y. Cell Sci. Suppl. 10, 231-242 (1988) Printed in Great Britain © The Company of Biologists Limited 1988
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Feedback regulators in normal and tumour tissues
B. I. LO RD Paterson Institute for Cancer Research, Christie Hospital, Manchester M20 9BX, UK
Summary
Regulation of cell behaviour and population size is presumed to be not unlike classical regulation in non-biological systems, i.e. it is controlled by the cybernetic principle of negative feedback whereby the performance of progenitor cells depends inversely on a signal from their product, the size of which is proportional to the mass of the product. This signal may be inhibitory, acting directly on the progenitor cells. Alternatively, it may operate via an indirect and integrated inhibitor/stimulator feedback loop in which the one influences the production of the other. Illustrations taken from the various phases of haemopoietic development show the operation of these loops. Haemopoietic stem cells are under the direct influence of both inhibitor and stimulator but it is a feedback signal from the stem cell population that dictates the production of the one rather than the other. A second inhibitor acting at the stem cell level is a low molecular weight tetrapeptide which blocks the entry of cells into DNA synthesis, thus protecting them during a regimen of treatment with an S-phase cytotoxic drug. Proliferation of the maturing cells is also inhibited by feedback products of their fully mature descendants. Here, the effect is one of cell cycle modulation, whereas in the stem cell population the inhibitor and stimulator effect an on/off switch. Attempts to characterize the molecules involved have been limited. A series of tri- to pentapeptides has been described for haemopoietic or epithelial cell inhibitors. A common feature of several is a pGlu-Glu end though whether this has any significance is not known. In tumours it has been shown that some ascites are self-limiting and treatment of small tumours with cell-free fluid from a mature growth blocks their further growth. It appears that many tumour cells produce the feedback signals characteristic of their normal counterparts but are themselves less sensitive to it. The same is true of transforming growth factor-/? which is produced and detected by virtually all cell types. In this case, the factor, inhibiting in most cases, is produced in inactive form and achieves its target specificity by a localized capacity to activate it. Some tumours, while responding to exogenous active T G F-¡3 are incapable of activating the latent molecule. It is concluded that the differential sensitivity of normal and neoplastic tissues to physiological feedback regulators is a potentially exploitable property in cancer therapy. Principles of feedback regulation
The cybernetic principle of negative feedback regulation to maintain a system in a steady state is one which has always been exploited by man, intuitively or systematically, to control life’s inventions. Science, rather belatedly, is coming to recognize that the same principle can operate physiologically to maintain cell populations. Thus, while the speed of Watt’s steam engine needed to be regulated by a govenor which reduced the input of steam in proportion to its speed, so the production of cells in a biological system needs to be limited by a feedback message(s) which depends on the size of the resultant population. Weiss & Kavenau (1957) introduced a theoretical concept of templates acting as catalysts for the Key words: feedback regulators, normal tissue, tumour tissue, haemopoiesis, stem cells.
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promotion of a process and antitemplates, produced by the product, to limit their function. When Bullough (1962) attempted to demonstrate the concept of feedback inhibition (the chalone hypothesis) in a biological system, however, it was not well received, mainly because of the difficulty of providing convincing ‘negative’ experimentation. Nevertheless, several groups did take the principle seriously and within a few years, inhibitors were described for epidermal and about a dozen more types of cell proliferation (see Forcher & Houck, 1973). In subsequent years, interest waned, probably due to the problems of obtaining pure factors. In 1979, Allen and Smith, reporting on attempts to purify the lymphocyte chalone demonstrated that its estimated molecular weight tended to be less with each successive publication and a graph of molecular weight plotted against the year of estimation would effectively have predicted its total disappearance by about 1982. They were not far wrong. Nevertheless, notably in haemopoietic and epithelial tissues, considerable efforts have been made to elucidate the significance of negative feedback processes in the regulation of cell proliferation, and cell population mass. Fig. la illustrates the simplest form of negative feedback regulation. The product, P, elaborates an inhibitory factor, I, to act directly on the generator, G, and thus limit its output. It may be noted that the principle of positive feedback, whereby more leads to even more which leads to ...ad infinitum is not a tenable concept in a control process. However, stimulatory processes may be involved as seen in Fig. lb and lc. In Fig. lb, the inhibitory factor operates via a ‘black box’ to limit the output of a stimulatory factor. Alternatively it is also possible that the product factor may act as a stimulator of inhibitor production (Fig. lc). Negative feedback regulatory loops in haem opoiesis
Stem cells Haemopoietic stem cells are conventionally assayed using the spleen colony technique described by Till & McCulloch (1961) and should strictly be identified by B LACK BOX
11 A W
I
BLACK BOX
Fig. 1. The principle of negative feedback regulation. P is the product of a generator, G. I and S represent inhibitory and stimulatory processes.
Feedback regulation in tissues 233 their functional properties as CFU-S (colony forming units in the spleen) or spleen colony forming cells, CFC-S. As a population, in vivo, at least some of these cells are self-maintaining and multipotent in their capacity for differentiation. However, there is considerable heterogeneity in the population and it now seems clear that some, having a higher individual self-renewal capacity, are developmentally younger than others (Schofield, 1978; Schofield et al. 1980; Magli et al. 1982). There is now some question as to whether the most primitive stem cell can itself develop as a spleen colony or whether it exists as a pre-CFU-S (Hodgson & Bradley, 1979). Neverthe less, work on stem cell regulation has been conducted on this CFC-S population and two proliferation inhibitors, specific for it, have been described (Lord et al. 1976; Frindel & Guigon, 1977). The first of these inhibitors is prepared by washing fresh, normal bone marrow (in which the majority of the CFC-S population is proliferatively quiescent) and obtaining a conditioned medium which contains the inhibitory activity. The background to this approach was laid by observations on mice that had been heavily irradiated but with one hind limb shielded (Gidali & Lajtha, 1972). In these experiments, it was found that the proliferative behaviour of CFC-S in the shielded and unshielded limbs is independent, leading to the conclusion that control is exercised locally. Rencricca et al. (1970) obtained a similar imbalance when following recovery from phenylhydrazine-induced anaemia. In their experiments, CFC-S migrated to the spleen and remained predominantly non-proliferating while increasing in number approximately fivefold. By contrast, a reduced number of CFC-S in the marrow was induced to proliferate rapidly. Again, localized CFC-S proliferation control was indicated. The conditioned medium obtained from normal marrow, used either directly or as an Amicon Diaflo, semipurified and freeze-dried fraction (Lord et al. 1976; Wright & Lord, 1977), is found to block the entry of CFC-S into the DNA-synthesis phase of their proliferative cycle. Furthermore, this inhibition is confined specifically to the CFC-S population. It has no effect on the mixed in vitro colony-forming cells (multipotential cells considered to occupy the mature end of the CFC-S age spectrum) or on the committed progenitors of granulocytes, macrophages or erythroid cells (Lord et al. 1976; Tejero et al. 1984). This inhibitor is not detected in marrow containing rapidly proliferating CFC-S but, by contrast, a stimulator is present (Lord et al. 1977a). Prepared and assayed in a similar way to the inhibitor, this stimulatory activity is capable of triggering cells from the Go-phase directly into DNA synthesis and it, too, is specific for CFC-S (Tejero et al. 1984). A search for the cells which produce these inhibitory and stimulatory activities, using sorting techniques which included density separation, plastic adherence and fluorescence activated cell sorting, showed that two distinct subpopulations of macrophages are responsible, the one for inhibitor and the other for stimulator (Wright et al. 1980, 1982; Simmons & Lord, 1985). Furthermore, although only either one of these activities can be detected in any source of haemopoietic tissue and irrespective of the proliferative status of CFC-S in that tissue, both types of producer cell are always present (Wright & Lord, 1979).
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Although only inhibitor or stimulator is produced at any one time, it was shown that each type of macrophage, on separation from the other, is capable of producing its specific factor (Wright & Lord, 1979). In addition, although the two factors are not mutually destructive, the presence of one blocks the synthesis of the other (Lord & Wright, 1982). A final feature of this interaction between inhibitor and stimulator is that while their effects are reversible, the proliferation reversion is not automatic on removal of the activity. The presence of the opposing factor is required to effect this proliferation switch (Lord et al. 1977a). In other words, the movement of CFC-S between a non-proliferative Go-state and cell cycle requires an On/Off switching mechanism, two separate operations which appear to be provided by the inhibitor and stimulator. Fig. 2 thus summarizes these observations. The CFC-S population generates a range of haemopoietic cell lineages, including the macrophage subpopu lations which produce inhibitor and stimulator. The inhibitor arm is identical to the mechanism illustrated in Fig. la. By contrast, since the stimulator on its own cannot act as a control mechanism, this arm must be considered a variation on that shown in Fig. lb and require a feedback regulator to limit its production. Furthermore, since the production of stimulator is limited to a subpopulation of macrophages w'hich is only one of many lines of CFC-S progeny, it is unlikely that this feedback can come from anywhere other than the mass of the CFC-S population itself. Thus, the macrophages become the black box in Fig. lb and the inhibitor, in this process, becomes a feedback factor from the CFC-S population (Fig. 3). By separating out a pure population of CFC-S (Lord & Spooncer, 1986) and treating a stimulator-producing bone marrow with the CFC-S or an extract from them, it was possible to demonstrate the existence of this feedback signal (Lord,
Range of haemopoietic cell lineages
Fig. 2. Inhibitory (I) and Stimulatory (S) factors, generated by two macrophage (M(/>) subpopulations, influencing the proliferation of spleen colony forming cells (CFC-S).
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Range of haemopoietic cell lineages
Fig. 3. The spleen colony forming cell (CFC-S) feedback factor (FBF) which deter mines the relative production of inhibitory (I) and stimulatory (S) factors (see Fig. 2).
1986). Furthermore, by separating the inhibitor-producing macrophages from the bulk population, it was shown that the effect of this feedback signal was directly to block stimulator production, rather than to induce inhibitor production. The second inhibitor of CFC-S proliferation (Frindel & Guigon, 1977) was obtained as a dialysate from frozen bovine calf marrow and subsequently from frozen bovine foetal liver. This activity has been tested largely in vivo and was shown to block the recruitment of CFC-S from Gq into cell cycle, a change which is normally induced by injection(s) of cytosine arabinoside (Guigon & Frindel, 1978; Guigon et al. 1980). It was originally considered to be specific for the CFC-S in the same way as the other inhibitor but recently, in its more purified form, some of this specificity appears to have been lost (Guigon, 1987), neonatal hepatocyte proliferation being inhibited, for example (Lombard et al. 1987). This group also reported stimulatory activity in damaged marrow (Frindel et al. 1976) but has not directly investigated any mechanism of action and interaction between the two activities. Nevertheless, they have, most importantly, demonstrated that the protection afforded by prevent ing CFC-S recruitment into cell cycle increases the survival from normally lethal single and multiple doses of S-phase cytotoxic agents (Guigon et al. 1982; Wdzieczak-Bakala et al. 1983). Committed and maturing cells While strictly outside the boundaries of stem cell regulation, feedback processes undoubtedly exist also in the more mature, single lineage compartments of haemopoietic tissue and, as will be shown below, may have some bearing on the processes involved at the stem cell level. In 1968, Rytomaa & Kiviniemi (1968a)
B. I. Lord reported an inhibitor of myelocyte proliferation present in medium conditioned by mature granulocytes. By a totally independent method, that of cell cycle-associated changes in fluorescence polarization (Cercek et al. 1973), it was shown that such conditioned media were able to induce changes specifically in the proliferating myelocytic cells (Lord et al. 1974a). Subsequently it was shown to reduce both the tritiated thymidine ([3H]dThd) autoradiographic labelling index of myelocytes when injected into mice (Lord, 1975) but had little or no effect on their committed progenitors (Lord et al. 1977). Unlike the On/Off mechanism operating for CFC-S, a mechanism perhaps reserved for cells which under normal steady-state conditions reside in an out-of-cycle Go-state, this inhibitor was reversible simply by washing it out (Lord et al. 19746). From repeated [3H]dThd-labelling experiments it was shown that the inhibitor merely reduced (not blocked) the rate of flow of cells into DNA-synthesis, thus limiting output by lengthening the Gi-phase of the cell cycle (Lord, 1975). Maurer et al. (1978) found that similar preparations did in fact prevent colony formation in vitro by the granulocyte/macrophage committed progenitors (GMCFC) and following its purification (Paukovits & Laerum, 1982) there is now some evidence that it may have an inhibitory effect on CFC-S similar to that described by Guigon (1987). It appears therefore, that this activity may affect a wider spectrum of cell stages, perhaps in a dose-dependent manner similar to that of some of the growth factors. As will be discussed below, this material is a pentapeptide but it is only in its monomeric form that it is inhibitory to GM-CFC. The oxidation product of it, however, is a dimer and has been found to be stimulatory. Laerum & Paukovits (1987) speculated that the producer cells, the granulocytes, through their strong oxidizing and reducing capacity (Weiss et al. 1983; Watanabe & Bannai, 1987) can maintain an unstable equilibrium between the monomer and dimer, which may bring about a rapid and efficient modulation of granulopoiesis. In parallel with this inhibitor(s) of granulopoiesis, two erythroid factors have been described. Kivilaakso & Rytomaa (1971) reported one obtained from mature erythrocytes and its activity was confirmed by the fluorescence polarization technique (Lord et al. 1974a) and autoradiography (Lord et al. 1977b). Its reversibility and mechanism of action appeared to be very comparable with that for the granulocyte extract. A second inhibitor, described by Axelrad and his colleagues (1987) was obtained from normal bone marrow. It appears to act very rapidly to block the erythroid committed burst-forming unit, BFU-E, in the S-phase of cycle. The blockage, however, is reversible, equally rapidly, on removing the factor. The best recognized regulator of erythropoiesis, however, is erythropoietin, EPO. Although EPO promotes rapid proliferation of maturing normoblasts, its primary function appears to be the induction of haemoglobin synthesis so that cells can proceed to maturity. Nevertheless, as a stimulating regulatory molecule, its concentration, varying with the demand for erythropoiesis, is dependent on negative feed back. Although indirect in its action, EPO is produced by the kidney in inverse proportion to the oxygen tension developed there by the red cell mass. 236
Feedback regulation in tissues 237 Lactoferrin and acidic isoferritins have been implicated as further feedback regulators of GM-CFC growth and development (Broxmeyer et al. 1978, 1982). Elaborated by the mature granulocyte macrophage populations, they are reported to block the production of the appropriate colony-stimulating factors for GM-CFC development. Although they appear to be active at very low concentrations (10-15 M or less) their role is somewhat controversial because of the relatively high concentrations normally found in vivo (Rich & Sawatzki, 1987). Characterization of feedback regulators
The chemical characterization of inhibitory molecules is very patchy and is limited to those shown in Table 1. Paukovits and his colleagues (1987) have isolated and characterized a pentapeptide as the so-called granulocyte chalone. Its structure differs in only one group from another pentapeptide inhibitor, that of epidermal keratinocyte proliferation (Elgjo et al. 1986), and also bears a striking similarity to a tripeptide with cell cycle inhibitory effects in the colon (Skraastad et al. 1987). Whether the PyroGglu component occurring at the end of each of these peptides has any common mechanistic significance is unknown. The inhibitor of CFC-S described by Guigon & Frindel has recently been defined as a tetrapeptide (Lenfant et al. 1987) but has no structural similarity with the pentapeptide structure. This is perhaps surprising since Guigon, working also with the pentapeptide material, has suggested that it has similar properties to their own tetrapeptide (Guigon, 1987). To date, there is no information on the proteinaceous inhibitors of CFC-S and BFU-E. Both have reported molecular weights in the range of 50K to 100K daltons (79000 for the BFU-E inhibitor) (Axelrad et al. 1987) and both are inactivated by trypsin. Currently they are both considered as glycoproteins but clearly they are still components of a fairly generalized ‘soup’. Feedback inhibitors in tumours
The fact that tumour cell populations grow without apparent regard for the normal feedback processes raises the question whether tumour development is the result of Table 1. Peptide inhibitors of cell proliferation
Peptide structure PyroGlu-Glu-Asp-Cys-Lys
Target cells Myelocytic? GM-CFC CFC-S
Author Paukovits et al. (1987)
PyroGlu-Glu-Asp-Ser-Gly
Keratinocytes
Elgjo et al. (1986)
PyroGlu-His-Gly Lys-Pro-Asp-Ser
Colonic epithelial cells
Skraastad et al. (1987)
CFC-S Neonatal hepatic cells
Lenfant et al. (1987) Lombard et al. (1987)
B. I. Lord their failure to recognize the feedback signals and, if so, can this failure be exploited to improve therapy for the condition? There is evidence that some ascites tumours, at least, can be self-limiting in their growth. For example, Bichel (1972) showed that the hypotetraploid ascites tumours (JB-1 and Erlich) each grow to a maximum size of 109 cells. The growth of each was quite independent because, grown together in one mouse, they reached a total of 2X109 cells. However, each could be limited independently by its own cell-free ascitic fluid (see Fig. 4). Treatment of a mouse bearing one of the tumours with cell-free fluid from the other tumour did not affect the flow of cells through mitosis. By contrast, cell-free fluid from a tumour of the same type did block entry to mitosis (see Bichel, 1972). Thus, a highly specific tumour product appears to be acting as a feedback regulator for that tumour. It seems probable, however, that the sensitivity of a tumour cell to the inhibitory factor is lower than that of its normal counterpart. Rytomaa & Kiviniemi (19686) found that Shay myelocytic chloroleukaemia cells generate large quantities of the granulocyte feedback inhibitor, release it very rapidly but are themselves consider ably less sensitive to its effects. Nevertheless, regression of this tumour was obtained by large-dose treatments with granulocyte chalone (Rytomaa & Kiviniemi, 1969). In a similar way, interleukin-3 (IL-3)-dependent, haemopoietic stem cell lines are inhibited, in a dose-related manner, by the bone-marrow extract described by Lord et al. (1976). A spontaneous leukaemic, IL-3-independent derivative of one of these lines, however, was highly resistant and continued to undergo rapid proliferation (Lord et al. 1987). In addition, Friend virus-induced polycythaemia was unrespon sive to the negative regulatory protein for BFU-E (Axelrad et al. 1987). Normal and neoplastic epithelial cells, too, appear to have a differential response to the epidermal inhibitory pentapeptide. Used in a dose range of 10“ 10 to 10-4 M, the autoradiographic labelling index of mouse tongue keratinocytes was reduced by an average of 28 %. That for a squamous carcinoma cell line (SCC-9) was reduced by only 3 % (Professor W. J. Hume, personal communication). Guigon & Frindel (1981) studied the effects of the haemopoietic tetrapeptide 238
Fig. 4. Mitotic activity of ascites cells (type A) treated with cell-free ascitic fluid from type A or type B tumours. Adapted from Bichel (1972).
Feedback regulation in tissues 239 inhibitor in mice bearing EMT6 mammary-derived tumours. The inhibitor had no effect on the tumour response to treatment with cytosine arabinoside and the number of survivors was somewhat increased (Guigon & Frindel, 1981; Guigon et al. 1986; Guigon, 1987). Transforming growth factor-/? (TGF-/J)
Owing to its ubiquitous appearance in many tissues, TGF-/3 has become a much investigated molecule (see review by Sporn et al. 1987). It is a polypeptide of 25 000 molecular weight and virtually all cell types both produce and bear receptors for it. In addition, its effects may be either stimulating or inhibitory, depending upon the cell type it encounters. Although its mechanisms of action are still somewhat speculative, TGF-/3 is being given serious consideration as a widely operative and physiologically regulatory molecule. Virtually all cells produce TGF-/3 in an inactive or latent form and it has been suggested that target specificity - one of the most important considerations for cell population control - may be critically determined by the ability of a cell to activate the latent complex. For example, unregulated epithelial growth may be the result of failure to activate the latent form of its autocrine feedback TGF-/3. Thus a human A549 lung carcinoma cell, which has abundant TGF-/3 receptors and secretes large amounts of inactive TGF-/3, continues to proliferate unless exogenous active TGF-/3 is made available. In contrast with the normal parent cell type, which is inhibited by the TGF-/3 that it generates, it appears that the tumour cell has lost its ability to activate the latent molecule. Osteoblasts, on the other hand, are stimulated by TGF-/3 and it has been found that osteosarcoma cells respond similarly to exogenous material (Pfeilschifter et al. 1987). It appears that bone remodelling is under autocrine feedback control, its highly acidic microenvironment providing a mechanism for activation of the latent TGF-/3 produced and secreted by the osteoblasts (Sporn et al. 1987). Conclusion
,
The differential sensitivity of normal and neoplastic tissues to normal physiological feedback regulators is potentially an exploitable property in cancer therapy. Not only should it be possible to protect the bone marrow while treating distant tumours as in the experiments with EMT6 tumours but also, particularly with bone marrow, the relative insensitivity of tumour tissue compared to its normal counterpart, suggests that this same protection could be utilized when treating malignancies of the same origin. A greater understanding of the mechanisms of TGF-/3 action and activation would appear to be necessary before its widespread effects can be harnessed to modify the performance of specific cell types. This work was supported by a grant from the Cancer Research Campaign.
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