.I. theor. Biol. (1977) 68, 101-137

Regulation of Cell Division and Malignant Transformation: A New Model for Control by Uptake of Nutrients PUSHPA M. BHARCAVA Division of Biochemistry, Regional Research Laboratory, Hyderabad 500 009, India (Received 13 July 1976, and in revisedform

21 February 1977)

A comprehensive model of regulation of normal growth and of malignant transformation is presented. The model postulates four chemical (f, Anti-Z, SF1 and SF2) and two structural (Sites A and Sites B) entities. Sites A and B are functionally different transport sites on the membrane for essential nutrients. Sites A are open in resting cells and need a serum factor, SFl, for operation at V,,,. Sites B are closed in resting cells but open in dividing cells; the “on+off” control of Sites B is achieved through Z, a protein with a high rate of turnover and two binding sites. Sites B are closed when Z is bound to them; the affinity of Z for Sites B increases when one molecule of I links two Sites B. A second serum factor (SF2), Anti-Z when released from the cells (e.g. as a result of tissue damage), and other external triggers for cell division (such as mitogenic hormones), destroy or inactivate f, or prevent its binding to Sites B. The opening of Sites B results in an enhancement of the rate of uptake of nutrients; the resulting increase in the intracellular concentration of one or more of the nutrients starts the programmed operation of events that culminate in cell division; two possible mechanisms for the initiation of this programme are suggested. Growth ceases as a consequence of re-establishment of I function on the membrane. Malignant transformation is defined as an inheritable intracellular event, spontaneous or induced, which interferes with the production or activity of Z and leads to a loss of the capacity for transition from the dividing to the resting state. Likely candidates for the various entities proposed are listed.

1. Introduction and the Rationale A model is presented here that attempts to explain regulation of normal cell division in terms of control of the uptake of essential nutrients; implicit 101

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in the model is also an explanation of malignant transformation. The model derives primarily from the following considerati0ns.t (1) All cell types in which the phenomenon of malignancy is encountered are auxotrophic for certain organic nutrients (the “essential nutrients”), and cannot grow on a single carbon source. For example, mammalian cells need, amongst other substances, about 10 amino acids for growth. (2) All cells which can be transformed malignantly can exist in two states : the resting (G,) state (Baserga, Costlow & Revera, 1973; Augenlicht & Baserga, 1974) and the dividing (G, + M) state. Resting cells may not divide--even under optimal nutritional conditions-for a prolonged period during which they continue to be functional; to enter the division cycle, they need to be triggered by an agent which does not serve as a nutrient. Examples of resting cells are the majority of parenchymal cells in adult mammalian liver, and cells in density-inhibited, non-growing cultures. A distinction, therefore, needs to be made for such cells between (a) a triggering agent required for initiation of growth; (b) auxiliary growth factors (such as carriers for certain nutrients, spreading factors, etc. [Holley, 1974; Gordon & Brice, 19741) which perform a defined function distinct from initiation of growth in specified cell types under given conditions, and which are not used for increase in cell mass; and (c) nutrients which undergo metabolic transformation in the cell and are used for increase in cell mass during growth. A triggering agent must be present for every cell cycle to begin but may not be required for the continuance of the cell cycle through its later stages, e.g. the S, Gz or M phase (Harding, Wilson, Wilson, Reddan & Reddy, 1968; Burk, 1970; Otsuka & Moskowitz, 1976; Hovi & Vaheri, 1976). (3) The essential nutrients need to be taken up by the cell for the maintenance of both the states, the resting and the dividing. (4) The rate of uptake of essential nutrients-specially those which arc needed for macromolecular synthesis-in dividing cells is several-fold higher than that in resting cells (Weber & Edlin, 1971; Weber & Rubin, 1971; Bose & Zlotnick, 1973; Plagemann, 1973; Harel, Jullien & Blat, 1975; Bhargava, Allin & Montagnier, 1976a; Bhargava & Vigier, 1976; Bhargava, Szafarz, Bornecque & Zajdela, 1976b; for other references, see Shodell & tThe references cited are only illustrative and not exhaustive. The rates of uptake or transport (the two terms are used interchangeably) of essential nutrients referred to here, are the nef rates, that is, they represent the difference between the rate of influx and the rate of efflux. The terms “transformed” and “malignant” are also used interchangeably, even though it is recognized that-under certain circumstances-the two phenomena could be separated (the same cell, for example, may or may not exhibit malignancy when injected into an animal, depending on the method of injection). The term “normalcy” is used to designate a state of cells in which they are not maligant or transformed; no distinction is made between diploid and aneuploid cells, or between immortal cell lines and cell lines which show a finite life span.

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lsselbacher, 1973). We have, for example, shown that (a) the rate of uptake of essential amino acids when used at extracellular concentrations required for growth, in synchronously growing BHK cells nearing the end of the division cycle is, on an average, eight times higher than in the resting cells (Bhargava et al., 1976~); (b) the rate of uptake of these amino acids in logarithmicalfy growing chick embryo fibroblasts is about five times higher than in the non-dividing density-inhibited cells (Bhargava & Vigier, 1976); and (c) the uptake (per mg acid-insoluble material) of these amino acids in Zajdela ascitic hepatoma cells in which the mitotic activity is high is, on an average, 3.4 times more than in normal adult liver cells in which the mitotic activity is low (Bhargava et al., 1976b). (5) Increase in the rate of uptake of essential nutrients such as amino acids and ions, is one of the earliest events to occur when a resting (G,) cell is triggered into the division cycle (Baserga et al., 1973; Costlow & Baserga, 1973; Augenlicht & Baserga, 1974; Plagemann & Richey, 1974; Parker, 1974; and the references that follow in this paragraph). This increase appears to be independent of the nature of the triggering agent and has been, for example, observed in the case of (a) an asparagine-requiring strain of BHK cells initiated into the division cycle from a simulated resting phase by asparagine (Bhargava et al., 1976~); (b) lymphocytes triggered by phytohaemagglutinin (PHA) or concanavalin A (Con A) (Peters & Hausen, 1971; Averdunk, 1972; Kay, 1972; Whitney & Sutherland, 1972, 1973; Vandenberg & Betel, 1973a,b, 1974a,b); (c) a variety of target cells triggered by mitogenic hormones such as growth hormone (Hjalmarson & Ahren, 1965; Drezner, Eisenbarth, Nellon & Lebovitz, 1975), insulin (Guidotti, Borghetti, Luneburg & Gazzola, 1971; Goldfine, Gardner & Neville, 1972), estrogens (Smith & Smith, 1971; Riggs & Pan, 1972),ecdysone(Kroeger, 1966)and erythropoietin (Hrinda & Goldwasser, 1969); (d) liver cells triggered by partial hepatectomy (Ferris & Clark, 1972; Ord & Stocken, 1972); and (e) density-inhibited cells in a monolayer culture initiated into cell division by a proteolytic enzyme (Sefton & Rubin, 197 1; Kubota, Ueki &Shoji, 1972; Mallucci, Wells &Young, 1972; Teng & Chen, 1975), neuraminidase (Vaheri, Ruoslahti & Nordling, 1972), serum (Cunningham & Pardee, 1969; Pariser & Cunningham, 1971; Sefton & Rubin, 1971; Hare, 1972a,b; Grimes & Schroeder, 1973; Lemkin & Hare, 1973; Bradley & Culp, 1974; Deasua & Rozengurt, 1974; Deasua, Rozengurt & Dulbecco, 1974; Kletzien & Perdue, 1974; Rubin & Koide, 1975; Jullien & Harel, 1976), or a polypeptide hormone or growth factor (Hollenberg & Cuatrecasas, 1975; Rubin & Koide, 1975; Hovi & Vaheri, 1976). (6) The primary event which initiates a resting cell into the division cycle appears to occur at the cell surface (for references, see Everhart & Rubin,

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1974). In support of this view, PHA and pokeweed lectin which stimulate division in lymphocytes and density-inhibited chick embryo fibroblasts (Vaheri, Ruoslahti, Sarvas & Nurminen, 1973) respectively, proteolytic enzymes which can initiate division in a variety of non-growing cell cultures (Burger, 1970; Ceccarini & Eagle, 1971; Yamamoto, Omata, Ohnishi & Terayana, 1973~; Noonan & Burger, 1973~; Kaplan & Bona, 1974), and certain mitogenic hormones such as growth hormone and insulin, do not enter the cell (Turkington, 1970; Burger, 1971~; Greaves & Bauminger, 1972). Surface stimuli can apparently generate the signal for DNA synthesis (Mallucci, 1972; Vittorelli, Cannizzaro & Giudice, 1973), and a genetic cell membrane anomaly has been reported to impede cell division (Verwilghen, Tan, Dewolf-Peeters, Orshoven & Louwagie, 1971). (7) Surface changes appear to be obligatory to, and precede, the expression of the malignant phenotype. Thus, virally-and, presumably, chemicallytransformed cells acquire Con A-agglutination sites, tumour-specific transplantation antigens (TSTA) and other cell surface changes before expression of the malignant phenotype (Gurtsevich, Mazurenko, Jarova, Probatova & Stepanova, 1970; Zarling & Tevethia, 1971; Ambros, Chen & Buchanan, 1975; Peitras & Szego, 1976). When normal cells are infected with temperature-sensitive mutants of oncogenic viruses, they exhibit normal cell-surface properties-e.g. in regard to antigenic behaviour-at non-permissive temperatures, and altered cell-surface properties characteristic of malignant cel1s-e.g. agglutinability with Con A and increased rates of uptake of nutrients-at the permissive temperature (Eckhart, Dulbecco & Burger, 1971; Burger & Martin, 1972; Noonan, Renger, Basiliev & Burger, 1973; Kletzien & Perdue, 1974). Further, the less tumorogenic mutants of adenovirus 12 are defective in the induction of cell surface changes (Yamamoto, Shimojo & Hamada, 1972), and in a study of hybridization of normal with malignant cells, a parallel correction of malignancy and of a genetic cellsurface defect in cell-to-cell communication has been observed (Azarnia & Loewenstein, 1973). The above observations suggest that the inability of resting cells to take up essential nutrients at rates required for cell division, may be due to a functional block at the cell membrane, and that triggers for cell division may act by removing this block. The model that follows is based on this premise. 2. A New Model for Regulation of Normal Cell Division

The model, schematically propositions.

presented in Fig. I, comprises the following

CELL

1 is speaflc for the cell type 8 but the some for all Sites on the some cell type. Its blnding constant may vary from celt system to cell systern, but is generally low. It turns over rapldly.

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o) Closed when 1 IS ‘0~’ (OS shown here) ( b) Respond to (1 trigger for cell dlwslon (e g SF2) which removes or wxtlvatesl EdI @pen when I’ 15 ‘off

_ -5tays !n !he cell; released on cell death, mactlvotlng I on other more stable thon I

LetI ,,

blocked.eo.bvi

FIG. 1. A model for the regulation of cell division through control of uptake of essential nutrients. EN1 and EN2, essential nutrients: Sites Al and A2, membrane sites for the uptake of various ENS in resting cells (these sites may also be open in dividing cells); Sites Bl and B2, membrane sites for the uptake of ENS in dividing cells, closed in resting cells; rate x, the maximal rate (V,,.J of uptake of an EN through a Site A; rate y, the maximal rate of uptake of an EN through a Site B, probably between 5x and 10x (see text); SFI, a serum factor necessary for transport of an EN through Site A at the maximal rate: I, an inhibitor of transport through Sites B, which comes out of the cell and acts from outside. Anti-I, an intracellular factor, functionally antagonistic to I and normdy incapable of coming out of the cell; SF2, a serum factor functionally antagonistic to I. The main figure shows a resting cell. Sites B (inset left) are postulated to consist of three subsites, named Type I, Type II and Type III receptors; morphological overlap of these receptors is not ruled out.

(1) Two types of uptake of essential nutrients are distinguished functionally: (a) the uptake obtained in resting cells (the “Type A” uptake); and (b) the uptake obtained in dividing cells over and abow the uptake in resting cells (the “Type B” uptake). Type B uptake of a nutrient proceeds at a rate several times higher than the rate of Type A uptake of the same nutrient; the two types of uptake occur through two functionally different sites on the cell membrane, called “Sites B” and “Sites A” respectively. A separate set of Sites A and B may exist for each essential nutrient, or the same set of sites may be used by a group of nutrients. (2) Sites A are open constitutively and are the only sites open for the uptake of nutrients in resting cells. However. for the uptake of an essential nutrient to occur at the maximal rate (V,,,) through its Sites A, a serum factor, SFI, which is not specific for the cell type, is required. (3) Sites B are blocked in resting cells by an inhibitor (I) of Type B uptake synthesized by the cell for translocation to the cell surface. Z is a protein

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the structure of which is prescribed by a single, non-repeated set of genes. Z can bind to all the Sites B for the various essential nutrients but exhibits histogenetic specificity, that is, it is specific for the type of cell which makes it. (It is not crucial to the argument whether or not Z is specific for the species.) Z has a short half-life and its affinity for Sites B is low. It is, however, bivalent, and can bind to two Sites B belonging to the same cell or to adjacent cells; when this happens, it is bound more tightly to Sites B than whenconly one of its valencies is utilized. A Site B (Fig. I, inset) is postulated as*a unit of structure on the cell surface, in which are integrated three functionally distinct entities: (i) a receptor (“Type I”) for Z or other growth-inhibitory substances acting at the cell surface; (ii) a receptor (“Type 11”) for growthstimulatory (mitogenic) substances which act at the cell surface; (iii) a receptor (“Type III”) for the substrate to be transported. The transport machinery is postulated to act through the last-mentioned sub-site (i.e. the Type III receptors). Type III receptors are operative only when the adjacent Type I receptors are unblocked; this unblocking may be achieved by destruction or inactivation of Z, or by blocking of the adjacent Type II receptors by growth-stimulatory substances. (4) An agent that triggers resting cells into division, destroys, inactivates or modifies Z, or prevents its binding to the membrane by binding itself to Type II receptors; in all such cases, Type III receptors would be unblocked and, consequently, Sites B opened. One such trigger is proposed to be a second serum factor, SF2, which can inactivate all Is for the various cell types; SF2 may be, therefore, in contrast to Z, made by just one type of cells in the organism. SF2 does not act on Sites A, nor SF1 on Sites B. V,,, for Sites B for an essential nutrient in the presence of SF2 is postulated to be greater than V,,, for Sites A for the same nutrient in the presence of SFl, by a factor equal to the ratio of the rate of uptake of the nutrient in the dividing cell to that in the resting cell. (5) The opening of Sites B and the resulting increase in the rates of uptake of the nutrients, are the crucial early events common to all triggering agents, which occur when a resting cell is triggered into the division cycle; these events control further progress of the cell through its division cycle. Unblocking of Sites B is thus akin to the putting on of the “switch” for a computer programme: once the unblocking has occurred, all subsequent events leading to cell division follow a predetermined sequence. (6) The cell also produces a substance called Anti-Z which can inhibit, inactivate or displace Z from Sites B. Z, however, is not rendered ineffective by Anti-Z ordinarily, as Anti-Z is not secreted under normal circumstances and, within the cell, Z and Anti-Z may not “see” each other. Z, a secreted protein, is likely to be synthesized at sites which are different from those

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used for synthesis of non-secreted proteins; apparently, proteins (like serum albumin) that are secreted by cells of higher organisms are synthesized on membrane-bound ribosomes and sent out of the cell directly from the site of synthesis, whereas those staying in the cell are synthesized on free ribosomes (Redman, 1969). Anti-Z, like Z, is not specific for a Site B or for an essential nutrient, but is specific for the cell type; in the latter respect, it differs from SF2 which it resembles functionally. Each Z can, therefore, be “neutralized” only by a specific Anti-Z. Anti-Z is more stable than Z, and can come out of the cell under special circumstances (discussed later) when it unblocks Sites B and triggers cell division. The key postulates of the model, therefore, are: (a) two different types ot sites, Sites A and Sites B, for the uptake of essential nutrients, one type (Sites A) open constitutively in resting (or in resting and in dividing) cells, and the other type (Sites B) open only in dividing cells; (b) a cell-type-specific factor, Z, that acts at the surface of the cells synthesizing it and inhibits transport through Sites B only; (c) another cell-type-specific factor, Anti-Z, that normally stays in the cell but can, when it comes out, nullify the effect of Z and open up Sites B; and (d) two serum factors, SF1 and SF2, that provide for positive regulation of Sites A and B, respectively. It is proposed that all triggers for cell division act in the manner suggested for SF2, that is, they interfere with the negative control exercised by I; the consequent opening of Sites B and the influx of nutrients, it is suggested, starts a programmed sequence of events which culminate in cell division. Several earlier workers (Pardee, 1964; Amos, 1968; Cunningham & Pardee, 1971; Holley, 1972) have also proposed that control of growth may occur through regulation of the uptake of nutrients; no detailed model, however, appears to have been presented so far. 3. The Model Explains Various States of Cells Different states of cells of higher organisms--embryonic, normal (adult) resting, normal (adult) dividing, and malignant-and the transitions normally encountered from one state to the other, can be explained by the model in terms of control of Z function. Ii phenotype would imply that Sites B are closed, and I- phenotype that Sites B are open. When Sites B are open and enough nutrients (and auxiliary growth factors (Gordon & Brice, 1974)) are present in the extracellular environment, resting (G,) cells would proceed through Gr to the M phase and then from one cell cycle to the other, without going into the resting phase in between two division cycles. When Sites B are closed (I+ phenotype), cells will normally stay arrested in the GO phase and not enter G, even if nutrients (and auxiliary growth factors) are available.

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Resting cells would thus exhibit a I’ phenotype, whereas all dividing cells (embryonic, adult dividing, or malignant) would show a I- phenotype; the various types of dividing cells would differ one from the other in regard to the causeleading to the Z- phenotype. I shall now consider certain specific transitions. TABLE 1

The proposed status of I and of Sites B in various types of cells Type of cell Embryonic Normal resting Normal (adult) dividing (a) viral carcinogenesis (b) chemical and other types of carcinogeneses, not involving an endogenous virus

Phenotype with respect to I function

Genotype with respect to I function

Status of Sites B

II+ I-

I+ I’ I+

Open Closed Open

I-

I’

Open

I-

I-

Open

(A) EMBRYONIC CELLS AND THEIR TRANSITION TO THE ADULT RESTING STAGE

The suggestedI- phenotype of embryonic cells could be a result of one of the following situations: (a) embryonic serum may contain a high concentration of SF2; or (b) production and release of active Z by the cells may not begin, or Anti-Z may be releasedfrom the cells, until they are fully differentiated and become adult resting cells. During the transition from embryonic to the adult resting stage, the normal process of differentiation could lead to an I+ phenotype through either a lowering of the concentration of SF2 in serum, or formation of functional Z, or inhibition of the release of Anti-Z. Alternatively, the higher cell density normally obtained in adult tissuesin comparison to embryonic tissues, could allow both the valencies of Z to be satisfied which, as already postulated, would lead to an increase in the affinity of Z for Sites B; this increase could reduce the susceptibility to Z to destruction or inactivation by SF2 [seesection 3(c)]. (B) TRANSITION FROM NORMAL RESTING CELLS TO NORMAL DIVIDING CELLS

Such a transition could be explained by a loss of Z function and the consequent opening of Sites B. Three examples may be considered.

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( 1) Liver wgeticrutioti

Regeneration of liver following partial hepatectomy could be a consequence of the release of Anti-Z from the cells due to damage or “shock” (shock, that is, sudden stress, is well-known to cause dramatic changes in membrane function); Anti-Z released would render Z unfunctional and open up Sites B in a proportion of cells to be determined by the amount of Anti-Z released. The opening of Sites B following partial hepatectomy, could also be a result of lowering of the extracellular @lasma) concentration of Z due to a reduction in the number of cells making Z and the high rate of turnover postulated for I; the lowered extracellular concentration would lead to a reduction in the amount of Z bound to the hepatic cell surface. In support of these suggestions the rate of DNA synthesis and, therefore, the number of cells triggered into division during regeneration of liver, have been shown to depend on the amount of liver excised (Bucher & Swaffield, 1964). Sera from partially hepatectomized animals exhibit a growth-stimulatory effect, and dilution of plasma in viva by plasmaphoresis (which should reduce the concentration of Z in the plasma) induces mitosis in intact rat liver (Glinos, 1952, 1958, 1967).

(2) Tissue culture

The primary cells for tissue culture are usually obtained by treatment of the tissue with an enzyme such as trypsin which could degrade Z and open up Sites B. This would explain why the rates of growth and the proportion of cells in mitosis, are generally higher in cells grown in tissue culture than in the same cells in the tissue of origin. The model also explains the requirement for serum that normal cells exhibit as a rule, for growth in vitro (Todaro, Lazar & Green, 1965; Amos, 1968; Holley & Kiernan, 1968; Dulbecco, 1970; Clarke, Stoker, Ludlow & Thornton, 1970; Castor, 1971; Dulbecco & Elkington, 1973; Holley, 1974). We have recently shown that for normal cells in tissue culture, serum is required to mainrain the rate of uptake of amino acids at the elevated level necessary for growth (Bhargava & Vigier, 1976); on replacement of the serum-containing growth medium with the serum-free medium, these rates were maintained only for 30 to 45 min, after which period they fell dramatically. Enough Z could be synthesized during 30 to 45 min of serum deprivation to block Sites B; Z would either be degraded or be unable to bind to Type I receptors in the presence of serum (i.e. SF2). As the model predicts, serum can be replaced in cell culture by low, non-toxic concentrations of trypsin (Sinclair, Reid & Mitchell, 1963).

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(3) Mitogenic

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hormones and plant agglutiniw

Such substances could bind to receptors of Type II; this binding would lead to the release of I from Type I receptors, which release would render Type III receptors functional and lead to the opening of Sites B. (C) TRANSITION

FROM

NORMAL

DIVIDING

CELLS

TO NORMAL

RESTING

CELLS

I shall consider two model systems here, that is, cessation of growth following tissue regeneration (an in vivo system), and density-dependent inhibition of growth in tissue culture (an in vitro system). (1) Cessation of growth after regeneration of tissues as is obtained, for example, after partial hepatectomy or during wound healing, would be explained by the build-up in the extracellular environment, as a result of the increase in the number of cells, of a concentration of I which is sufficiently high to allow reblocking of all Sites B, e.g. by neutralizing the effect of Anti-I released at the time of the original tissue damage. (2) Amongst factors (Gordon & Brice, 1974), besides nutrients, that limit the growth in tissue culture of normal cells which show anchorage dependence, two appear to be specially important: (a) certain macromolecular substance(s) contained in serum; and (b) cell density, that is, the number of cells per unit surface area available for growth (Dulbecco & Elkington, 1973). Existing data (inter alia, Todaro et al., 1965; Macieira-Caelho, 1967; Holley & Kiernan, 1968; Ceccarini & Eagle, 1971; Castor, 1971; Dulbecco & Elkington, 1973; Bradley & Culp, 1974; Ellem & Mironescu, 1974; Nair, 1974; Taylor, 1974; Froehlich & Anastassiades, 1975) support the view that the extent of growth of normal cells in tissue culture starting from a small inoculum, depends initially on the amount of serum present in the medium; when a high cell density characteristic of the cell type is reached, no growth occurs even if serum is not limiting and no cytotoxic product has accumulated in the culture medium (that is, the medium removed from the monolayer is still capable of sustaining growth of a fresh inoculum of cells (Sekhan, 1976)). The growth of normal cells in tissue culture when nutrients are not limiting may, therefore, cease as a result of (a) depletion of certain factor(s) contained in serum, or (b) acquisition of a certain cell density (it would be, perhaps, appropriate to use the term “density-dependent inhibition of growth” (cf. Oren & Kohn, 1969; Stoker & Rubin, 1969), only in the latter case and not in the former, irrespective of the cell density required for the inhibition). In either case, the transition from growing to resting cells is explained by the model in terms of a competition between SF2 and I. As the cells grow in the presence of serum, they would release

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in the medium increasing amounts of Z till the SF2 originally present in the culture medium is “neutralized” by Z, and I function re-established on the surface of cells; when this happens growth would cease. Addition of more serum (as a source of SF2) would reinitiate growth that would continue (Dulbecco & Elkington, 1973; Mauck & Green, 1973; Holley, 1974) till a cell density high enough to allow I to “link” cells is reached. With both the valencies of Z now satisfied and its affinity for Sites B increased, I could become resistant or inaccessible to SF2 (e.g. due to slower diffusion of the latter (Lengerova, 1972)) so that growth would not be possible even though enough serum may be available (cf. Castor, 1970; Nair, 1974). There is evidence suggesting that competition between a cell factor and a serum factor acts as a determinant of growth in tissue culture (Bellanger, Jullien & Harel, 1970; Engelhardt, 1971; Bartholomew, Neff & Ross, 1976~); the observed correlation between cell density and mitotic index in uivo, e.g. in chick wing, and the progressive decrease in the rates of uptake of certain nutrients with increasing cell contact, e.g. in kidney epithelial cells, also support this view (Summerbell & Wolpert, 1972; Zetterberg, Auer & Moore, 1974). Kinetic evidence too suggests, in support of the model, that the reduction in the rates of uptake of nutrients in density-inhibited cells is a consequence of decrease in the number of functional transport sites (Plagemann, 1973), and that cell growth is correlated with an increase in the number of such sites (Kletzien & Perdue, 1974). The recent demonstration (Canagaratna & Riley, 1975) that a density-dependent, partial inhibition of growth can be exhibited in the presence of 10% serum in sparse cultures occupying only 5% of the surface area available for growth, is also in accordance with the view that inhibition of growth of normal cells in tissue culture may be mediated by a factor produced by the cells and capable of acting on the other cells in a trans manner. Stimulation of growth by agents like proteolytic enzymes (Sefton & Rubin, 1971; Blumberg & Robbins, 1975), neuraminidase (Vaheri et al., 1972) and insulin (Yarnell & Schnebli, 1974; Teng, Bartholomew & Bissell, 1976), in dense cultures in which growth has stopped due to depletion of serum, could be explained by the destruction of I, or by the inability of I to bind to Type I receptors at Sites B due to occupation of the adjoining Type II receptors by the agent. The density-dependent inhibition of growth reported for certain non-malignant cell lines in suspension culture (Glinos & Werrlain, 1972; Glinos, 1973; Glinos, Vail & Taylor, 1973; Glinos & Bartos, 1974) under conditions in which limitation of serum or nutrients, including oxygen, is apparently not the cause of cessation of growth, could similarly be explained by accumulation of I in a dense culture to a concentration sufficiently high to block Sites B.

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4. The Malignant

Transformation

In the model, the transition from normalcy to malignancy, i.e. the malignant transformation, is explained as an inheritable intracellular event,

spontaneous or induced, which interferes with the production or activity of 1. Such an event may be a consequence of (a) modification of cellular genetic information controlling normal Zfunction, following mutation in a structural gene for Z or in a gene controlling the formation of functional Z (specially if Zis a conjugated protein) or of a constituent of Sites B (that is, the Type 1 receptor or a component controlling the binding of Z to the receptor); or (b) introduction of new genetic information which makes the cells impermeable to Z or permeable to Anti-Z, or leads to production of a substance which acts like Anti-Z at the cell surface. Viral carcinogencsis would bc explained by possibility (b), and those malignant transformations in which no virus--exogenous or endogenous (Huebner & Todaro, 1969; Temin, 1970, 1971, 1974; Gross, 1974; Sarin & Gallo, 1974; Gillespie & Gallo, 1975; Lazar, Schlesinger, Horowitz & Heller, 1975)-appears to be involved, such as those obtained with chemicals (Rapp, Nowinski, Reznikoff & Heidelberger, 1975; Jones et al., 1976), by possibility (a). Evidence supporting this view is now presented. Chemical carcinogens, or their active metabolites, bind or interact with DNA (Miller & Miller, 1966; Farber, 1968; Bhargava, 1970; Chauveau, Meunier & Benoit, 1974; Brookes, 1975), induce erroneous base pairing (Sirover & Loeb, 1974), and act as mutagens (Ames, Durston, Yamasaki & Lee, 1973). The mutagenicity of members of a chemical carcinogenic series correlates well with their carcinogenicity (Grover, Cookson & Sims, 1971), and available information on the nature of the transformants (e.g. of BHK cells (Bouck 6r. diMayorca, 1976)) obtained by chemical carcinogens suggests strongly that chemical carcinogenesis is a consequence of a somatic mutation. If chemical carcinogens were to cause a point, deletion or frame-shift mutation (Ames, Gurney, Miller & Bartsch, 1972; Isono & Yourno, 1974) leading to a I- genotype, one would expect the DNA of the tumour cells to be devoid of at least one carcinogen-binding site. This appears to be so: carcinogens bind to a lesser extent to the DNA of tumour cells than to that of homologous normal cells (Brookes & Duncan, 1971), and tumour cells are less susceptible to the toxic action of carcinogens-which action, at least in some cases, could be a consequence of their interaction with DNA-than homologous normal cells (Borenfreund, Krim, Sanders, Sternberg & Bendich, 1966; Takaoka et al., 1968; Evans, Price, Kerr & Deoca, 1970). Transformation with oncogenic viruses leads to the appearance on the host-cell surface, of what are believed to be virus-coded proteins (Wickus

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& Robbins, 1973; Gilead et al., 1976) and surface antigens (e.g. TSTA) (Burger, 197lb; Eckhart, 1972; Lengerova, 1972; Benjamin, 1974; Kurth & Bauer, 1975; Kurth, 1975; Kurth & Macpherson, 1976; Martin, 1977); these substances are not structural components of the virus and their presence on the cell surface seems to be correlated with, and obligatory for, the expression of the malignant phenotype (Burger, 1971b; Benjamin, 1974; Kurth, 1975; Kurth & Macpherson, 1976; Martin, 1977). One of these substances could have an Anti-Z-like activity, and be identical to the overgrowth stimulatory factor isolated from certain virally transformed cultures (Rubin, 1970). The inhibition of development of transformation by Rous sarcoma virus in cultures of high cell density (Clark & Bader, 1974) also suggests the possibility that in virally transformed cells a competition may exist between two factors, one coded by the cell (according to the present model, I) and the other by the virus. Studies with temperature-sensitive mutants have shown that an early viral gene product is required for the increase in the uptake of sugar obtained on transformation of resting cells with oncorna viruses (Eckhart & Weber, 1974; Siddiqi & Iype, 1975). The model requires the continued presence in the cell of the transforming agent when possibility (b), in which the genetic capacity to make Zis retained, holds true; in the case of possibility (a) in which the genetic capacity to make Zis lost, the carcinogenic agent is not required to be present in the transformed cells. It is established that in viral carcinogenesis [possibility (b)], the presence in the cell of at least a part of the viral genome, and its continued expression, are necessary for the manifestation of the malignant phenotype. On the other hand, chemically induced tumours [possibility (a)] do not appear to contain the original carcinogen or an active metabolite of it. The suggestion that the primary causative event which leads to malignant transformation following interaction of a carcinogenic agent (including an oncogenic virus) with the target cell, occurs on the cell membrane, has been made earlier by several authors (Wallach, 1968; Holley, 1972, 1974; Levine & Burger, 1972). 5. The Model Explains the General Characteristics of Neoplastic Cells (A)

LACK

OF DENSITY-DEPENDENT LOWERING

INHIBITION

OF THE REQUIREMENT

OF GROWTH FOR

AND

A

SERUM

The model invokes a loss of Z function in malignant cells. These cells, therefore, should neither show an obligatory requirement for SF2 for growth, nor exhibit the phenomenon of density-dependent inhibition of growth. This seems to be generally true. Malignant cells, irrespective of their origin 8 7.8.

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(virally transformed, chemically transformed, or of unknown etiology), require a much lower concentration, if any, of serum for growth than do normal cells (Temin, 1968; Clarke et al., 1970; Dulbecco, 1970; Scher & Todaro, 1971; Smith, Scher & Todaro, 1971; Vogel, Risser & Pollack, 1973; Vogel & Pollack, 1973; Dulbecco & Elkington, 1973; Bradley & Gulp, 1974; Oshiro & Dipaola, 1974; Yamane, Murakami & Katu, 1975); in the case of virally transformed cells, loss of the requirement for serum is controlled by the same viral gene the expression of which is required for maintenance of the transformed state (Bell, Wyke & Macpherson, 1975). (The requirement for a smallamount of serum exhibited by many transformed cell lines may be due to the possibility+ompatible with the model-that the uptake of certain essential nutrients needed in trace amounts may occur only through Sites A; it would then be necessary for Sites A to operate at V,,,,, even when Sites B are open. Alternatively, serum may serve as a source of certain nutrients such as essential fatty acids, which are not normally present in the growth media (Yamane et al., 1975.)) Further, under conditions in which nutrients or serum are not limiting, malignant cells grow to higher saturation densities than are obtained with homologous normal cells (Holley & Kiernan, 1968; Pollack, Green & Todaro, 1968; Clarke et al., 1970; Dulbecco, 1970; Castor, 1971; Dulbecco & Elkington, 1973; Oshiro & Dipaolo, 1973; Reznikoff, Bertram, Brankow & Heidelberger, 1973; Mishra & diMayorca, 1974; Holley, Baldwin, Kiernan & Messmer, 1976). In fact, a correlation appears to exist between the extent of density-dependent inhibition of growth and the degree of malignancy as adjudged by other criteria (Pollack et al., 1968). Apparently, growth of malignant cells in culture is limited only by the availability of nutrients or the accumulation of a cytotoxic product in the medium. Malignant cells also do not show densitydependent inhibition of transport exhibited by normal cells (Temin, 1968; Scher & Todaro, 1971; Smith et al., 1971; Weber & Edlin, 1971; Weber & Rubin, 1971; Plagemann, 1973; Bose & Zlotnick, 1973 ; Vogal et ul., 1973 ; Oshiro & Dipaolo, 1974; Bhargava & Vigier, 1976), whereas revertants from virally transformed cells show density-dependent inhibition of both growth and transport (Schultz & Gulp, 1973). (B)

LOWER

CELL

ADHESIVENESS

Malignant cells generally exhibit a lower degree of adhesiveness (Edwards, Campbell & Williams, 1971; Gersham, Drumm & Culp, 1976). This would be explained by the absence of Z on the cell surface. Z could be one of the materials “linking” the cells, e.g. at or near the tight junctions (Staehlin, 1974); in the model, Z is postulated to be bivalent and its affinity for Sites B to increase when both its valencies are satisfied by binding to adjacent cells.

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In support of the above view, trypsin-releasable material which would include Z, has been reported to confer adhesiveness upon cells, and the cellaggregation factors have been shown to be tissue specific (Deman, Bruyneel & Mareel, 1974; Balsam0 & Lilien, 1975; Hausman & Moscona, 1975). (C)

ALTERED

CELL

SURFACE

CHARACTERISTICS

Non-virally transformed ceils would have either no f or inactive Z on the cell surface, or would possess altered Sites B; virally transformed cells would have, instead of Z, an Anti-Z-like substance on the surface. The cell surface of malignant cells would therefore be, as a rule, different-chemically and structurally-from that of homologous normal cells, and show “additions” or “deletions”, depending on the probe used. This is supported by a variety of studies on both chemically and virally transformed cells (Wallach, 1968; Ambrose, Batzdorf, Osborn & Stuart, 1970; Emmelot, 1973; Scott, Furcht & Kersey, 1973; Hogg, 1974; Kim, Issacs & Perdomo, 1974; Patterson, 1974; Terry & Culp, 1974; Hakomori, 1975; Kolata, 1975a; Shin, Ebner, Hudson & Carraway, 1975; Torpier, Montagnier, Biquard & Vigier, 1975). Two types of specific, widely studied differences between the surfaces of normal and malignant cells, namely immunological differences and differences in the response to Con A, are now briefly discussed. The postulated presence of altered Z or Sites B, or of virus-coded material or exposed Type I receptors, on the cell surface of malignant cells, implies that malignant transformation should result in the appearance of neM* surface antigens not detected in normal resting cells; some of these antigens (such as Type I receptors and other membrane components which can be blocked by Z) should be, in fact, present in normal resting cells but in a masked form. This appears to be generally true. It is now well established (Burger, 1971b; Benjamin, 1974; Kurth, 1975) that new surface antigens appear following chemical (Baldwin & Moore, 1968), viral (Tevethia & Rapp, 1965; Aoki, Stephenson & Aaronson, 1973) or spontaneous (Mukherji & Hirshaut, 1973) transformation. Several of these antigens have been shown to be present in a masked or cryptic form in normal, non-growing cells (Hakomori, Teather & Andrews, 1968; HByry & Defendi, 1970; Burger, 1971~; Denechaud & Uriel, 1971; Dyee & Hoverback, 1974; Wolf & Robbins, 1974; Hellstrom, Hellstrom & Nishioka, 1975); treatment of such cells with proteases (e.g. trypsin) exposes, for example, certain antigenic hematosides (Hakomori et al., 1968), the Forssman antigen (Burger, 1971~; Ankerst, Steele & Sjogren, 1974; Silinska, Fang & Mukkur, 1974) and the S antigen (HPyry & Defendi, 1970), all of which are normally exposed only in malignant cells. Cross-reactivity (Ankerst et al., 1974; Silinska et al.. 1974) of chemical carcinogen and oncogenic virus-induced tumour-associated

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transplantation antigens also suggests that at least some of the surface changes occurring on malignant transformation are independent of the nature of the transforming agent and are, as would be expected on the basis of the model, characteristic of the transformation phenomenon per se. Con A and several other lectins, at low concentrations, generally agglutinate virally and spontaneously transformed cells but not normal cells (Burger, 1969, 1971b; Inbar & Sachs, 1969b; Cline & Livingstone, 1971; Ozanne & Sambrook, 1971; Burger & Noonan, 1972; Inbar, Ben-Bassat & Sachs, 1972; Kapeller & Doljanski, 1972; Sharon & Lis, 1972; Salzberg, Robin & Green, 1973; Benjamin, 1974; Nicolson, 1974; Vannest 8c Grimes, 1974; Kataoka, Tsukagoshi & Sakurai, 1975; Gollard & Temmink, 1976); agglutinability with Con A and tumorogenicity appear to correlate well in such cells (Vannest & Grimes, 1974; Kataoka et al., 1975). Although it appears likely that the lateral mobility of Con A-binding sites, which may be greater in malignant cells in comparison to normal resting (e.g. densityinhibited) cells (Barnett, Furcht & Scott, 1972; Roth, 1974), may be involved in the above phenomenon (Nicolson, 1971; Sharon & Lis, 1972; Shoham & Sachs, 1972; Nicolson & Lacorbiere, 1973; Noonan & Burger, 1973b,c; Garrido, Burglen, Samolyk, Wicker & Bernhard, 1974; Guerin et al., 1974), such mobility is probably not the sole determinant of agglutination (Sharon & Lis, 1972; Depetris, Raff & Mallucci, 1973). The possibility that the observed specific biological and biochemical effects of binding of Con A to malignant cells are due to the blocking of Sites B by Con A in a mannei analogous to that suggested for I in normal cells, is supported by several observations. Binding of Con A to malignant cells leads to a lowering of the rates of uptake of nutrients (Inbar, Ben-Bassett & Sachs, 1971) and to a loss of the malignant phenotype (Burger &Noonan, 1970; Demicco, Pagis &Meyer, 1973; Mannino & Burger, 1975). Con A-binding sites appear to exist in untransformed cells in a cryptic state; thus trypsin, papain and pronase which would remove 1 and expose Sites B, convert normal cells that are poorly agglutinable by Con A, to tightly agglutinable cells. Trypsin does not increase the Con A-agglutinability of malignant cells, and the agglutinability of normal cells, following trypsin treatment, does not exceed that of malignant cells (Burger, 1969, 1971b; Inbar & Sachs, 1969a,b; Pollack & Burger, 1969; Kapeller & Doljanski, 1972; Nicolson, 1972; Sharon & Lis, 1972; Depetris et al., 1973; Glynn, Thrash & Cunningham, 1973; Noonan & Burger, 1973a: Roth, Meyer, Neupert & Bolck, 1973; Weber, 1973a; Benjamin, 1974). (D)

INABILITY

OF MALIGNANT

CELLS

TO ENTER

THE

Go PHASE

The model defines malignant cells as those in which Sites B cannot be blocked by endogenously produced I. Consequently, malignant cells would

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be incapable of entering the true Go or the resting phase which the model defines as the cellular state in which Sites B are blocked by I. Therefore, in the absence of an external influence such as that of normal cells, and given enough nutrients and auxiliary growth factors, malignant cells must proceed towards cell division, from one mitotic cycle to another, sometimes slowly but always surely. This appears to be so (Baserga et al., 1973; Bush, 1973; Augenlicht & Baserga, 1974; Burstin & Basilica, 1975; Pardee & James, 1975; Bartholomew, Yokota & Ross, 1976b; Rozengurt & PO, 1976). (E)

SIMILARITY

TO OTHER

TYPES

OF GROWING

CELLS

The model implies a basic similarity between all types of dividing cells, normal or malignant, embryonic or adult, growing in duo or in vitro. In all dividing cells, Z would be functionally ineffective and Sites B would be open ; therefore, all such cells should resemble each other in regard to properties which would be a consequence of an I- phenotype. Available evidence supports this view. The rate of growth and the rates of uptake of amino acids and other nutrients during growth, are substantially the same in transformed cells as in homologous untransformed cells (Burger, 19716; Roman0 & Colby, 1973; Venuta & Rubin, 1973; Weber, 1973a,6; Benjamin, 1974; Colby & Romano, 1975; Hare1 et al., 1975; Bhargava & Vigier, 1976) with the possible exception of the rates of uptake of certain hexoses which may be slightly higher in cells transformed by oncorna viruses than in the untransformed cells (Eckhart & Weber. 1974; Siddiqi & Iype, 197.5). There are strong immunological resemblances between malignant and embryonic (or foetal) cells, and tumour development is apparently inhibited in animals sensitized to embryonic tissues (Huebner et al., 1970; Alexander, 1972; Ting, Lavrin, Shiu & Herberman, 1972; Artzt et al., 1973; Avis & Lewis, 1973; Castro, Lance, Medawar, Zanelli & Hunt, 1973; Kurth & Bauer, 1973; Lemevel & Wells, 1973; Mukherji & Hirshaut, 1973; Turberville, Darcy, Laurence, Johns & Neville, 1973; Weinhouse, 1973; Baldwin & Vose, 1974: Dyee & Hoverback, 1974; Fritsche SCMach, 1975; Hirai & Alpert, 1975; Pees, Shah & Baldwin, 1975); foetal antigens, possibly including those which are found in malignant cells, have also been shown to appear transitorily during normal cell division, e.g. in liver regeneration (Denechaud & Uriel, 1971; Wolf & Robbins, 1974; Hellstrom et al., 1975). Agglutinability of embryonic and foetal cells and of dividing oocytes by Con A is similar to that of malignant cells (Moscona, 1971; Weiser, 1972; Becker, 1974; Pienkowski, 1974), and normal cells growing in tissue culture become agglutinable with Con A for a certain period during the cell cycle (Shoham & Sachs, 1974). The degree of exposure of glycolipids on the cell surface of normal cells during the division cycle is similar to that found in malignant

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cells, and mitotic untransformed cells exhibit endogenous non-contactdependent surface galactosyl-transferase activity similar to that observed in transformed cells (Gahmbarg & Hakomori, 1974; Webb & Roth, 1974). 6. Other Implications (A)

BINARY

NATURE

OF THE

MODEL

AND

CELLS

of the Model THE

FATE

OF TRANSFORMED

IN VIVO

The model is binary: Sites B are proposed to be either open or closed, and a cell either normal or malignant, either resting or dividing. While the model does not allow prediction of what the precise fate of a transformed cell might be in the organism, it suggests several possibilities that are normally encountered. For example, the transformed cell could be rejected immunologically or multiply, depending on the immunogenicity of the new antigens appearing on the cell surface; the model predicts considerable variation in the immunogenicity of these antigens. The rate of multiplication of the transformed cell may be slow or fast; the extent of the tram effect of homologous normal (I+) cells on the transformed (I-) cells would be an important factor which would determine this rate. Normal cells are known to inhibit the growth of malignant cells in vitro (Stoker, 1967). (B)

INVOLVEMENT

OF A SMALL

NUMBER

OF GENES

IN REGULATION

OF

GROWTH

The model requires that at most a few genes should be involved in regulation of growth and in the control of the transitions from one cellular state to another discussed earlier; malignant transformation must be a consequence of a single event affecting one of these genes or their products. This view is supported by several observations. Studies on somatic cell hybridization between normal and malignant cells have shown that the malignant state is controlled by a small number of genes distributed over at most a fewin some cases, probably just one-linkage groups (Bregula, Klein & Harris, 1971; Klein, Bregula, Wiener & Harris, 1971; Wiener, Klein & Harris, 1971; Yamamoto, Rabinowitz & Sachs, 1973b; Croce & Koprowski, 1975). In the case of oncogenic viruses, only a small proportion of the viral genome (probably just one gene-at least for polyoma (Dulbecco, 1973) and sarcoma viruses (Anderson & Martin, 1976; Graessmann & Graessmann, 1976; Weiss, 1976)) appears to be essential to allow the maintenance of the malignant phenotype (Martin, 1970; Kawai & Hanafusa, 1971; Bader, 1972; Lewis & Rowe, 1973; Botchan, Ozanne, Sugden, Sharp & Sambrook, 1974; Gallimore, Sharp & Sambrook, 1974; Graham, Vandereb & Heijneker, 1974; Kimura & Itagaki, 1975; Flint, Sambrook, Williams & Sharp, 1976;

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Levine, 1976; Sugden, Summers & Klein, 1976). The ability to obtain temperature-sensitive mutants of chemically transformed cells, which mutants show culture morphology characteristic of normal cells at non-permissive temperature, suggests that mutation in one gene may be enough to cause malignancy (Yamaguchi & Weinstein, 1975). The dose-response data with chemical carcinogens also indicates that malignancy is a single hit phenomenon (for references, see Reznikoff et al., 1973; Misra & dihlayorca, 1974; Huberman, Mager & Sachs, 1976). Further, there is evidence that the oftobserved development of malignancy during cell culture in vitro (Hammerman, Todaro & Green, 1965; Aaronson & Todaro, 1966; Oshiro, Gerschenson & Dipaolo, 1972) is a result of selection, under appropriate conditions (in which the transformed (I-) cells could grow in preference to the parent (I+) cells), of a small number of cells, each transformed spontaneously as a consequence of what would appear to be a single mutational event. Thus, in one study (Hgyry & Defendi, 1970), mouse embryo lines when grown under conditions of minimal cell contact continued to be non-tumorogenic for at least 200 generations, whereas the same cell lines grown, before subculturing, to high cell densities (at which only the mutant I- cells with a low requirement of serum would multiply) with extensive cell contact, produced tumours after the thirtieth generation. Lastly, spontaneous tumours appear to generally originate from a single cell (Knudson, 1973); this observation would be most easily explained by a single mutational event. (C)

TRANSITION

FROM

MALIGNANCY

TO NORMALCY

On reversion to normalcy (Macpherson, 1971; Vogel & Pollack, 1973), as adjudged by loss of the characteristics of malignant cells mentioned earlier and of their ability to cause tumours in vivo, malignant cells should regain the ability to make functional Z irrespective of the mechanism of reversion. For example, where the capacity to exhibit normal Z function was lost permanently-a possibility suggested here for chemical carcinogenesis-Z” genotype (normalcy) would be dominant over I- genotype (malignancy); in such cases, fusion of malignant cells with normal cells (not necessarily homologous) would lead to hybrids which would behave, phenotypically, like a normal cell, unless the gene for Z donated by the normal parent was lost as a result of chromosomal segregation during development and growth of the heterokaryon. By a similar argument, the hybrid obtained in the case of virally transformed cells would be malignant, unless (a) the viral gene(s) responsible for ontogeny were lost during development and growth of the heterokaryon ; or (b) the anti-Z-like product coded for by the viral genomc cannot be translocated to-or, if translocated, cannot act at-the hybridcell surface due to certain features of the cell surface which are determined

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by the normal cell genome. The results reported in the various published studies (Barski & Cornefert, 1962; Silagi, 1967; Ephrussi, Davidson 8c Weiss, 1969; Harris, Miller, Klein, Worst & Tachibana, 1969; Bregula et al., 1971; Harris, 1971; Klein et al., 1971; Wiener et al., 1971; Sol1 & Krooth, 1972; Vandernoordaa, Vanhaagen, Walboomers & Vansomeren, 1972; Barski, Blanchard, Youn & Leon, 1973; Levisohn & Thompson, 1973; Croce & Koprowski, 1974; Meyer, Berebbi & Klein, 1974; Wiener, Klein & Harris, 1974a,b; Jami & Ritz, 1975; Stanbridge, 1976; Klein, 1976) on somatic cell hybridization between normal and malignant cells or between two malignant cell lines, can be explained in the above fashion; as the model would suggest, such hybridization has been reported to yield, depending on the origins of the parent cells and the nature and activity of the chromosomes retained during growth of the heterokaryon, a normal phenotype or a malignant phenotype. 7. Further Rationale for the Proposed Entities Two different sites are postulated for the transport of essential nutrients, as they are envisaged to perform non-overlapping functions and to be regulated in a basically different manner, Sites A being under a positive control (of SFI) and Sites B under a negative control (of I). (Their partial overlap-even identity-in morphological terms is not ruled out.) A Site B is conceived as an allosteric unit in which three distinct functions (transport, its positive regulation, and its negative regulation) are integrated; this proposed property of Sites B represents a minimal requirement for any model of regulation of growth based on regulation of transport of nutrients. Z should have a high rate of turnover and a low affinity for Sites B to allow rapid operation of the on t, off “switch” by a variety of mitotic agents; Z, if it exists, would appear to be capable of being replaced on normal cells in tissue culture within 30 min (Bhargava & Vigier, 1976). The postulates that Zis bivalent and that when Zacts as a bridge between two Sites B its affinity to either of the Sites B is increased so that it is no longer displaceable by SF2-specially at low concentrations of the latter-provide a mechanism for cessation of net growth in vivo as well as for the density-dependent inhibition of growth in vitro in the presence of SF2. Z must be specific to the cell type as in organisms where growth regulation of the type discussed here occurs, growth of histogenetically different cell types can be controlled separately; for example, only the cells of liver proliferate following partial hepatectomy. Lastly, only one Z is postulated for all Sites B, as available evidence (cited earlier) suggests that (a) there is a co-ordinate rise in the uptake of essential nutrients when a resting cell is triggered into the division

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cycle, and (b) a single mutagenic event appears to be capable of converting a normal to a malignant cell and enhancing the rate of uptake of the nutrients. To keep Sites B open, two types of factors should exist: one non-specific to make an integrated growth of the organism possible, and the other cell type or tissue-specific to allow for the growth of one particular cell type or tissue as occurs following partial hepatectomy or during wound healing. SF2 and Anti-Z satisfy these two criteria, respectively. The mechanism of action of SF2 and Anti-Z may be different; for example, SF2 may act by inactivating Z, and Anti-Z by displacing Z from Type I receptors on Sites B. Anti-Z ought to be more stable than Z as it should continue to be present ir, the system for at least one division cycle when released from the cells, e.g. following partial hepatectomy. Postulation of SF1 is based on the observation that serum is generally required not only for growth but also for the maintenance of normal, nondividing cells (Paul, Lipton & Klinger, 1971; Lipton, Paul, Henahan, Klinger & Holley, 1972; Hosick & Nandi, 1974), and for the uptake of essential nutrients in these cells at optimal rates (Bhargava & Vigier, 1976).

8. Contenders for the Proposed Entities

6’) I Some possible contenders for Z would be: (a) chalones (Bullough & Laurence, 1968; Maugh, 1972; Hennings & Houck, 1973; Yamaguchi, Hirobe, Kinjo & Manaka, 1974; Simard, Corneille, Deschamps & Verly, 1974; BrugaI & Pelmont, 1975; Bullough, 197&b; Marks, 1975; Thornley & Laurence, 1975; Bell, 1976) and similar growth inhibitors (Parshley, 1965; Parshley & Mandl, 1965; Nilsson & Philipson, 1968; Darzynkiewicz & Balazs, 1971; Chany & Frayssinet, 1971; Lozzio, Lozzio, Bamberger & Lair, 1975) isolated from normal tissues; as postulated for Z, these substances have been reported to exhibit histogenic specificity and to inhibit transport of nutrients (e.g. amino acids) and the division of normal as well as tumour cells, both in uioo and in vitro; (b) the amino acid transport-inhibitory proteinoid material released from liver cell suspensions at 37°C that brings down the rates of transport of essential amino acids in low-concentration liver cell suspensions or in Zajdela ascitic hepatoma cells, to the value obtained in normal adult resting liver cells, but no further (Bhargava, Siddiqui, Kumar & Prasad, 1975; this material acts like Z and is being purified in my laboratory; it appears to be present mostly in the extracellular compartment

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of the tissue as would be expected for 1);f (c) the inhibitors of protein and DNA synthesis shown to be present in tissues (Nadal, Lombard & Zajdela, 1976; Vinet & Verly, 1976), in media from cells grown in a monolayer (Hovi & Vaheri, 1976) or a suspension culture (Bellanger et al., 1970), and in extracts of density-inhibited cells (Engelhardt, 1971; such inhibitors could act through inhibition of transport of nutrients such as amino acids); (d) the short-lived tissue-specific mitotic inhibitors, such as those recently described for liver (Nadal, 1973; Yoshikawa & Onda, 1973), present in plasma of normal adult animals but not of young animals or of those undergoing a regenerative process; (e) the specific, trypsin-sensitive, high (N 250,000) molecular weight proteins present on the plasma membrane of normal cells-e.g. chick embryo fibroblasts and 3T3 cells-that turn over rapidly and are not found on the surface of malignant cells (Gahmberg & Hakomori, 1973; Hynes, 1973; Wickus & Robbins, 1973; Hogg, 1974; Hynes & Humphreys, 1974a,b; Robbins et al., 1974; Kolata, 1975b; Rieber, Bacalao & Alonso, 1975); (f) the high molecular weight protein factor released by normal melanocytes that can restore density-dependent inhibition of growth in malignant melanocytes (Lipkin & Knecht, 1974); (g) the protein present on the surface of normal fibroblasts that partially restores morphology, adhesiveness and contact inhibition of movement in transformed fibroblasts (Yamada, Yamada & Pastan, 1976); (h) tissue-specific aggregation factors, or the trypsin-releasable material which confers adhesiveness upon cells (Deman et al., 1974; Balsam0 & Lilien, 1975; Hausman & Moscona, 1975); (i) the macromolecular factor (mol. wt 40,000 to 80,000) present in foetal serum which factor depresses DNA synthesis in primary monolayer cultures of foetal hepatocytes (Leffert, 1974); (j) the proteasesensitive, tissue-specific surface antigens of normal cells that are absent in transformed cells (Ruoslahti, Vaheri, Kuusela & Linder, 1973; Ruoslahti & Vaheri, 1975). (B)

ANTI-I

Some possible candidates are : (a) multiplication-stimulatory factors like those secreted by mutant cell lines (derived from normal cells such as strain L and liver cells) that do not require serum (Shodell & Isselbacher, 1973; Smith & Temin, 1975; Gusky & Jenkin, 1976); such factors have been reported to replace serum in the case of the serum-requiring parent strain and cause a five- to sixfold increase in the rate of uptake of amino acids (cf. Donta, 1973) (the appearance of these factors in the growth medium could be explained by the leakiness of the serum-independent cell lines for ?A similar factor affecting transport of phosphate and apparently absent from tumor cells, as would be expected for Z, has been recently described (Hare1 ef al., 1975).

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Anti-I); (b) cell-type-specific growth factors present in serum-such as the polypeptide fibroblast growth factor or the epidermal growth factor-that can replace serum, at least partially, for the growth of cells and trigger nondividing cells into the division cycle (Gospodarowicz, 1974; Rudland, Eckhart, Gospodarowicz & Seifert, 1974; Rose, Pruss & Hersschman, 1975; Carpenter & Cohen, 1976; Kamely & Rudland, 1976); (c) factors present in “conditioned media” which factors stimulate growth of cells in tissue cultures (Austin, McCulloch $ Till, 1972); (d) factors like the one present in the serum of partially hepatectomized animals that stimulates protein and DNA synthesis in liver cells (Sakai, 1970; Paul, Leffert, Sato & Holley, 1972; Morley & Kingdom, 1973; Sakai & Kountz, 1975). (C) SF1 AND SF2 Several studies (Bucher & Swaffield, 1964; Leffert & Paul, 1973; Oshiro & Dipaolo, 1973; Nishikawa, Armelin & Sato, 1975; Bhargava & Vigier, 1976) suggest the existence of two types of growth-regulatory factors in serum, one necessary for the survival of normal cells or for the growth of malignant cells, and the other necessary for the growth of normal cells; these factors may be the same as SF1 and SF2, respectively. SF1 and SF2 could also be identical with some of the other factors shown to be present in serum, that stimulate transport, cell survival and/or growth (Hulser & Frank, 1971; Paul et al., 1971; Jullien, Blat & Harel, 1972; Dulak & Temin, 1973a,b; Heffman, Ristow, Veser & Frank, 1973; Houck 8c Cheng, 1973; Houck, Sharma & Cheng, 1973; Leffert & Paul, 1973; Oshiro & Dipaolo, 1973; Pickart & Thaler, 1973; Strauss & Berlin, 1973; Nishikawa et al., 1975; Bhargava 8c Vigier, 1976; Groelke & Baseman, 1976). Out of these factors, the more likely candidates for SF2 would seem to be (a) the mitogenic sialoprotein (mol. wt 120,000) obtained from blood plasma, that apparently has anti-chalone activity (Houck & Cheng, 1973; Houck et al., 1973); (b) the macromolecular serum factors (mol. wt.)- 120,000) that have been shown to stimulate amino acid uptake, DNA synthesis and the rate of cell division in foetal hepatocytes (Leffert, 1974; Paul 8c Walter, 1975); and (c) the recently purified serum factor that stimulates DNA synthesis and cell division in confluent cultures, and is distinct from factors with insulinlike activity (Scher, Stathakos & Antoniades, 1974). (D) SITES A AND SITES B In several types of mammalian cells (Rosenberg, Albrecht & Striver 8~ Wilson, 1967; Bittner, 1972; Otsuka & Moskowitz, malignant and normal, two different systems of transport of have been demonstrated; in one study (Otsuka & Moskowitz,

Segal, 1967; 1975), both amino acids 1975) only

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one of the two transport systems-the less “efficient” one-was shown to be operative in non-dividing (confluent) normal cells, while the other-the more efficient-system operated in malignant and in dividing (sub-confluent) normal cells. These two systems could correspond to the two systems of transport postulated in the present model: the Site A-SF1 system and the Site B-SF2 system. The same appears to be true for the transport of sugar (Eilam & Vinkler, 1976). The model suggests that the following functional sites on the membrane may be identical to-or overlap topologically with-Sites B to which, it is proposed, I is bound (in most cases, only references not cited earlier are cited here): (i) sites to which bivalent Con A binds in malignant cells or in trypsinized normal cells, leading to their agglutination; (ii) sites to which PHA or other lectins bind in cells in which they initiate cell division; (iii) sites to which mitogenic hormones that act from outside, bind, (iv) sites on malignant cells at which chalones (or other similar growth-inhibitory substances) act; (v) sites at which the proposed serum factor, SF2, depletion of which would lead to cessation of Type B transport and of growth, would act; (vi) the antigenic sites that are present on the membrane of normal cells in an unexposed, cryptic form, but are exposed in malignant cells; (vii) sites at which tissue-specific surface antigens (Ruoslahti et al., 1973; Ruoslahti & Vaheri, 1975) are present; (viii) sites to which the antigenic materials and proteins that are specified by oncogenic viruses and that migrate to the membrane (Eckhart, 1972; Lengerova, 1972; Wickus & Robbins, 1973; Kurth, 1975; Kurth & Bauer, 1975), are bound; (ix) sites to which certain components of the intercellular material involved in regulation of transport of essential nutrients (Bhargava et al., 1975) or in cell adhesion, bind; (x) sites to which tissue-specific serum factors responsible for enhancing mitotic activity, like the one contained in the serum of partially hepatectomized animals, may bind; and (xi) sites to which tissue-specific growth inhibitory serum factors, such as the one contained in the serum of normal adult animals that inhibits hepatic cell division (Nadal, 1973), may bind. Available evidence supports the view that at least some of the above sites may be overlapping, even identical. For example, the sites on the membrane to which Con A is bound seem to be spatially close to the sites to which insulin binds (Cuatrecasas, 1973; Cuatrecasas & Tell, 1973). Glucocorticoids, like serum, stimulate cell division in density-inhibited fibroblasts as well as lead to an increase in Con A agglutinability (Connor & Marti, 1966; Thrash & Cunningham, 1973); these observations suggest that the site of action on the membrane, of glucocorticoids, Con A, and serum (SF2) may be the same. Treatment of normal cells with cycloheximide for short periods makes them agglutinable by Con A (Baker & Humphreys, 1972); as in the presence of

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the drug, I would not be synthesized and Sites B would be open, Con Abinding sites may overlap with the proposed Sites B. Protease action at the cell surface releases cells from density-dependent inhibition of growth and enhances their agglutinability by Con A (Burger, 1969; Inbar & Sachs, 1969b; Sefton 8c Rubin, 1970), suggesting that the site of action of proteolytic enzymes and the site to which Con A is bound, may be the same. Con A reacts with purified carcinoembryonic antigen (CEA) (Chu, Holyoke & Murphy, 1974); some of the antigenic sites exposed in malignant cells may, therefore, represent Con A-binding sites. A fragmented, non-agglutinating form of Con A inhibits spontaneous aggregation of dissociated embryonic cells, suggesting that the cell-surface sites involved in cell adhesion may overlap with those involved in cell agglutination with Con A (Evans & Jones, 1974). Recent work indicates that the following sets of sites, too, on the cell surface may be identical or overlap substantially: (a) PHA and Con Abinding sites in thymocytes (Ozato, Ebert & Alder, 1975); (b) receptor sites for mirogenic hormones; (c) sites at which proteolytic enzymes act; and (d) sites at which serum or purified multiplication-stimulatory factors from serum, act (Hayashi & Sato, 1975; Holley, 1975; Rechler, Podskalny & Nissley, 1976). It should be emphasized that identity of any two or more of the sites enumerated above would not imply identity of the receptors at these sites for the concerned substances. As already mentioned, those receptors may be of two types: one (Type II receptors) to which growth-stimulatory substances (e.g. (ii), (iii), (v) and (x) in the above listing) would bind, and the other (Type I receptors) to which I or other growth-inhibitory substances (e.g. (ii, (iv), (ix) and (xi)) would bind. It should be possible to locate Sites A once the serum factor, SFI, which is postulated to be specific for these sites, has been isolated. 9. Programme

of the Division Cycle

In the model, the control of cell division is proposed to operate through regulation of Sites B, open Sites B representing the “on” position of the control “switch” and closed Sites B the “off” position. An agent (such as serum, a mitogenic hormone, a lectin or a proteolytic enzyme) that triggers cell di.vision, would then operate by putting this switch on, and all subsequent events (such as a change in the concentration of cyclic AMP (Breckenridge & Sheppard, 1972; Burger, Bombik, Otten, Johnson & Pastan, 1972; Byron, 1972; Sheppard, 1972; Anderson, Russell, Carchman & Pastan, 1973; Dulbecco, 1973; Kram, Mamont & Tomkins, 1973; MacManus, Braceland, Youdale & Whitfield, 1973; Rozengurt & Deasua, 1973; Russell

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& Pastan, 1973; Oey, Vogel & Pollack, 1974; Berridge, 1975; Furuno & Matsudaira, 1975; Lawrence & Jullien, 1975; Miller, Lovelace, Gallo & Pastan, 1975; Moens, Vokaer & Kram, 1975; Pastan, 1975) which appears to be a universal early event, and the initiation of DNA synthesis, a later event) of which the programme of the division cycle is comprised, would be under parental control of this switch. For the model to be established, the nature of this control must be defined. Although the model does not provide a mechanism for this control which would be supported by hard evidence, several possibilities suggest themselves, two of which are mentioned below. (1) One (or more) of the proteins needed for early events in the cell cycle (the “early-event proteins”) may not be synthesized in a resting (G,) cell in sufficient amounts due to limited availability, in the intracellular free amino acid pool, of one (or more) of the amino acids present in the protein in relatively large amounts; when the proportion of this amino acid in the free pool is disproportionately low in relation to the other amino acids, proteins which are not rich in the deficient amino acid are likely to be synthesized in preference to the abovementioned early-event protein rich in this amino acid. The avaiiability of the deficient amino acid following the opening of Sites B by the agent triggering the cell division, would lead to the synthesis of the early-event protein. Once the regulation of the synthesis of such a protein is explained, it is not difficult to envisage how a programmed synthesis of other proteins and intracellular constituents (including DNA: Mitchison, 1969) could cccur during the cell cycle; the programme could be, for example, comprised of a “sequential induction” system. A good candidate for the early event protein suggested here would be the regulatory protein proposed by Dulbecco which may control DNA synthesis through monomer-oligorner transition (Dulbecco, 1975). (2) When the intracellular concentration of one or more of the amino acids (or of another essential nutrient the transport of which is under control of I) reaches a critical value in the cell, following the opening up of Sites B, the nutrient could begin to act as an allosteric activator of one or more of the early-event proteins which may be inactive in resting cells due to the concentration of the allosteric activator being below the critical value. Possibilities such as the above are supported by five types of observations : (a) the free amino acid pools of rapidly dividing cells are higher than those of homologous resting cells (Christensen, Rothwell, Sears & Streicher, 1948; Holden, 1962; Ryan & Carver, 1966; Bhargava, 1970; Bhargava et al.,

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19766); (b) the profile of the amino acid pool changes in a characteristic manner during the cell cycle (G, + M) (Bhargava et al., 1976a); (c) the amino acid pool rises as cells go through G1, and is saturated in the S phase (Bhargava et al., 1976~) at about the same time at which the cells appear to be committed to division and no longer require the continued presence of the trigger, e.g. serum; (d) a correlation has been shown to exist in several cell types between the rates of protein synthesis and the intracellular concentration of the required free amino acids (Munro, 1968; Morgan et al., 1971; Griffiths, 1972); and (e) a parallelism appears to exist in animal cells between the uptake of essential amino acids and the composition of the protein that is synthesized (&use, Miedema & Carter, 1967).

10. Proofs to be Sought Verification of the model presented here, towards which current work in my laboratory is directed, would require the following to be accomplished; (a) isolation and identification of I and Anti-I for at least one tissue, and of SF1 and SF2 for one species; (b) localization of Sites A and B; and (c) elucidation of the control exercised by the opening of Sites B on operation of the programme of the division cycle. Further, all such changes in the surface properties of the cell as are obligatory to malignant transformation, should be shown to be a consequence of the same unitary event, that is, a constitutive opening of Sites B following either the deletion of an existing material CZ) from these sites or the acquisition of a new material (e.g. a virus-coded protein) by these sites. Another possible experimental approach towards verification of the model would be to determine its compatibility with other systems not discussed here, in which control of growth or of related cellular activities has been described. For example, interferon (or an inducer of it) which inhibits the mitotic response of liver cells to partial hepatectomy (Jahiel, Taylor, Rainford, Hirschberg & Kroman, 1971), and macromycin which inhibits DNA synthesis without apparently penetrating the cells (Kunimoto, Hori & Umezawa, 1972), may act by blocking Sites B; such a block would cause a rapid decrease in the rate of uptake of essential nutrients in the target cells which decrease could be experimentally verified. Isopentenyladenosine inhibits growth and the transport of nucleosides, both in virally and in chemically transformed cells (Hare &Hacker, 1972); it too may act by binding to Sites B. The reported reversal of density-dependent inhibition of growth by urea (Weston & Hendricks, 1972) could be a result of denaturation of I by urea.

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11. Deficiencies of the Model The model does not provide a satisfactory biochemical basis for leukemia, although it is not inconsistent with the postulate that normal I function is a necessary albeit insufficient condition for cellular differentiation-blocked in leukaemia--to occur without concurrent cell division (cf. Inbar, Ben-Bassat, Fibach & Sachs, 1973; Fibach & Sachs, 1974). The model also does not adequately explain anchorage-dependence of normal cells. I thank Drs L. Montagnier, Patricia Allin, D. S. Szafarz, P. Vigier, C. Nadal, J. Mandelstam, M. R. Pollock, J. M. Mitchison, T. S. Work, J. R. Tata, H. Gutfreund, D. R. Trentham, M. Stoker, C. Heidelberger, R. C. Gallo, A. Glinos, A. Sibatani, G. S. Sidhu, and my present co-workers at Hyderabad, for reviewing the first drafts of this paper and for helpful suggestions. A part of my work referred to here was carried out under the award of an Eleanor Roosevelt International Cancer Research Fellowship at Institut du Radium, Orsay, France.

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U.S.A. 70, 2281. AMES, B. N., GURNEY, E. G., MILLER, J. A. & BARTSCH, H. (1972). Proc. natn. Acad. Sci. U.S.A. 69, 3128. AMOS, H. (1968). Natn. Cancer Inst. Monograph No. 26, p. 23. ANDERSON, J. L. & MARTIN, R. 6. (1976). J. cellul. Physiol. 88, 65. ANDERSON, W. B., RUSSELL, T. R., CARCHMAN, R. A. & PASTAN, I. (1973). Proc. natn.

Acad. Sci. U.S.A. 70, 3802. ANKERST, J., STEELE, G. & SJOGREN, H. 0. (1974). AOKI, T., STEPHENSON, J. R. & AARONSON, S. A. 70, 742. ARTZT, I