ABSTRACT
Certain growth factors act by stimulating the hydrolysis of inositol lipids to yield putative second messengers such as diacylglycerol (DG) and inositol trisphosphate (IP3). One function of the former is to stimulate C-kinase, which may act by switching on a sodium/hydrogen exchanger to induce the increase in pH that appears to have a permissive effect on DNA synthesis. Studies on Swiss 3T3 cells have revealed that growth factors stimulate an increase in two separate isomers of IP3. In addition to inositol 1,4,5-trisphosphate there was a large increase in inositol 1,3,4-trisphosphate. While the former functions to elevate intracellular calcium, which has been implicated in the control of growth of many different cell types, the function of the latter is unknown. Since the 1,3,4 isomer turns over very slowly, it may control long-term events and thus could play a role in cell growth.
There are other growth factors such as insulin and epidermal growth factor (EGF), which apparently do not work through the inositol lipids but they may initiate ionic events similar to those just described for calcium-mobilizing receptors. The bifurcating signal pathway based on IP3/Ca2+ and DG/C-kinase provides an interesting framework within which to consider the mode of action of oncogenes.
INTRODUCTION
A critical period in the life of a cell occurs immediately after each mitosis when a decision must be made as to whether or not to remain within the cell cycle. During early development cells opt to remain in the cell cycle but as cell numbers increase groups of cells stop growing and enter a Go phase during which they differentiate to perform some specific function. In some cases, such as in nerves and muscle, such cell differentiation represents a terminal decision in that the cells are incapable of returning to the cell cycle. However, there are many differentiated cells that retain the ability to return to the cell cycle if provided with an appropriate mitogenic signal usually in the form of a specific growth factor. Cell growth is carefully regulated to ensure that sufficient cells are produced during development and that cells re-enter the cell cycle at appropriate times commensurate with the requirement for new cells for body repair and other functions. This carefully orchestrated control of cell growth breaks down in cancer when individual cells break free of their normal constraints and begin to divide continuously. Such cell transformation appears to arise from a subtle alteration in some step of the normal control pathway resulting in the cell receiving an inappropriate growth stimulus delivered through the normal signal pathways. The genes responsible for these signal pathways have been referred to as proto-oncogenes, which when altered become the oncogenes responsible for cell proliferation.
The behaviour of cells in tissue culture provides a convenient model system for studying the control of cell growth in the intact organism. When first seeded in a culture dish cells will grow continuously until they become confluent and deplete the medium of growth factors, at which point they enter a stationary phase. Such quiescent cells can be induced to grow either by the addition of growth factors similar to those that are thought to be released in the intact animal or by the introduction of oncogenes through transfection or a viral vector. Such oncogenic transformation of cultured cells appear to be an excellent model system for studying the etiology of cancer because these cells, which have been transformed in vitro, are tumorigenic when injected back into mice. When trying to understand how cell growth is regulated, therefore, we have to consider two closely related questions. First, what are the signal pathways utilized by growth factors when they stimulate DNA synthesis? Secondly, how do oncogenes subvert this normal control pathway to lock cells in continuous bouts of cell division? The inference is that in order to find out how oncogenes work it will be necessary to understand first how growth factors act to stimulate DNA synthesis. This notion is supported by the fact that the two oncogenes sis and erbB, whose functions have been identified, have been linked with a specific growth factor and a growth factor receptor. In this article we examine a dual signal hypothesis of cell growth, which attempts to explain how many growth factors might use the inositol lipids to generate a bifurcating signal pathway ultimately responsible for stimulating DNA synthesis. This hypothesis provides an interesting framework within which to discuss how oncogenes might function.
GROWTH FACTORS AND IONS
One of the important simplifying assumptions of the dual signal hypothesis is that growth factors stimulate cell growth by means of second messenger pathways similar to those used by neurotransmitters and hormones to stimulate cellular processes such as secretion and contraction. Until recently the action of growth factors has tended to stand apart on a temporal basis in that the onset of DNA synthesis occurs after many hours whereas hormones and neurotransmitters act immediately. However, this temporal gap has been dramatically reduced by the observation that growth factors such as platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) can switch on DNA transcription within minutes (Greenberg & Ziff, 1984; Kruijer, Cooper, Hunter & Verma, 1984; Muller, Bravo, Burckhardt & Curran, 1984). However, it is important not to lose sight of the fact that growth factors do require time to act. Although they can switch on the transcription of certain genes within minutes, such short stimulations are usually not adequate to induce DNA synthesis. The growth factor must act for several hours, which presumably means that it must be capable of activating the signal pathway for a protracted period. This need for a prolonged stimulus could possibly be the basis for the synergistic interactions that exist between various growth factors. Further evidence for supposing that growth factors might use conventional messenger pathways comes from studies on salivary gland hyperplasia where DNA synthesis can be stimulated using the same neurotransmitters that stimulate amylase secretion (Guidotti, Weiss & Costa, 1972; Durham, Baserga & Butcher, 1974). Similarly, norepinephrine can stimulate DNA synthesis in hepatocytes using -adrenoreceptors (Cruise, Houck & Michalopoulos, 1985).
The actions of many hormones and neurotransmitters on cellular processes such as secretion, metabolism and contraction are critically dependent upon second messengers such as cyclic AMP, inositol trisphosphate (IP3), diacylglycerol and calcium, all of which have been implicated in the control of cell growth. The role of cyclic AMP is something of an enigma in that it can function as either a negative or a positive regulator of growth (Boynton & Whitfield, 1983). Chinese hamster ovary cells are normally inhibited by cyclic AMP, but once the cells have become infected with Rous Sarcoma virus cyclic AMP begins to promote growth (Gottesman, Roth, Vlahakis & Pastan, 1984). A possible explanation for the action of cyclic AMP is that it functions as a modulator of the calcium signal pathway, as argued previously (Berridge, 1975). In this article we will concentrate on the role of calcium, which appears to be a major ionic signal for cell proliferation. The dual signal hypothesis attempts to account for how certain growth factors use the inositol lipids as part of a bifurcating transduction mechanism in the cell membrane to deliver mitogenic signals such as calcium to the cell interior.
PHOSPHOINOSITIDES AND THE DUAL SIGNAL HYPOTHESIS
The basis of the dual signal hypothesis is that growth factors act by stimulating the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP3) (Fig-1). This hydrolysis of PIP2 represents a bifurcation point, in that the two products act as second messengers to initiate two separate signal pathways (Nishizuka, 1984; Berridge, 1984a; Berridge & Irvine, 1984). The neutral diacylglycerol acts within the plane of the membrane to stimulate C-kinase (Nishizuka, 1984), which phosphorylates a specific set of proteins. The precise role of this signal pathway remains to be uncovered because as yet no function has been ascribed to any of the proteins phosphorylated by C-kinase. However, this pathway does seem to be important in controlling cell growth because it appears to be the site of action of the potent tumour-promoting phorbol esters. Castagna et al. (1982) showed that these phorbol esters stimulated C-kinase and subsequent studies showed that this enzyme was the cellular receptor for these tumour promotors (Kikkawaet al. 1983; Leach, James & Blumberg, 1983).
Just how the activation of C-kinase in the plasma membrane by DG results in the formation of a signal to convey information to the nucleus is still a mystery. One interesting possibility is that C-kinase activates the Na/H exchanger, which results in the rapid alkalinization of the cytosol that accompanies the action of many growth factors (L’Allemain, Paris & Pouyssegur, 1984; Moolenaar et al. 1984; Heskethetal. 1985). The link between C-kinase and pH was suggested by the observation that phorbol esters, which had been shown to act through C-kinase (Castagna et al. 1982), could activate the Na/H exchanger (Burns & Rozengurt, 1983; Rosoff, Stein & Cantley, 1984). What remains to be determined is whether the resulting increase in pH plays a role in stimulating DNA synthesis as proposed by some authors (Schuldiner & Rozengurt, 1982; L’Allemain et al. 1984; Moolenaar, Tsien, van der Saag & de Laat, 1983) but refuted by others (Besterman, Tyrey, Cragoe & Cuatrecasas, 1984). Despite this uncertainty, the fact that phorbol esters can stimulate early transcription of certain oncogenes (Greenberg & Ziff, 1984; Kruijer et al. 1984), in the same way as growth factors, indicates that the C-kinase pathway does play some role in transferring information to the cell interior but whether or not this depends upon a change in pH remains to be seen.
The other limb of the signal pathway is controlled by IP3, which is released to the cell’ interior to function as a second messenger to mobilize intracellular calcium (Berridge, 1984a; Berridge & Irvine, 1984). A complication in the IP3 story is the recent description of a 1,3,4 isomer (Irvine, Letcher, Lander & Downes, 1984). As yet the source of this isomer is unknown, but the most likely possibility is that it originates from inositol 1,3,4,5-tetrakisphosphate (1,3,4,5—IP4), which has been identified in brain slices (Batty, Nahorski & Irvine, 1985) and in GH4 cells, Swiss 3T3 cells and blowfly salivary gland (Heslop, Irvine, Tashjian & Berridge, 1985). There are two possible sources of 1,3,4,5-IP4; it may originate either from PIP3 or it may arise by phosphorylation of 1,4,5-IP3 (Fig. 2). Although both 1,3,4-IP3 and 1,3,4,5-IP4 are potential second messengers (Irvine et al. 1984; Batty et al. 1985) no such function has been identified and we shall concentrate, therefore, on the proposed calcium mobilizing action of 1,4,5-IP3 (Berridge & Irvine, 1984).
An increase in the intracellular level of calcium has been implicated in the control of growth of many different cell types (Boynton, Whitfield, Isaacs & Morton, 1974; Berridge, 1975; Metcalfe, Pozzan, Smith & Hesketh, 1980). Measurement of either calcium fluxes (Lopez-Rivas & Rozengurt, 1983) or levels of intracellular calcium with quin 2 (Moolenaar et al. 1984; Hesketh et al. 1985) indicate that many growth factors appear to act, at least in part, by mobilizing calcium from intracellular stores. Some of these growth factors, e.g. PDGF and vasopressin, cause a breakdown of PIP2 to give DG (Habenichtet al. 1981) and IP3 (Berridge, Heslop, Irvine & Brown, 1984). The latter has also been shown to mobilize calcium from intracellular stores in Swiss 3T3 cells (Berridge et al. 1984). The inositol lipid transduction mechanism thus plays an integral role in generating the calcium signal, which appears to be important in mitogenesis. However, most of the evidence linking calcium and cell proliferation is correlative in nature and there is no direct evidence to show that an increase in intracellular calcium is a sine qua non for the onset of DNA synthesis. Perhaps the most direct evidence implicating calcium is the observation that the early steps leading to DNA synthesis in lymphocytes can be induced by a calcium ionophore, particularly if the latter is combined with an activator of C-kinase such as a phorbol ester (Mastro & Smith, 1983; Truneh, Albert, Golstein & Schmitt-Verhulst, 1985; Kaibuchi, Takai & Nishizuka, 1985). It is relevant to point out that an increased level of calcium together with the activation of C-kinase are not sufficient to stimulate mitogenesis, which also requires receptor activation by either inteleukin-2 (Truneh et al. 1985) or a small amount of phytohaemoagglutinin (PHA) (Kaibuchi et al. 1985). Perhaps such receptor activation provides some essential messenger other than DG and calcium. It would be fascinating if this additional signal was related to one of the IP3 isomers. The first indication that the two limbs of the bifurcating signal pathway might act synergistically with each other emerged from studies on blood platelets where near-maximal rates of serotonin secretion could be achieved by combining threshold doses of a calcium ionophore and an activator of C-kinase (Kaibuchi et al. 1983). The importance of the inositol lipid pathway is highlighted by the fact that such synergism has been demonstrated in many other cell types including neutrophils (Robinson, Badwey, Karnovsky & Karnovsky, 1984; Dale & Penfield, 1984), adrenal glomerulosa (Kojima, Lippes, Kojima & Rasmussen, 1983), insulin-secreting islet cells (Zawalich, Brown & Rasmussen, 1983), liver (Fain, Li, Litosch & Wallace, 1984), pancreas (de Pont & Fleuren-Jacobs, 1984), smooth muscle (Rasmussen, Forder, Kojima & Scriabine, 1984) parotid (Putney, McKinney, Aub & Leslie, 1984), parasympathetic nerve (Tanaka, Taniyama & Kusunoki, 1984), pituitary (Delbeke, Kojima, Dannies & Rasmussen, 1984), and the lymphocytes mentioned earlier. The second messenger pathways concerned with cell growth are probably very similar if not identical to those used by hormones and neurotransmitters to activate other cellular processes.
So far, attention has been focussed on those growth factors that stimulate inositol lipid breakdown and can thus activate C-kinase and mobilize intracellular calcium. However, there are other growth factors such as insulin and EGF that do not appear to act through the inositol lipids, yet they clearly have a profound effect on cell growth and it is of interest to consider whether they may function indirectly through the bifurcating signal pathway used by other growth factors or whether they have a completely independent mode of action perhaps based on their tyrosine kinase activities. EGF is a particularly interesting case because, apart from an effect on lipid labelling in A431 cells (Sawyer & Cohen, 1981; Smithet al. 1983), it has very little or no effect on inositol phospholipid breakdown in other proliferating cells yet it is capable of inducing changes in calcium and pH that resemble those obtained with other growth factors. Upon closer inspection, however, it is apparent that there are subtle differences in the way in which EGF brings about these ionic events, particularly concerning calcium. While the ability of other growth factors to raise intracellular calcium can occur in a calcium-free medium, the effect of EGF is totally dependent on external calcium (Hesketh et al. 1985). The binding of EGF to its receptors results in entry of external calcium apparently independently of PIP2 breakdown and IP3 formation. Similarly, EGF can stimulate an increase in pH, which again must use some mechanism independent of the C-kinase pathway (Moolenaar et al. 1983; Hesketh et al. 1985). One interesting possibility is that EGF may activate the Na/H exchanger through tyrosine phosphorylation. Such a mechanism might account for the observation that vanadate, which is particularly effective in inhibiting tyrosyl phosphatases, can stimulate an increase in intracellular pH (Cassel, Zuang & Glaser, 1984). Although EGF does not appear to stimulate inositol lipid breakdown it operates, at least in part, by activating the same second messengers operating in the bifurcating signal pathway. The mode of action of insulin is still an enigma. It certainly does not stimulate inositol lipid breakdown yet it acts synergistically with those hormones that do. For example, the concentration of bombesin necessary to stimulate DNA synthesis in Swiss 3T3 cells is greatly reduced if administered in the presence of insulin (Rozengurt & Sinnett-Smith, 1983). A possible basis for such synergism may reside in the observation that insulin can stimulate phosphatidylinositol (PI) synthesis in cells (Fareseet al. 1984), suggesting a site of action before the bifurcation step. Alternatively, insulin could act after the bifurcation point to enhance the activity of one or other of the two signal pathways. This suggestion is based on the observation that insulin can potentiate the effect of growth factors on intracellular pH (Moolenaar et al. 1983) while apparently having no effect on the calcium pathway (Hesketh et al. 1985).
It is clear from the above discussion that we are still a long way from understanding how growth factors such as EGF and insulin contribute to the onset of cell proliferation. However, there are indications that they might fit into the framework provided by the dual signal hypothesis of cell growth. The latter also provides a framework for considering the way in which oncogenes might function to cause uncontrolled cell growth. The hypothesis is that certain oncogenes may amplify various aspects of the inositol lipid pathway so leading to an inappropriate formation of second messengers and uncontrolled cell growth (Berridge, 1984a, b;Sugimoto, Whitman, Cantley & Erikson, 1984; Macara, Marinetti & Balduzzi, 1984; Macara, 1985).
ONCOGENES
As the function of each oncogene is unveiled it is becoming more and more apparent that many of these can be neatly slotted into either the inositol lipid signal pathway or related pathways such as that used by EGF (Fig. 1). The first indication that oncogenes might encode proteins that function in the signal pathways used by growth factors was the discovery that the sis gene produced PDGF (Doolittle et al. 1983; Waterfield et al. 1983), which we now know to be a potent activator of PIP2 hydrolysis leading to the formation of DG (Habenicht et al. 1981), and IP3, calcium mobilization (Berridge et al. 1984), and an increase in the intracellular level of calcium (Moolenaar et al. 1984; Hesketh et al. 1985). TheerfcB gene is similar to the EGF receptor except that the external EGF binding site is missing (Downward et al. 1984). This truncated receptor may lead to the continuous formation of those intracellular second messengers normally associated with EGF, which might partly resemble those generated through the PI mechanism as argued earlier. Various oncogene products that are associated with the membrane have been implicated in the process of signal transduction. The ras gene product might function as a G protein to couple surface receptors to the phosphodiesterase (PDE) that cleaves PIP2 to IP3 and DG (Berridge & Irvine, 1984). By analogy with the adenylate cyclase system, the information transduction activity of the normal ras gene product would presumably be terminated by the hydrolysis of GTP. The absence of GTPase activity in the activated ras gene product (McGrath, Capon, Goeddel & Levinson, 1984; Sweet et al. 1984) suggests that the latter may cause continuous formation of second messengers in the absence of growth factors. The transforming gene products pp60v-src and pp68v-ros both appear to stimulate the phosphorylation of PI and can increase PI turnover in vivo (Macara et al. 1984; Sugimoto et al. 1984). Of particular interest is the finding that similar changes in PI metabolism can be observed in cells transformed with polyoma middle T antigen (Whitman et al. 1985). Since the middle T antigen is known to associate with pp60c-src, which is the normal cellular homologue of v-src, Whitman et al. (1985) have made the intriguing suggestion that middle T might act by stimulating the PI kinase activity in association either directly or indirectly with c-src. Such a model may neatly explain why over-expression of c-src by itself is unable to transform cells whereas mutated v-src can (Parker, Varmus & Bishop, 1984; Shalloway, Coussens & Yacuik, 1984). The c-src protein may normally require some endogenous activator, which can be mimicked by middle T, whereas v-src may have lost this requirement for an activator and becomes constitutive with regard to enhancing PI turnover.
As described earlier, the stimulation of cells with growth factors such as PDGF results in the early transcription of the myc and fos oncogenes (Greenberg & Ziff, 1984; Kruijer et al. 1984; Müller et al. 1984). Since these genes can also be induced by phorbol esters it is conceivable that they may function further down the signal pathway to mediate the second messengers spawned by the cell surface receptors. Since myc and fos encode nuclear proteins it is conceivable that their activation may be part of the nuclear events responsible for initiating DNA synthesis.
CONCLUSION
When growth factors combine with cell surface receptors they entrain a sequence of events that gradually commits the cell to enter DNA synthesis. There is growing evidence that a signal pathway operating through the inositol lipids may provide a way of generating second messengers to convey information from the cell surface into the nucleus. The key event is the agonist-dependent hydrolysis of PIP2 to give DG and IP3, both of which seem to function as second messengers. While it is known that IP3 mobilizes calcium and DG activates C-kinase, which appear to raise pH by stimulating an Na/H exchanger, just how these two signal pathways function to stimulate DNA synthesis is unknown. Whether these ionic events are themselves sufficient to initiate DNA synthesis or whether other signals are required is also a mystery. A possible solution to this problem may emerge as we begin to understand the precise function of each oncogene. At present many of the oncogene products whose functions are being revealed appear to be intimately linked with the inositol lipid signal pathway thus confirming that DG and IP3 might be of central importance in regulating DNA synthesis.