resulted in inositol lipid hydrolysis which was followed by early events of T cell activation, including IL2 production and IL2-receptor 6 chain expression. Importantly, the physiological stimulation of the T-cell receptor with specific antibodies triggered identical biochemical events [Desai DM et at., 1990].

In conclusion, selective activation of inositol lipid hydrolysis, following stimulation of PI-PLC-coupled neuroreceptors was able to initiate several biochemical events that normally precede and prepare the cell for the mitosis. These observations suggest that activation of inositol lipid turnover constitutes one of the critical steps that precede and trigger G/S-phase transition. The observation that PI-PLC microinjection induced normal fibroblasts to enter the S phase and, in some instances, to undergo neoplastic transformation, appears entirely consistent with this hypothesis [Smith IR et a/., 1989]. Furthermore, transfection of mouse fibroblasts with cDNA encoding PKC isozymes was sufficient to confer multiple growth abnormalities to these cells including formation of neoplastic foci in vitro and increased tumorigenicity in nude mice. [Mousey GM et al.,

1988], [Persons DA et al., 1988]

1.6.3.b. Experiments in which the ligand-stimulated operation of inositol lipid turnover was selectivelv blocked.

Ligand stimulated PI-PLC activity as well as the biological effects of PdtIns(4,5)P2- derived second messengers, may be blocked by various agents. Unfortunately none of these agents act specifically on inositol lipid turnover, making the interpretation of these experiments difficult. Neomycin inhibited PI-PLC activity stimulated by Ca^^, guanine nucleotides or by the chemotactic peptide fMetLeuPhe probably by binding to PdtIns(4,5)P2 [Cockcroft S & Gomperts B, 1986], [Cockcroft S & Stutchfield J, 1988]. Exposure to neomycin also inhibited thrombin stimulated mitogenesis, however, neomycin

is toxic and its specificity for Pdtlns(4,5)?2 remains unclear [Whitman M & Cantley L, 1988].

In certain cell systems, pertussis toxin causes inhibition of ligand stimulated PI-PLC activity [Cockcroft S & Stutchfield J, 1988]. Thrombin, as well as bombesin-stimulated mitogenesis was abolished by pertussis toxin treatment, consistent with the proposed role of inositol lipid breakdown in cell proliferation [Chambard JC gf <2/., 1987]. However, because of pertussis toxin interactions with other biochemical pathways, it cannot be established whether the inhibition of inositol lipid hydrolysis alone is sufficient to abrogate ligand-induced mitosis [Gilman AG, 1987].

The development of a monoclonal antibody to Ptdlns?2, has provided the possibility of selectively blocking the generation of inositol lipid-derived second messengers. This antibody, by binding to Ptdlnsf2, inhibited ligand stimulated Ptdlnsf2 hydrolysis in vitro. Microinjection of the anti-PtdIns?2 antibody in NIH 3T3 fibroblasts caused inhibition of the mitogenic effects of PDGF and bombesin. Conversely, the same anti-PtdIns?2 antibody did not inhibit FGF, EGF, insulin or serum-stimulated mitosis [Matuoka K et at., 1988]. These results were partially explained by the observation that EGF and FGF, by contrast to PDGF and bombesin, did not stimulate PdtIns(4,5)P2 hydrolysis in NIH 3T3 cells [Matuoka K et a i, 1988].

Molecular approaches have also been used to selectively abolish ligand stimulated inositol lipid hydrolysis. A mutated FGF receptor, unable to bind PLCy and therefore unable to activate phosphoinositide hydrolysis upon ligand stimulation, retained the capacity of transducing mitotic signals independently from the activation of the inositol lipid signalling pathway in a myoblastic cell line [Peters IG et al., 1992] [Mohammadi M et al., 1992]. These observations, although restricted to one cell line (L6 myoblasts), demonstrated that, FGF-stimulated inositol lipid breakdown did not play a critical role in signalling cell division. These data also suggested that phosphoinositide hydrolysis may not be directly involved in the regulation of mitogenesis triggered by growth factors acting

through receptors with tyrosine kinase activity.