Action of growth factors in the cell cycle

91

Biochimica etBiophysicaActa, 560 (1979) 9 1 - 1 3 3 © Elsevier/North-Holland Biomedical Press

BBA 87058 ACTION OF GROWTH

FACTORS

IN THE CELL CYCLE

PHILIP S. R U D L A N D a and LUIS JIMENEZ DE ASUA b,*

a Imperial Cancer Research Fund, P.O. Box 123, Lincoln's Inn Fields, London WC2A 3PX (U.K.J and b Friedrich Miescher-lnstitut, P.O. Box 2 73, CH-4002 Basel (Switzerland) (Received April 3rd, 1978) Contents I. II.

III.

IV.

V.

VI.

VII.

VIII.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Isolation of positive and negative growth factors . . . . . . . . . . . . . . . . . . . . . . . . A. Positive growth factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Negative growth factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interactions between growth factors and h o r m o n e s . . . . . . . . . . . . . . . . . . . . . . A. Positive growth factors and h o r m o n e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Synergistic interactions between positive growth factors and h o r m o n e s . . . . . . . . . C. Negative growth factors and h o r m o n e s . . . . . . . . . . . . . . . . . . . . . . . . . . . The cell cycle during constant proliferation rates and m e t h o d s of altering these rates . . . A. Models for the cell cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Theoretical considerations when altering proliferation rates . . . . . . . . . . . . . . . Regulation o f the cell cycle when cellular proliferation rates are increased or decreased . A. Relationship o f rates o f cellular entry into S phase with proliferation rates after stimulation of the growth o f cultured fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . B. Kinetics for the stimulation o f the rate o f cellular entry into S phase . . . . . . . . . . C. Regulation o f the cell cycle upon reduction o f proliferation rates . . . . . . . . . . . . Temporal interactions o f growth factors and h o r m o n e s during the lag phase of cultured 3T3 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Organisation o f the lag phase after addition o f growth promoting substances to quiescent fibroblastic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Possible ways o f organising intracellular pathways in the lag phase generated by two interacting growth promoting substances . . . . . . . . . . . . . . . . . . . . . . . . . . C. Temporal nature of the interaction of growth factors and h o r m o n e s . . . . . . . . . . D. Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role o f the early biochemical events generated after additions o f growth factors and synergising h o r m o n e s to cultured 3T3 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Identification o f the early changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Use o f growth factors and synergising h o r m o n e s to establish possible correlations with rates o f cellular entry into S phase . . . . . . . . . . . . . . . . . . . . . . . . . . . Specific and general changes in protein synthetic and degradation rates and their relationship to altered rates o f cellular proliferation . . . . . . . . . . . . . . . . . . . . . . . . A. Changes in general rates of protein accumulation during transition from one proliferative state to another . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. IntraceUular m e c h a n i s m s which cause increased rates o f protein accumulation during the lag phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

* To w h o m reprint requests should be addressed.

92 93 93 96 97 97 99 100 101 101 103 104 104 107 108 109 109

110 112 113 114 114

115 118 118 119

92 C. The production of specific proteins during the lag phase prior to increase in rates of cellular entry into S phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Possible relationships of specific and general protein production in controlling cellular proliferation rates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Speculations and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Possible mechanisms for a growth factor to generate two independent intracellular pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The nature of the step which eventually governs the rate of initiation of DNA synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. The relationship of growth factor and hormonal control of fibroblast proliferation rates in other cell systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

120 121 122 122 124

126 127 128

"Man does n o t realize h o w that which varies is a unity. There is a harmony o f opposite tensions as there is one b o w and lyre'" Heraclitus the Obscure [ 1]. I. Introduction

One o f the most remarkable characteristics of living organisms is that nearly all their activities are highly ordered and regulated. This implies that for each function there must be an efficient regulatory mechanism [2,3]. However, most of the success achieved in establishing and understanding the operative mode of specific regulatory circuits has been provided by studies in single cells, particularly bacteria. At a higher level of evolution, an organism consists of an ordered collection of cells, the regulation of their multiple activities being exerted at several levels o f complexity. However it may be that those seemingly more complex mechanisms are simply derived from pre-existing mechanisms present in single cells [2,3]. Basically, mammalian cells have to regulate two separate functions, (1) their frequency of replication and (2) the expression of their functional phenotype [4]. The former resolves into a study of the cell cycle, the time it takes for a cell to replicate and produce two daughter ceils. In microorganisms, and to a lesser extent in mammalian cells the study of the regulation of the cell cycle has been vigorously examined using three main approaches, (1) the use of specific inhibitors, (2) changes in physiological conditions and (3) the use of temperature sensitive mutants [5]. In this review, we shall discuss a fourth approach, the use of growth factors and hormones to analyse specific events in the mammalian cell cycle. Virtually every process of growth and metabolic activity of tissues and organs in the young and adult animal seems to be regulated by hormones. However, most hormoneresponsive tissues do not possess a uniform population o f cells and the control of tissue growth often involves interactions between endocrine glands. Hence the study of the hormonal action directly on the isolated cell in tissue culture is of paramount importance in understanding the detailed steps involved at the cellular and molecular level. The use of clonal lines o f mammalian cells in tissue culture has overcome these difficulties. Perhaps the best studied o f these are the Swiss mouse and BALB/c mouse 3T3 fibroblastic cell lines, since their proliferation rates can readily be regulated by the concentration of serum in the medium [6]. However, the use o f a complex mixture such as serum, which contains many growth-controlling agents, has its limitations, since the direct effects of the major proliferogenic substances in serum cannot be separated from effects of other hormonal agents. Nevertheless, it is possible to circumvent this difficulty by the use of purified growth factors (Section IIA). In this review, we shall examine the action o f growth

93 factors and hormones in the fibroblastic cell cycle, except where insufficient evidence warrants including additional results from other cell systems. Numerous reviews have appeared on the effect of different hormones on cellular proliferation of tissues in whole animals and of the effect of serum on fibroblastic cells in tissue culture [6-10]. There is some evidence that in vivo and in vitro replication of mammalian cells occurs by an orderly expression of a sequence of signals and events involving DNA synthesis, a doubling of cell mass and cell division [11-13]. In micro-organisms the sequence of events which involves the 'DNA-division cycle' may be partially separable from a second sequence that constitutes the 'the growth cycle'. The latter includes the main processes of protein and RNA synthesis which cause cell growth [14]. However, in mammalian cells the extent of this separation is less clear. Here we shall cover the identification and definition of positive and negative growth factors and the effects of their interacting hormones, the organisation of the cell cycle for cells growing under constant environmental conditions, and alterations in the cell cycle induced by growth factors and hormones when proliferation rates change. Since the majority of work with cultured ceils has centred upon stimulating them to increase their proliferation rates, we shall deal mainly with this topic. We shall show that changes in proliferation rates are mainly brought about by changes in the rate of cellular entry into the DNA synthetic or S phase of the cell cycle. Hence the remainder of the review will mainly concentrate on events which increase the rate of this process. The times when growth factors and hormones are required during the transitional period or lag phase (Sections IV and V) before the occurrence of increased rates of cellular entry into S phase leads to models for the organisation of events in this period. At the biochemical level the initial changes and the molecular mechanisms required to generate increases in RNA and protein will be described and their relevance to increasing proliferation rates will be discussed. Finally, we shall compare the general organisation of the intracellular sequences discussed here for cultured fibroblasts with those obtained in other systems, and discuss hypothetical biochemical models to account for these observed changes. This represents a somewhat personal view of the action of growth factors and hormones in the cell cycle, and the authors can only apologise in advance for any omissions of fact or fancy that may occur.

II. Isolation of positive and negative growth factors IIA. Positive growth factors The study of the effects of known hormones on the rates of replication of cells in tissue culture has been mainly disappointing. There are some examples of hormones which can modulate growth rates in neoplastic cells [ 15,16], normal tissues previously primed in vivo with the same or other hormones [17,18] or even established fibroblastic lines [19], but the effects are small compared with those obtainable with serum. This suggests that the effects of serum are produced chiefly by other components and there is evidence that small molecular weight proteins or growth factors may be important amongst these. Unfortunately only small quantities of such growth factors can be produced from serum with the current techniques available. This is due in part to the great complexity of serum and to their extremely low concentrations (2 • 10 -2 to 5 • 10-6% by weight of proteins [20]). The great complexity of serum has caused special problems for the isolation of the positive growth factors in that several different positive factors for the same target cell (e.g., 3T3) can be present in serum. In addition, there are also components which increase their activity but which have little or no effect alone in promoting cell multiplication (Section IIIB). Thus, when serum is fractionated, the resulting fractions are much less

94 active than the starting material and the activity is spread over a large range of fractions [6]. However, more progress was made when growth factors were isolated from endocrine glands [21] or from the culture medium after incubation with certain cells [22-24], where they occur in higher concentrations. For the purposes of this review, we shall define a positive growth factor for a given cell as a component which will stimulate the multiplication of cells in a nutritionally and otherwise complete medium for that cell (see below) at concentrations which are considered to be near its physiological level. A negative growth factor is a component which inhibits the positive growth factor. These are definitions only strictly applicable to cells grown in tissue culture in a completely serum-free medium where potential growth factors can be tested (Section VA). A clear distinction can then be made between growth factors and agents which maintain cell metabolic processes (e.g. insulin, as opposed to fibroblast growth factor [25]), cell survival [26] and attachment of the cells to the substratum [27]. However, few cells can be grown in serum-free condition and then this distinction becomes important for two reasons. First, methods used to assay growth factors involve allowing the cells to attain a very low proliferation rate either by allowing them to exhaust the medium of essential serum components or by lowering the serum concentration in the medium. These cultures are termed quiescent, although in fibroblastic cultures there is a slow rate of cell division in all cells (Sections V and VI). The potential growth factor is then tested for its ability to increase cellular proliferation rates usually by observing the increase in the fraction of cells synthesising DNA in a given time after addition of the test material [28]. If, however, additional components in serum (metabolic, survival, attachment, see ref. 6) are also lost, then the potential growth factor may be ineffective in the assay [25]. Second, when the assays are performed in serum to maintain cellular viability, certain hormones, e.g., glucocorticoids, can, under certain conditions, interact with growth factors (Section IIIA) present in serum to increase 3T3 cellular proliferation rates [29]. However, they are ineffective when serum is removed and other steps are taken to maintain cellular viability [28]. Only growth factors which can satisfy the above definition and can be considered pure, or nearly pure, will be discussed in this review article; other reviews have appeared on less well characterized growth promoters [6.30]. The dei~mitive test that distinguishes whether a growth factor has a physiological role in vivo is to demonstrate its activity in the whole animal. The growth factors are probably best classified as to their source of origin since relatively little is known about their site of action in the animal at present. The classification naturally divides up into 5 main, though not entirely mutually exclusive, groups: growth factors in plasma, platelets, serum, endocrine glands (notably pituitary and submaxillary glands), and from cultured cells (Table I). Many growth factors may be related. Thus, nonsuppressible insulin-like activity, somatomedins in plasma and multiplication stimulating activity in serum may represent a single class [56], while the platelet factor, Antoniades human serum factor, and fibroblast growth factor are possibly the same [20]. The growth factor, however, need not come from a single source. Thus multiplication stimulating activity is produced by rat liver cell lines [57] as well as occurring in serum, and fibroblast growth factor is found in brain [47] as well as in pituitary glands. Nerve growth factor activity has beeh found in mouse sarcomas, snake venom, rat granuloma, chick embryo tissues and cultured mouse L and 3T3 cells as well as mouse submaxillary glands [20]. A smaller epidermal growth factor than that isolated from mouse submaxillary glands, of molecular weight 5400 and now thought to be identical to urogastrone, has been isolated from human urine [20]. Colony stimulating factors of differing molecular

95 TABLE I SUMMARY OF SOME GROWTH FACTORS FOR MAMMALIAN CELLS Abbreviations used: NSILA-S: non-suppressible insulin-like activity soluble peptide; MSA: multiplication stimulating activity, NGF: nerve growth factor, EGF: epidermal growth factor, OGF: ovarian growth factor, FGF: fibroblast growth factor, MGF: myoblast growth factor, MF: mesenchymal factor, CSF: colony stimulating factor, PGF2a: prostaglandin F2a , BHK: baby hamster kidney. Principal source

Name

Target tissue tested

Molecular weight

Plasma

NSILA-S Somatomedin C Erythropoietin

chick embryo fibroblasts cartilage cells proerythroblasts

7 000 human 7 000 human

Platelets Serum

Platelet factor Bovine serum factor Human serum factor Bovine serum factor Bovine MSA

Mouse submaxillary gland

Fetal calf $2 NGF EGF

Bovine pituitary gland

OGF FGF

Chick embryos Cells in culture Mouse L-cells

MGF MF CSF

L x factor Virus transformed BHK fibroblasts

Migration factor

Human fibrosarcoma cells

MSA-like activity

Virus transformed BHK fibroblasts

PGF2a

* May be aggregated.

46 23 smooth muscle cells, 13 Swiss mouse 3T3 ceils 25 BALB/c mouse 3T3 13 human WI-38 fibro120 blasts chick embryo fibro4 blasts rat fibroblasts 26 embryonic sympathetic ganglia 26 epithelial and fibro6 blasts ovarian tumour cell 10 line 3T3 and mesodermal 13 cells myoblasts ? pancreatic epithelium ? immature mouse bone marrow and spleen cells producing growing colonies of mature granulocytes and macrophages 3T6 and BHK fibroblasts migration and mitogenic activity in fibroblasts receptor competition and mitogenic activity in fibroblasts Swiss mouse 3T3 cells

References [31,32] [33-35]

000 sheep 000 human 000 monkey 0 0 0 - 3 0 000 000 000 *

[36] [37] [38] [39] [40]

0 0 0 - 5 000

[41]

000

[42]

000 000

[43] [44,45]

0 0 0 - 1 3 000

[ 46 ]

400

[47,48] [49] [50]

40 0 0 0 - 7 0 000

[51,52]

40 000

[23]

?

[ 24 ]

?

[53]

450

[54,55]

96 weights are found in a wide variety of normal tissues, the richest source being the male mouse submaxillary glands, but they are also present in mouse and human serum, human urine and also released into the culture medium by many cells including mouse L cells and human peripheral leucocytes [20,51 ]. The prostaglandins are produced by many cell types [58,59] and probably are the most ubiquitous growth controlling agents in the animal. The prostaglandin F2~ is notable for being the first predicted growth fact6r for fibroblasts based on changes in glucose uptake [54,96] (Section VIIB). Although their comparatively short half-lives in the circulatory system exclude them from the role of circulating hormones in blood, nevertheless, they may have a role as short range signals [60]. The reason for the relatively large number of different growth factors active for a single cell type is unclear. However, they appear capable of independent action since of those tested non appears to crossreact with each other's cell surface receptors in fibroblastic lines [20] and failure on one growth factor, the fibroblast growth factor, to stimulate proliferation of cold temperature sensitive BALB/c 3T3 cells at the non-permissive temperature does not prevent serum and hence presumably other growth factors from acting [611. The fact that cultured cells, and in particular transformed fibroblasts [24,53] and human tumour cells [62] produce growth factors has led some authors [24,53] to speculate that the mechanism of both transformation and formation of neoplasia is an increased ability of cells to produce their own growth factors, so ensuring a degree of cellular autonomy from external control by growth factors present in serum. This hypothesis fails to explain some other changes which accompany transformation in fibroblastic cells (e.g. cell surface glycoprotein changes, increased protease activity, reduced cell adhesive properties) which can be observed independently of changes in the regulation of the cell cycle and in proliferative rates in temperature-sensitive mutant BALB/c 3T3 cells [63, 64], and the extended time course of changes brought about by carcinogen-induced transformation of cultured cells or formation of tumours in experimental animals [65]. This hypothesis may, however, explain some of the changes in response to serum (Section VIIA) and proliferation rates seen upon transformation of cultured fibroblasts. The identification and purification of growth factors has mainly progressed only when growth factor responsive 'normal' cells can be grown in tissue culture as clonal cell lines. These cell lines can then be used as the assay system for growth factors by observing their effect on cellular proliferation rates. Thus, at present, the only systems available to investigate in detail the effects of growth factors are based on the original cell lines used for their isolation (mainly BALB/c 3T3 and Swiss mouse 3T3), and any conclusions drawn may not have general applicability. Also, the uncertain identity of many of the tissue culture cell lines (e.g. whether BALB/c 3T3 are of endothelial or of fibroblastic origin [66]) has largely precluded their use as a means to determine the cell and tissue specificity of the different growth factors. Hence, the true target cells of many of the different growth factors are as yet unknown. The isolation of new, well-defined cell culture systems from different normal tissues in the future should act as an impetus for the discovery of new growth factors, and serve to clarify the target cell specificity of present growth factors.

liB. Negative growth factors Since positive regulators of" cell proliferation exist, it has been suggested that negative regulators must also exist to maintain a balanced control of cellular proliferation. The chalone concept envisaged that the rate of new cell production in a tissue was controlled by a tissue-specific diffusable agent which inhibited cell proliferation within that tissue

97 [67,68]. However, the evidence for hormonal agents with properties similar to those described for chalones in the regulation of the regenerative process of tissues and organs is contradictory [67]. The majority of the experiments performed during liver regeneration to prove that a depletion of the activity of hypothetical chalones in the blood and liver remnant occurs after partial hepatectomy [69] can be interpreted just as easily on the basis of an increased production of a positive regulator for cell proliferation. Hence, the negative feedback control when tissues and organs such as the liver, kidney [70-71 ] and mammary gland [72] undergo cellular proliferation in processes of regeneration and repair can be explained on the basis of a diffusable substance in two not necessarily mutually exclusive ways, either (1) by the production of a negative regulator or chalone or (2) by the effective loss of the positive regulator either by reducing its concentration or by preventing it from reaching its target cell. A process akin to wounding, however, can also be critically examined in tissue culture. This is brought about when a portion of a confluent monolayer of 3T3 cells is removed, the cells in the vicinity of the wounded region are stimulated to enter S phase at higher rates than those in the undisturbed monolayer. The increased rates of cellular entry into S, however, are dependent on the concentrations of a pure growth factor in the medium [73]. Similar results have been obtained with epidermal growth factor and wounded monolayers of BDC-I epithelioid cells [74]. Thus, in these examples of increased proliferation rates after wounding, there is no obvious requirement for the involvement of chalone-like agents. Nevertheless, in other less well defined systems 'mitotic' inhibitors have been postulated to control rates of cellular proliferation; the greater the tissue mass within the body space, the higher the levels of the mitotic inhibitor [67]. The isolation of negative regulators has not proceeded at the same pace as the isolation of their positive counterparts, since it is difficult to differentiate between a decrease in growth rate and a simple toxic effect. No chalone, therefore, seems to be at the same state of purity as growth factors such as the fibroblast or epidermal growth factor. The most pure preparations seem to be the epidermal chalones, the G1 chalone which leads to accumulation of cells in G1 [75], the G2 chalone [76] and the granulocyte chalone [77] which is reported to be purified l0 s times over the starting material. Chalones were seen some years ago as a selective means to control the growth rate of neoplastic tissue. However, the findings of wider target cell specificities for the epidermal chalones other than epidermal cells [78], and that the chalones from either normal mature granulocytes, malignant leukemic granulocytes [79] and epidermis [80] were much less effective against their malignant tumour cells than the normal ceils have possibly reduced the promise of this approach. Once pure negative regulators of cell proliferation are isolated from tissues, then this should place the chalone concept on a much more secure scientific basis.

III. Interactions between growth factors and hormones

IliA. Positive growth factors and hormones Most of the growth factors for fibroblastic cell lines have their growth-promoting activity altered by certain metabolic hormones such as the glucocorticoids and insulin (Table II). This is usually quantified by measuring the increase in the fraction of cells stimulated to synthesize DNA in a given time (labelling index) after adding saturating concentrations of growth factor and hormone to quiescent fibroblastic cells. This measurement is usually a good and much more accurate reflection of the increase in proliferation rates than mea-

98 TABLE II GROWTH FACTORS AND SOME OF THEIR INTERACTING HORMONES Abbreviations used: OGF: ovarian growth factor, FGF: fibroblast growth factor, EGF: epidermal growth factor, PFG2c~: prostaglandin F2~, +: positive effect, - : negative effect, O: no significant change. Growth factor

Tissue or cell

Insulin

Glucocorticoids

References

OGF

ovarian tumour line rat mammary epithelial cells Swiss mouse 3T3 BALB/c 3T3 human diploid fibroblasts Swiss mouse 3T6

+ + + 0 0 +

+ + + + 0 +

[46 ] [81] [82] [28] [ 83,84 ] [85 ]

Swiss mouse 3T3 human diploid fibroblasts Swiss mouse 3T6 rat mmnmary epithelial cells human mammary epithelial

+ + + + 9

9 9. 9 9

[86 ] [ 87 ] [88] [ 81 ] [89]

FGF

EGF

cells

PGF2c~

Swiss mouse 3T3 BALB/c 3T3 Swiss mouse 3T6

+ 0 +

0 -

[54,90] [60] [60,85 ]

Prolactin

rat mammary cells in vivo ** rat mammary ceils in vitro *

+ +

+ +

[91,92] [18]

Growth hormone

rat liver cells in vivo *** rat liver cells in vitro *,***

+ +

+ +

[93] [94]

* Designates that high concentrations are required in vitro. ** Additional sex steroids also involved. *** Triiodothyronine and glucagon were also reported to be involved.

suring cell n u m b e r s (Section VA). The metabolic h o r m o n e s alone usually have little or n o effect o n the labelling index w h e n measured under the stringent conditions described in Section IIA. Hence we shall only discuss effects o f interacting h o r m o n e s which have been measured u n d e r the defmed c o n d i t i o n s which approximate to those in Section IIA. This will then exclude a large a m o u n t of conflicting literature on the effect o f h o r m o n e s , particularly that of the glucocorticoids on cellular proliferation rates measured in less chemically defined conditions. For a particular h o r m o n e this interaction on a given fibroblastic cell line can be of 3 types, (1) positive with all growth factors it interacts with, causing an increase in the labelling indices, e.g. insulin with the fibroblast and epidermal growth factors and prostaglandin F2a in fibroblasts [85] or with prolactin in m a m m a r y cells [81] (Table II), (2) positive or negative depending on the i d e n t i t y of the growth factor (e.g. hydrocortisone with fibroblast growth factor or prostaglandin F2a in Swiss 3T3 cells [90] (Table II) and (3) negative at all times, although this is difficult to distinguish from a negative growth factor, and as yet no clear example has emerged. However, w h e n the stringent conditions for the assay o f the growth factor are no longer observed and the culture m e d i u m is artificially depleted o f specific n u t r i e n t s (e.g. glucose, phosphate, certain amino acids) in 3T3

99 cells [25] or of arginine in rat liver epithelial cells rather than serum factors [94], a complete dependence for the stimulation of cell growth is seen on those hormones (e.g., insulin) which can stimulate uptake of the deficient nutrient into the cell. Thus, the small positive effects of known hormones on increasing labelling indices may be explained in part by their ability to increase cellular metabolic rates rather than by eliciting the same response that is provided by growth factors such as fibroblast growth factor. Metabolic hormones other than insulin and glucocorticoids have yet to be tested in such a stringent manner, although thyroid and parathyroid hormones amongst others are implicated in supporting the growth of baby hamster kidney fibroblastic cells [95]. If they can affect the labelling indices in the assays above they may be candidates for either of the first two classes of interacting hormones.

IIIB. Synergistic interactions between positive growth factors and hormones In the cases examined so far in 3T3 and 3T6 cells, positive interaction between growth factors and a hormone upon the stimulation of DNA synthesis is synergistic or co-operative, since the increase in the labelling index is greater than the sum of the labelling indices produced by their separate additions. Thus, addition of increasing concentrations of prostaglandin F2a to quiescent 3T3 cells produced increasing labelling indices, reaching a maximum value of 20% labelled cells in the given time at a concentration of 200-300 ng/ml. Addition of insulin at physiological concentrations, which had little effect alone, produced a synergistic or co-operative effect by increasing the maximal value of the labelling index to 40% in the same time period and decreased by about 10-fold the concentration of prostaglandin F2a required for its maximum effect [96]. Similar results have been observed with fibroblast growth factor, hydrocortisone in Swiss mouse 3T3 cells [47,82] and epidermal growth factor, insulin in 3T6 cells [88]. A complex co-operative interaction between fibroblast growth factor, and insulin and hydrocortisone is also observed in Swiss mouse 3T3 cells [82], while hydrocortisone markedly inhibits the increase in the labelling index produced by prostaglandin F2a with or without insulin in the same Swiss mouse 3T3 cells [90]. A tentative explanation for these two effects of hydrocortisone is offered in Section IXA. When prostaglandin F2a was added to quiescent Swiss mouse 3T3 cells and the incorporation of [3H]thymidine into DNA was measured after a given time as a function of different concentrations of prostaglandin (Fig. 1) [97] a sigmoidal curve was observed, while in the presence of physiological concentrations of insulin the curve was hyperbolic. Precisely the same curves were seen if the fraction of cells synthesising DNA measured by radioautography were plotted as a function of different prostaglandin F2a concentrations. However the use of scintillation counting methods reduced the variability between duplicate samples since these methods sampled the entire population of cultured cells rather than small subpopulations observed by radioautographic methods. The nature of the sigmoidal relationship between the incorporation of thymidine and the concentration of prostaglandin F2a suggests that the stimulation of a cell population by a growth factor may be a co-operative phenomenon by analogy with enzyme kinetic analysis [98]. When the results were plotted according to the Hill equation, the coefficient of interaction for prostaglandin F2~, alone was 1.8 and for prostaglandin F2~ with insulin was 0.98 (Fig. 1). Similar results were obtained by radioautographic means. The changes of value in the Hill coefficient produced by the presence of the polypeptide hormone suggest that insulin may have a positive co-operative effect upon the stimulation of DNA synthesis by prostaglandin F2u as shown in Fig. 1. Similar results were observed with insulin and increasing

100 1,0 20

0,6 0,4

/

PGF2 o( + // / ~ insulin

o x

0,2 u

:0

S

/c E

.=

10

/ O,O6

o,o,[

o/' ~ . . PGF2 ~

/ L

¢

PGF2 oc + insulin

..=

~/ ~

/ ' /

PGF2 c~

f 0,02

0,01 0,1

0,2 PGF2c((nM)

04

0.6

0,004

0008 0,01

0,02

0,04

pGF2 ~(n M )

Fig. 1. Stimulation of DNA synthesis by different concentrations of prostaglandin F2a with or without insulin. (A) Increasing concentrations of prostaglandin F2a (PGF2a) (D) or prostaglandin F2a with a fixed concentration of insulin (50 ng/ml) (s) were added to quiescent Swiss 3T3 cells, the cells were exposed to [3H]thymidine for 28 h and the cpm incorporated into DNA per 2.5 • 105 cells were recorded [97]. (B) The above results are expressed according to the Ilill Plot, log 10 [PGF2a] against log10 m i / M i - m i [98]. m i = incorporation of [3H]thymidine into DNA during 28 h for each concentration of prostaglandin F2co Mi = maximum incorporation of [3H]thymidine during the same time period for saturating concentrations of prostaglandin F2a. Similar, although not such detailed results were obtained if the corresponding fractions of cells synthesising DNA were plotted instead of [3H]thymidine incorporation into DNA.

concentrations of fibroblast growth factor [97]. Although this analysis and the Hill equation were formulated for the quantification of the interaction between ligands in enzyme kinetics, their application may also be useful here, although at which step the interaction takes place between prostaglandin F2~ and insulin has not been ascertained. It could, for example, be at the level of binding to the receptor or equally on some subsequent metabolic step.

IIIC Negative growth factors and hormones Hormones such as adrenalin and glucocorticoids can exert inhibitory effects on the proliferation of mouse epidermal cells [99], adrenalin can selectively depress the proliferation rates of lens epithelial and corneal cells, while both hormones have little or no effect in some tissues composed of cells with higher proliferation rates [67]. From this, Bullough has suggested that these hormones co-operate with tissue-specific chalones and have little effect alone, and hence they are more active inhibitory agents in those tissues of slowly proliferating cells which are thought to produce more chalone-like material [67]. This hypothesis would then place adrenalin and glucocorticoids in the role of synergising hormones to the negative growth regulators or chalones, just as insulin and glucocorticoids synergise with positive growth regulators such as fibroblast growth factor. However, the loss of adrenalin and/or glucocorticoid receptors from the more quickly growing tissues could also explain their lack of effect without the need for the involvement of tissue-specific chalones. Once pure negative regulators of cell proliferation are

101 isolated from tissues, then this should allow a study of their interaction with hormones in defined tissue culture systems. IV. The cell cycle during constant proliferation rates and methods of altering these rates

IVA. Models for the cell cycle The cell cycle can be divided up on a temporal basis into specific phases. The G~ period is the time after mitosis and before the initiation of DNA synthesis, while S, G2 and M are the periods cells spend in the DNA synthetic phase, in the gap between the DNA synthetic phase and mitosis, and the time in mitosis, respectively. A number of relationships are theoretically possible between observable events in the cell cycle. (1) A direct causal connection between successive events, or 'domino theory' [100] (dependent sequence). (2) The events are independent but there is a clock which initiates them in sequence (independent, single timer sequence) [14]. (3) The events are independent but each has a separate timing mechanism starting at the same time (independent multiple timer sequence or 'race track theory') [100]. In any of the above, two or more pathways can run in parallel and pathways can branch or converge [14]. The cell cycle of cultured fibroblasts often approximates to a single dependent sequence of DNA synthesis, nuclear and cell division (Section VA). Cell cycle sequences, however, must involve timers. In dependent sequences the timers may be the steps themselves, but in independent sequences the timers may have to run for the length of the cell cycle and then become synchronised for the next cycle. Two models for timers have been proposed, (1) linear reading theory, a linear transcription along the genome [101] with the timings being dependent on the movement of RNA polymerase molecules along the DNA and (2) the transition theory, where the build up of critical substances or structures triggers important events in the cell cycle [102-108]. These two models may not be mutually exclusive and may apertain to different parts of the cell cycle. The latter model has, however, proved more popular, the simplest form of which, a single first order transition or stochastic model has been postulated for mammalian cells in different situations [ 1 0 9 113] and is described below. Most of the variability of cell cycle times for many cells growing under varying or constant conditions arises in the time they spend in the G1 interval, the remaining phases of the cell cycle are of relatively fixed duration [4,114-I 17]. In a detailed analysis, Burns and Tannock [112] have proposed that this variability can be described by assuming that such cells enter an indeterminate phase ('Go') which is part of GI and from which they proceed to the determinate phase ('C', or S, G~, M and remainder of G~ of constant duration T) at random with a constant specific rate 3'. That is the random loss of cells from 'Go' to 'C' phase follows first order kinetics and the rate, dNGo/dt = --3'NGo- NGo is the number of cells in Go, and 3' is then the first order rate constant. Some experimental evidence in support of this was obtained from the fraction of cells continuously radioactively labelled with [aH]thymidine and from labelled mitoses in the hamster cheek pouch epithelium, when cells were slowly growing under constant conditions. A major advantage of this model is its simplicity, only two parameters 3' and T are required for a complete kinetic description of the cell population, if no cell loss occurred. Smith and Martin [113] late[ proposed a similar model with the following nomenclature changes 'Go' = 'A', ' C ' = ' B ' , 3' = k t r a n s , and the random but constant loss of cells from the A state to the B phase per unit time was termed the 'transition probability' (P). P = (1 - e-'r). Support

102 for this model was obtained from the distribution of differences in the intermitotic times of sibling cells [ 118]. Detailed experimental evidence for a probabilistic or stochastic theory was obtained from the distribution of intermitotic times of an androgen responsive mouse mammary tumour cell line [119] or fibroblastic cells [120] grown at constant rates in media containing different concentrations of androgens or serum, respectively. The P or 7 values increased with increasing concentrations of serum. However, in simian virus 40 transformed fibroblasts considerable variations in the 'B' of 'C' phases were observed ( 7 - 1 2 h) at very low serum concentrations [120] and the average duration of the 'B' or 'C' phase of the cell cycle was also reduced in cells with shorter average intermitotic times. Thus the generality of a simple two parameter model [112] may be open to question even when great care is taken to maintain constant environmental conditions and an additional parameter describing variability in 'B' or 'C' phases may sometimes be required. Normally, however, it is difficult to maintain constant environmental conditions, even in a simple tissue culture system with cells grown in monolayers, for the following reasons: (1) critical components are removed from the medium in a growing population of cells, (2) the cells themselves can produce components which can modify their own growth rates, either in a positive or a negative way, and their production depends on the number of cells present, (3) when cells come into close contact with one another, then their growth rate becomes lower, although whether this is caused by contact inhibition of growth [ 121 ], topo-inhibition of growth [122], density dependent inhibition of growth [123], reductions in available cellular receptors for growth controlling agents [74], alterations in diffusion boundaries at the cellular peripheries [124] or any combination of the above has been much debated. Nevertheless, even with the above reservations the transition probability model represents at present the simplest way of rationalising much of the observed kinetic data, although simplicity itself does not constitute a proof. If first order kinetics govern the loss of cells from the 'A' or 'Go' state, then this suggests that one step (a single transition) controls the overall rate of cell proliferation. The actual relationship of the 'A' or 'Go' state to the G1 phase is as yet not entirely clear, but since a small fraction of constantly proliferating cells can enter S phase with no detectable G1 [ 125], then the 'A' or 'Go' state can be equivalent to GI. If G~ is completely equivalent to the 'A' or 'Go' state then the exponential nature of the distribution of the cells in GI would mean that a very small fraction of the cells would spend no time in GI, but would enter the determination phase of the cell cycle immediately. This may not always be the case, however, since there is also some evidence for a part of G1 being present in the 'B' or 'C' phases [112,113,126]. Thus the time a cell spends in G1 would then be a summation of a deterministic period of duration T1 (the 'true' G1 phase) plus the time spent in Go [112]. The duration of the deterministic 'C' phase would then be T~ plus the time spent in S, G2, M phases of the cell cycle [112]. The positioning of the 'true' G1 phase before or after Go is unknown [112,126]. There is no a priori reason why two or more first order transitions should not occur in the cell cycle, however, although none has as yet been detected, where the data was sufficiently extensive to test for this possibility [ 120]. The duration of the 'B' or 'C' phase varies from 9.5 h for rat sarcoma cells to a value of 2 3 h for HeLa cells [113]. Mouse fibroblastic cell lines have values of 11 to 13h, although some cells of SV3T3 show as short a time as 7 h [120]. The problem of timing the determinate ('B' or 'C') phase of the cell cycle remains, and whether this is controlled by a linear reading theory or a deterministic transition theory is unknown. However, since

103 the 'B' or 'C' phases contain the DNA synthetic, nuclear division, mitotic and cell division phases which are themselves linked in a single dependent pathway in yeast [127], then this may suggest that the determinate phase is composed of a series of sequential steps perhaps timed by a linear reading programme. The complete cell cycle may then be timed by a combination of both types of timers. IVB. Theoretical considerations when altering proliferation rates If the cell cycle for cells growing at different rates can be generally described in terms of a variable 3' and a constant phase for a given type, then alterations in proliferation rates, both in a positive or negative direction could be viewed as a switching to higher or lower values of 3' (Fig. 2). Four categories can be envisaged: (1) increasing proliferation rates by the addition of a positive regulator, (2) increasing proliferation rates by removal of a negative regulator, (3) decreasing proliferation rates by addition of a negative regulator, and (4) decreasing proliferation rates by the removal of a positive regulator. The lag phase will be tentatively defined here as the time taken for cells to go from the old to the new value of 3'. This will be defined rigorously in the next section. Whether or not this time is constant and the form of the kinetics when 3' changes is identical for regulators in one category will be dealt with in Section V, suffice it to say that this seems to be so for several stimulatory agents (category 1) (Fig. 2). Similarly, there is no a priori reason to suppose that these four different categories trigger the same intracellular pathways to elicit changes in cellular proliferation rates, and hence one is unable to predict whether the time and form of the kinetics when 3' changes will be the same in all four cases or not. It seems likely, however, that different intracellular pathways would be required in all four cases and hence the time and kinetics of the change in 3' may well be different for each case. All that is required to change growth rates is that the cells end up with an altered but constant growth rate defined by a value of 3'. Most of the work involved in detecting and purifying growth controlling agents has concentrated on agents, which when added to slowly proliferating cells, increase their growth rates (category 1 agents) and hence more information concerning their mode of action is available. Also, since the majority of the slowly proliferating (quiescent) fibro-

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Fig. 2. Schematic representation o f the transition from a low to a high rate o f cell proliferation. Cells growing either (a) slowly or (b) quickly under constant environmental conditions are represented pietoriaUy by a diagram o f the cell cycle. The fixed period B, or S, G 2 and m, ( m ) and the variable period A or G 1 (m,.,.) are shown. An increase o f the growth rate is accomplished by cells traversing an out-of-cycle pathway o f fixed duration (lag 1) leading to the new A or GI state o f the faster growing population.

104 blasts collect conveniently in the same region of the cell cycle (G1), then most biochemical work on growth control has concentrated on an analysis of the changes occurring after addition of these category 1 agents to synchronised cell populations (Section VA). These two areas will form the bulk of the remainder of this review. However, some studies have been performed under category 4 by removing serum and nutrients from cells already replicating at high rates (Section VC), while the isolation of pure negative regulators or chalones (Section IIB) is at present in its infancy. V. Regulation of the cell cycle when cellular proliferation rates are increased or decreased

VA. Relationship of rates of cellular entry into S phase to proliferation rates after stirnution of the growth of cultured flbroblasts In theory, the attainment of the final steady state rates of cellular proliferation after addition of growth regulators to cells could be achieved in one of two ways, either (1) by slowly altering proliferation rates over several cell cycles or (2) by altering the proliferation rate within one cell cycle. At present, sufficiently detailed experiments have not been performed to distinguish between these two possibilities, although the fact that large changes in proliferation rates can be achieved within one cell cycle by adding serum to or removing it from the medium of cultured fibroblastic cells has been taken as circumstantial evidence in support of the second hypothesis. Since the majority of the experimental work in changing rates of cellular proliferation has centred upon the addition of positive regulators to slowly proliferating cultures of fibroblastic cells (quiescent cultures), for the reason outlined in Sections IIA and IVB, we shall concentrate on this, but the principles probably equally apply to decreasing cellular proliferation rates. From considerations in the previous section increasing the rate of cellular proliferation in one cell cycle could arise through two, not necessarily exclusive mechanisms, either (1) by increasing the rate of cellular entry into S phase and/or (2) by increasing the rate of progression of cells through the remainder of the cell cycle ('B' or 'C' phases). In experiments performed to date the former alternative is the more usual [128]. Thus, as an example [28] (Fig. 3), when fibroblast growth factor and hydrocortisone or 10% serum were added to quiescent cultures of BALB/c 3T3 cells grown with trace amounts of serum and bovine serum albumin, the kinetics for the increase in the fraction of cells which entered S phase (cumulative labelling index) were identical to the kinetics for the increased number of cells, except that the latter were delayed by 9 - 1 0 h. Increasing concentrations of fibroblast growth factor or serum caused identical increases in labelling index and in cell number (Fig. 3). Hence, the kinetics for increased cell production were governed entirely by the kinetics of cellular entry into S, and the duration of the remainder of the cell cycle up to division (S, G2 and M) was constant. This duration was also independent of serum and growth factor concentrations [28]. Similar results have been reported with epidermal growth factor-stimulation of Swiss mouse 3T6 cells grown in the presence of trace amounts of serum [88]. Is this a universal mechanism or can cells also regulate rates of progression through the S phase and the remainder of the cell cycle before cell division occurs? There have been numerous reports of agents which can control the rate of progress through the remainder of the cell cycle e.g., low and high density serum lipoproteins [129], oligopeptides from peptone [130], a fetal bovine fraction for cultured hepatocytes [ 131 ], T3 [ 132], conditioned medium from Lx cells [23], serum fractions for 3T3 cells stimulated to synthesize DNA by fibroblast growth factor, hydrocortisone and insulin

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[133] and serum fractions and fetuin for Vero kidney fibroblastic cells grown in 'conditioned' medium [134]. However, without the use of cells which survive in completely serum-free medium, it is difficult to distinguish between the following three possibilities: (1) an agent which synergistically interacts with components in serum to increase the rate of cellular entry into S and thereby increases cellular proliferation rates, (2) an agent which promotes cell attachment since detachment of most fibroblastic cells occurs in medium without any serum and (3) a genuine factor which controls progress of the cells through S and into division. This difficulty can be overcome for Swiss mouse 3T6 cells, since the cells can be exposed to trypsin in suspension to remove adhering serum components and then plated in the complete absence of serum [135]. The usual requirement of most fibroblastic cells for adhesion and other factors in serum [27] for ceil proliferation has been lost in this cell line. Under these conditions either fibroblast or epidermal growth factors of prostaglandin F2a in combination with insulin, can stimulate the majority of the 3T6 cells to enter S phase and nearly double their average DNA content [85] provided the medium also contains vitamin B-12 [135,88]. However, the ceils do not divide but eventually die. Iron salts and/or transferrin [136] at physiological concentrations, or 1% serum, are required to be concurrently present with the growth factors before an increase in cell numbers is observed [85]. If these components are added when the cells have entered S phase and have nearly doubled their mean DNA content [85], the components are ineffective (Fig. 4). However, the precise time(s) when iron salts and/or transferrin are required remains to be elucidated. The results obtained after the original trypsinisation of the 3T6 cells are unlikely to be due to the possible loss of surface receptors for factors

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required for progress through the remainder of the cell cycle, since 1% serum, or iron salts and transferrin, added together with the growth factors immediately after the trypsinisation step can cause normal cellular proliferation. Similar findings with Vero monkey kidney fibroblasts plated in conditioned medium (previously exposed to growing Vero cells), suggested that they slowly progressed through S phase increasing both DNA and protein content and that they accumulated in the G2 phase of the cell cycle without dividing. These blocked cells were gradually dying [ 134]. In the case of the 3T6 cells in the above conditions, the rates of cellular entry into S govern rates of cell division, since the lack during the G1 phase of specific components required for progress through later stages of the cell cycle merely leads to cell death and the removal of that cell from the growing population. However, it would seem from the 3T6 data that under certain conditions two dependent pathways diverge prior to S phase and their convergence is required before cells will divide. This may be similar to the requirement of two dependent sequences to explain the cell cycle of bacteria [137] or budding yeast [127]. Whether a genuine controllable step exists to regulate the rate of progression of cells through the later stages of the cell cycle in other ceil systems is still debatable, although starvation for DNA or mitotic apparatus precursors may be expected, and is indeed observed during starvation for thymidine [133], to alter cellular rates of progression through S, G2 and M. It seems difficult, however, to reconcile independent regulation of the S, G2 and M phases of the cell cycle during transition from a low to a higher proliferation rate with the fact that under different steady-state conditions these phases are of relatively fixed duration, at least for nontransformed fibroblasts (Section IVA).

107

VB. Kinetics for the stimulation o f the rate o f cellular entry into S phase The total fraction of cells which are initiated to synthesize DNA (enter S phase) (labelling index) can be followed with time after addition of a growth stimulus. A general pattern emerges. In the case of the addition of serum, fibroblast growth factor or prostaglandin F ~ to quiescent 3T3 cells, few cells enter the S phase in the first 15-16 h, and then at the end of this period the labelling index abruptly increases. The time from the addition of the growth stimulus to the time when the abrupt increase in labelling index is observed is here defmed as the lag phase (see Sections I and IVB) (Fig. 3). In this and other fibroblastic cells the duration of the lag phase is independent of serum [82,96,128,139] or fibroblast growth factor concentrations [277]. The length of the lag phase is usually invariant for a given cell type stimulated to grow under specifed conditions, e.g., epidermal growth factor in human diploid fibroblasts [140] and in 3T6 cells [88], prostaglandin F2~ in 3T3 cells [96], and when proliferation rates are limited by deprivation of essential nutrients and the nutrients are then restored [141]. The length of the lag phase can differ between different cell types grown under approximately the same conditions, e.g., 6 - 7 h for confluent, quiescent cultures of chick fibroblasts [128], 7 - 8 h for baby hamster kidney fibroblasts [125,139], 15-16 h for 3T3 or BALB/c 3T3 fibroblasts [28,96]. The following manipulations, however, alter the length of the lag phase after addition of serum in the same cell system; (1) keeping WI38 fibroblasts for 2 - 3 weeks as a confuent monolayer without a change of medium lengthened the lag phase [142], the main alteration being in the decreased amount of cytoplasmic and nuclear transcriptional activity and (2) adding protein synthetic inhibitors lengthened the lag phase [143]. Thus, the length of the lag phase appears to depend on the type and conditons of the cell and not on the identity of the growth factor. The effect of a growth factor and interacting hormones on the length of the tag phase will be discussed in Section VIB. The increased rates of cellular entry into S seem to be those of a first order process [139]. In experiments shown in Fig. 5 most of the quiescent cells are in the G1 phase of the cell cycle [144], hence the gradient of the curves in this figure of a plot of the logarithm of the percentage of cells not synthesising DNA against time gives the specific rate, r, at which cells were leaving the G1 phase, i.e. df/dt = - r , where f i s the fraction of non DNA synthesizing cells. Normally the curve is a straight line, i.e., the specific rate, r, is a constant, and is then described as a first order rate constant, k. A detailed statistical analysis of data similar to that in Fig. 5 has shown that straight lines fit the semi-logarithmic plots well, with co-efficients of determination varying from 0.93 to 0.99, unity implies a perfect fit [93]. First order kinetics have been observed after addition of serum to quiescent cultures of baby hamster kidney fibroblasts with adenosine [139], Swiss 3T3 cells with inosine [145], Swiss 3T3 cells with prostaglandin F2a [96] fibroblast and epidermal growth factors [146], and Swiss 3T6 cells with epidermal growth factor [88]. However, without adenosine serum yields more complicated kinetics in baby hamster kidney fibroblasts [139], but not in some Swiss 3T3 cells [96]. Increasing concentrations of serum or prostaglandin F2a yield increasing values of k. Quiescent cultures of 3T3 are slowly synthesising DNA and have a low value of k [96,120]. These results are compatible with the single transition model (Section IVA): a single rate-limiting step or event is then suggested by the first order kinetics of cellular exit from Gx. The general organisation of events in the lag phase will be discussed in Section VIA, where additional evidence for a single rate-limiting step will be presented. However, it should be remembered that although the increased rates of cellular exit from G1 appear to be those of a first order process, nevertheless, the data may not be sufficiently extensive at this stage to exclude

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Hours Fig. 5. Kinetics of cellular exit from G 1 after addition of prostaglandin F2~. The logarithm of the percentage of cells that remains unlabeUed with [aH]thymidine (% unlabeUed nuclei) (% in G1) is plotted against time in hours. (A) Prostaglandin F2a (PGF2a) at either 60 ng/ml (:), or at 300 ng/ml (o) was added at the start or (B) prostaglandin F2a at 60 ng/ml was added at the start and then 240 ng/ml was was added after 5 h (o) or 15 h (,) (15a) [150]. The lag phase is the time taken to change the rate of cellular exit from G 1. A detailed statistical analysis of this type of data showing that the kinetics for exit from G 1 are first order is given in ref. 96.

all other possibilities. The failure to observe first order kinetics without purines under certain conditions may be due to an additional purine requiring process involved in the regulation of the limiting step [139]. It is uncertain if the value o f k obtained from the stimulated rate o f cellular entry into S for the first round o f cell division is the same for subsequent rounds o f cell division, or whether additional changes take place before constant growth conditions are reached. The relationship between the measurement r, the specific rate o f loss of the fraction o f [aH]thymidine unlabelled (non DNA-synthesising) cells and the measurement 7, the specific rate of loss o f cells from the ' A ' or 'Go' state is not yet entirely clear. Even if the approximation that all quiescent cells are in the G1 phase is valid, r and 3, may still not be identical for the following reasons: (1) the cells in G1 may not all be in ' A ' or 'Go' state since the possibility exists that a part o f GI may be in the determinate 'B' or 'C' phase [112,126] and (2) the time o f the step which changes 3, in the lag phase may not be the same as that for cellular entry into S, but occur at time At earlier (Section VIA). Then r at time t will be 3' at t - At. For these reasons we shall use the terminology r for the specific rate o f cellular exit from G1 rather than 3' or the related transition probability, P, in this review. I f these complications do not materialise then, in the particular examples above, increasing proliferation rates can-be viewed as a switching o f the cell population from one ' A ' or 'G~' state to another o f higher value ofT. The time to elicit the change in 3' would then represent the lag phase (Fig. 2).

VC. Regulation o f the cell cycle upon reduction o f proliferation rates When we turn to the other methods for altering the growth rates o f cells (categories 2 - 4 , Section IVB), few experiments have been performed with purified growth regulators

109 in tissue culture systems on clonal cell lines. A few experiments have been performed to lower proliferation rates by reducing the serum concentration in the medium of fibroblastic cells which were already exposed to higher concentrations of serum (category 4 of Section IVB). Thus, the rate of cellular entry into S phase was reduced in 3T3 [145] and WI-38 cells [147] after a constant period of 5 to 6 h, respectively. However, this time interval is probably an overestimate of the true time for the reduction to take place, since removal of the medium, or even washing the cell monolayers several times with isotonic medium, fails to remove growth factors and hormones bound at their receptors [148]. Thus, the kinetics of the reduction of cell proliferation measured under these conditions will always have a component which is due to the loss or destruction of the growthinducing agents at their receptors. Nevertheless, this method has established the important point that the time taken to reduce proliferation rates by removal of the positive stimulus is considerably shorter than the time taken to increase them. This suggests that the two intracellular pathways which are triggered to elicit the change are at least partially different. Since the duration of 'B' phase shows little change for different proliferation rates of 3T3 reduction cells [120], it would seem that the kinetics for the reduction in cellular proliferation rates must be those for the reduction in k. Few defined tissue culture experiments have been performed by altering proliferation rates by removal (category 2) or addition (category 3) of a negative regulator. The negative regulators seem at first sight to divide up into two classes, those which reduce growth rates by increasing the average time spent in the G1 phase of the cell cycle (G1 cha!ones: epidermal chalone) and those which lead to an accumulation of cells in the G2 phase (G2 chalones [68]). The actual kinetics of the change are unknown, but these results imply that in epidermal tissue at least, other portions in addition to the G1 phase of the cell cycle are regulatable, notably the G2 phase. If the above results with the G2 chalone were to be true, then epidermal cells would not fit the simple model outlined in Section IVA. However, experiments with pure chalones and clonal lines of epidermal cells have to be performed before this supposition becomes scientific fact [149].

VI. Temporal interactions of growth factors and hormones during the lag phase of cultured 3T3 cells VIA. Organisation of the lag phase after addition of growth-promoting substances to quiescent fibroblastic cells Most of the investigations carried out to identify growth factors and to establish their mode of interaction with conventional growth-controlling hormones upon changing the value of the specific rate of exit from G1, r, have been made by adding these mitogenic agents simultaneously to quiescent fibroblastic cells. Further information on the relative roles of these agents in determining the lag phase and the value of r can be obtained by adding them at different times. Here we shall first discuss the organisation of the lag phase obtained after adding growth-stimulating agents to quiescent fibroblastic cells and then the possible and observed modes of interaction of growth-promoting substances during this period. After the addition of serum and/or growth factors to quiescent cultures of fibroblastic cells, two distinct kinetic phases are observed: a lag period and a period in which there is an increased rate of cellular entry into S. The former period is independent and the latter dependent on serum or growth factor concentrations. The asynchronous nature of cellular

110 exit from G1 can often be described as a first order process. During the lag period cells still leave G1 at a low basal rate with first order kinetics (Section VB). In theory, the organisation of events in the entire period which are required to generate the new asynchrony of cellular exit from G~, or entry into S phase, could occur through two extreme processes or any intermediates, The new asynchrony could be generated gradually (a modified linear reading theory, Section IVA) or by changing the rate of one limiting step (single transition theory, Section IVA). Since both the old and the new kinetics of entry into S phase are first order under specified conditions, this suggests that a change in the rate of a single step rather than a gradual change is responsible. Thus, all processes before this rate-limiting step will be relatively synchronous, all processes afterwards will be asynchronous. This information can be used to position this step in the lag phase. The time of the change in the rate of the limiting step [ 126] occurs towards the end of the lag phase in 3T3 cells for the following reasons: (1) The asynchrony in the inhibition of the growth-stimulating effects of prostaglandin F2a by hydrocortisone after 3 - 4 h [150] (Section VIC) were all much less than the asynchrony of cellular entry into S phase at the end of the lag interval. (2) Addition of inhibitors of protein synthesis at the end of the lag phase caused a reduction in r within 1-3 h [143] (Section VillA). (3) Addition of insulin or saturating concentrations of prostaglandin F2a at the end of the lag phase, caused an increase in r within 2 - 4 h [96,150]. Thus, certain steps during the lag phase are probably traversed in a more or less synchronous fashion before alterations in r occur. These facts also argue against the earlier idea that the proportion of cells committed to synthesise DNA depends on the duration of the original serum pulse [ 128,151 ]. It should be noted that progress through the lag phase is not related to progress through the cell cycle.

VIB. Possible ways of organising intracellular pathways in the lag phase generated by two interacting growth-promoting substances Assuming that a linear sequence of events governs the change in r during the lag phase at a step close to the end of this period, then for two growth-promoting substances A and B, four types of organisation of intracellular pathways can be envisaged. (1) The same single pathway of events for A or B (Fig. 6al). A or B will be capable of independently triggering all the events necessary to change r and also increasing concentrations of A or B will ultimately produce a maximum value. Thus, the combined effects of A and B will be merely additive provided that the concentrations of A and B are below those required to saturate the pathways used. There is evidence that this may be the case for two growth factors prostaglandin F2~ and fibroblast growth factor in 3T3 and in 3T6 cells [152]. Inhibitory agents may produce the opposite effects along the same pathway e.g., hydrocortisone inhibits the effect of prostaglandin F2~ (Section IIIA). (2) Two separate but independent sequences (Fig. 6a2). This implies that either A or B can saturate its own independent pathway but their combined effects (A + B) even at saturating concentrations will be additive. No examples of this type are known at present. (3) Two dependent sequences (Fig. 6a3). The addition of either A or B will not be capable of changing r when added separately but only when added together e.g., fibroblast growth factor and specific nutrients [25] ; fibroblast and epidermal growth factors, prostaglandin F2c~with vitamin'B-12 in 3T6 cells [85,88]. (4) One independent and one dependent sequence (Fig. 6a4). In this type of organization the leading signals and events necessary for changing r are organized in a single major sequence initiated solely by one component A. The events triggered by the minor compo-

111

(a)

(b)

A B

B i I

A T

D

"1

D

B I I I

A

B

A

/

B [ I I

A

/ // /

B ......

r" / ] I

I

I L

I

"h

Fig. 6. Theoretical pathways generated by two growth-promoting factors. The schematic diagram represents a linear sequence of steps in an intracellular pathway generated by two growth-promoting factors A and B. (a) The two intracellular pathways can be identical (la), totally independent (2a), equal but dependent, converging before the final process or (3a) non-equal, one independent the other dependent, converging before the final process (4a), minor pathway (. . . . . -) and major pathway ( ). In (b) three possibilities for one dependent and one independent interacting pathways are represented, hormone B only interacts at the first step with growth factor A (Ib) at a specific step after growth factor A (2b) or at any step after growth factor A (3b).

nent B are arranged in a second minor sequence which is incapable o f increasing the value o f r alone but can converge with the main pathway. The events and signals operating through the second sequence may only increase the effectiveness o f the events already required along the major pathway (synergistic interaction) (Section IIIB). Hence, the presence o f the minor component B, which has no effect alone, but which will increase the maximal effect observed with A will also decrease the concentration o f A required for maximum stimulation o f r. F r o m the previous section, the interaction between a growth factor and other stimulating and/or inhibiting agents can result in three different possibilities: (1) in a contraction or extension o f the lag phase, (2) in changes in r or (3) in alterations o f b o t h parameters. In the simplest case, when A = B, a subsaturating and a saturating concentration o f prostaglandin F2a yield two values o f k without changing the duration o f the lag phase (Section VB) (Fig. 5a). When a synergising hormone (e.g. insulin), nutrients or inhibitory

112 PGE 1

l L._G

Effect on

,Jl~ DNA Synthesis , II II IIII

iII~

7: ° Insutin

I

0

I

5

I

10 Time (hrs)

I

15

Fig. 7. Schematic representation of the times during the lag phase when prostaglandin F2a and hormones can interact. - - , times of additions of the growth-controlling agents, which give immediate positive (=) or negative ([]) alterations in the value of the rate constant k, at the end of the lag, - . . . . . times of additions which, at the end of the log phase, provide gradual increases in the specific rate of cellular exit from G1 (r). These became constant again after a few hours. The numbers represent the hypothetical signals delivered by prostaglandin F2tx. PGF2a , prostaglandin F2a , HC, hydrocortisone. Experimental data on lag times and k values can be found in refs. 96 and 150.

agents (e.g. hydrocortisone and prostaglandin El) are added at the start of the lag phase with prostaglandin F2a under specified conditions in Swiss mouse 3T3 cells, the net result is always to alter only the value of k [96] (Fig. 7).

VIC Temporal nature of the interaction of growth factors and hormones The two categories of dependent intracellular pathways (categories 3 and 4) can each be further subdivided into 3 subcategories according to the time of interaction of A and B (Fig. 6b): (1) At the start (Fig. 6bl). Thus, the cell has only one temporal option to integrate and co-ordinate the signals delivered by A and B. This is basically the model of the single restriction point proposed by Pardee [141], based on the evidence that the same time is required after alterations in nutrient or serum concentrations before increases in r are observed. (2) At a specific time in one pathway (Fig. 6b2). Thus, in category 4, the growth factor A triggers the sequence of intracellular events leading to changes in r, while positive or negative minor agents B can only act at specific times during the major pathway. (3) At any time in one pathway (Fig. 6b3). Thus, in category 4, the growth factor A triggers the sequence o f intracelluiar events leading to changes in r, while positive or negative minor agents B can interact at any time in this pathway. As an example, in the simplest case when A = B, a subsaturating concentration of prostaglandin F2~ will cause a small increase in r. Addition o f a saturating concentration alone or 5 h after a subsaturating concentration produces the same lag phase measured from the time of the first addition. Addition of the saturating concentration o f prostaglandin F2~ at the end of the lag, however, produces a gradual increase in r until it reaches a constant value, k and this is approximately the same final value as obtained above

113 (Fig. 5). These results can be interpreted by postulating the existence of two signals that are required to change r. The chemical nature of these signals is discussed later. One signal, signal (~) initiates an out-of-cell cycle phase, the lag phase, of 15 h duration which is independent of the concentration of prostaglandin F2a in the range tested. A second signal, signal (~), determines the new value of k. Furthermore, signal (2) need not necessarily be delivered at the start of the lag phase with signal C) (Fig. 7). An example of the second type of interaction, that at a fixed time during the lag phase, is the inhibitory effect of hydrocortisone with prostaglandin F2a. The glucocorticoid only inhibits the effect of prostaglandin F2a if added during the first 3 h after the addition of prostaglandin F2~; at later times it is without effect. An example of the third type of interaction, that at any time during the lag phase, is the effect of insulin with prostaglandin F2a. Addition of insulin up to 8 h after prostaglandin F2a produced the same kinetics for changing r as when they were added together. When insulin was added near the completion of the lag phase, the value of r gradually increased until after 9 to 10 h it was nearly the same as if insulin had been added at the start (Fig. 7). An increase was observed as early as two hours after addition of insulin, however. Thus, the two separate events triggered by prostaglandin F2a and insulin can be rapidly integrated when insulin is added at the end of the lag phase. The growth factor, prostaglandin F2~, however, determines the start of the lag phase, since if insulin is added at varying times before the prostaglandin the change in r still occurs at a fixed time after the addition of prostaglandin F2a [150]. The times of interaction of hormones with prostaglandin F2a during the lag phase are summarised in Fig. 7. Details of permitted times of addition, the .lag phase duration, and k values can be found in ref. 96 and 150. As the increased rate of cellular entry into S is still probably first order after simultaneous additions of prostaglandin F2a and insulin, this suggests that the two separate pathways generated by these agents integrate before an alteration occurs in the rate of one limiting step for entry into S. An alternative model, whereby prostaglandin- and insulin-triggered events converge after changes in the rates of two separate limiting-steps, would not follow simple first order kinetics. Even more complicated interactions between 3 growthpromoting agents can occur. Hydrocortisone inhibits completely the stimulating effect of prostaglandin F2a on r only within the first 4 h. However, insulin reverses this inhibition even when added 8 h after prostaglandin F2a and hydrocortisone. The reasons for this are unknown. VID. Future perspectives Little is known about the interaction of growth factors other than prostaglandin F2~ in the cell cycle. It is not clear if all the known growth factors will operate through the same intracellular pathway, i.e., if saturating concentrations of one growth factor will prevent any further increase in r after addition of another. It is also not clear if a description of the interaction between a growth factor and its interacting hormones is unique for all cell systems or whether different modes of interaction occur in different cell types. Preliminary experiments suggest that the mode of interaction of epidermal growth factor and insulin in Swiss mouse 3T3 cells and that in a cell variant (3T3L) may be different [153]. In the parent cell line, the mode of interaction resembles that between prostaglandin F2a and insulin, while in the cell variant neither epidermal growth factor nor insulin alone detectably increase r, but when they are added together, increases are observed after 15 h. This mode corresponds to two dependent sequences as discussed previously (Fig. 6a3). Prostaglandin F2a behaves normally in the variants in promoting DNA synthesis but epi-

114 dermal growth factor synergises with prostaglandin F2~ in increasing r, acting like insulin. This suggests that the variants allow only partial expression of the usual interaction of epidermal growth factor with the cell, the remaining part is then permitted only in the presence of insulin, for reasons unknown. These results support the contention for a two (or more) step interaction of a growth factor with its target cell, rather than a simple interaction at a unique surface receptor being responsible for the delivery of the complete mitogenic stimulus. Isolation of further mutants of this type should clarify this point. The study of the interaction of synergising hormones with different growth factors is only in its infancy. Interesting situations may arise, for example, hydrocortisone offers different possibilities of temporal interaction. It inhibits the action of prostaglandin F2a only during the first 4 h in the lag phase, but it is unknown if it can interact with fibroblast growth factor only in this discrete time period or at any time in the lag phase. It may be anticipated that since insulin acts in a synergistic manner with fibroblast and epidermal growth factors and prostaglandin F2a in 3T6 cells [85], then its temporal mode may possibly be the same with fibroblast and epidermal growth factors as with prostaglandin Fza in this system. Two points concerning the interaction of a single growth factor with its target cell require further elucidation: (a) the nature of the concentration-dependency of prostaglandin F2a in eliciting signal C), the signal which initiates the lag phase; (b) the time during the lag phase when prostaglandin F2a can be removed and the cells can still be stimulated to enter S by a second addition of a higher concentration of prostaglandin F2a at the end of the lag phase (signal (~)). On point (a), it is not clear if the time of the lag phase can be increased by reducing the concentration of prostaglandin F2a below those tested so far or whether the lag is constant for concentrations above a critical thresholdconcentration. Since there is a different concentration-dependence of prostaglandin F2a for initiating the lag phase (signal C)) and for increasing r (signal (~)), then this may argue for two separate cellular receptors for prostaglandin F2a with different affinities. For point (b), it is unknown whether the cells can retain the 'memory' of the first interaction with prostaglandin Fza (signal (~)), if prostaglandin F2a is removed and then replaced, for the delivery of signal (~). However, since a brief 2 h exposure of quiescent BALB/c 3T3 cells to platelet factor made them 'competent' to respond to platelet-poor plasma at a later time to increase rates of cellular entry into S [154], then this concept of cellular 'memory' may have some validity. VII. Role of the eady biochemical events generated after additions of growth factors and synergising hormones to cultured 3T3 cells

VIIA. Identification of the early changes Growth factors and polypeptide hormones were originally only thought to interact with the surface of the cell. Hence considerable effort has been directed towards identifying the initial biochemical events and the relationship of these events to alterations in proliferation rates [155-158]. Increases in the activity of several nutrient-uptake systems occur when quiescent fibroblastic cells are stimulated to increase their proliferation rates after addition of serum, while a reduction in the influx of nutrients occurs when these rates are reduced [155-158]. Indirect evidence suggesting that some medium components may have a role in determining proliferation rates has been provided by experiments in which the concentration of certain nutrients is reduced in the culture medium.

115 Thus, reducing the concentration of phosphate, glucose, some amino acids, or omitting vitamin B-12 in the presence of serum, reduced the fraction of cells synthesising DNA in a given time to that observed in confluent and quiescent cultures. Growth rates could be increased by re-addition of the depleted nutrient, the kinetics of the increase in rates of DNA synthesis and cell division being similar to those observed after the addition of serum to quiescent cultures [25,60,88,141,159]. However, this does not necessarily imply that changes in nutrient influx and intracellular concentrations per se have an obligatory role in altering the value of r after addition of growth factors to cultured cells (Section IIA) [25], although they may enhance the effect of the growth factor in a similar permissive manner to that of insulin [60]. Here we shall discuss the identity and organisation of the early events after the addition of growth factors and hormones to quiescent fibroblastic cells and the use of such components to test for correlation between the degree of changes of these early events and the eventual increases in r. After addition of serum or growth factors and hormones to quiescent 3T3 cells, a strikingly complex array of changes in the activity of several uptake systems and in the levels of cyclic nucleotides takes place. These include stimulation of the (Na÷/K~) membrane ATPase [160-162], increases in the uptake of pyrimidin6 nucleosides [163,164], phosphate [163,165,166] and glucose [167], decreases in the levels of cyclic AMP [168172] and changes in cyclic GMP [172-175]. The above events can be classified according to their mechanism of control into four different categories [167]: (1) changes which reflect a primary interaction between growth factors and synergising polypeptide hormones, such as insulin, with the cell, e.g. the early change in the activation of (Na÷/K +) membrane ATPase, phosphate ion and glucose uptake, intracellular changes in cyclic AMP and cyclic GMP. In lymphocytes changes in fluxes of calcium ions after addition of different mitogens have been reported, and may be involved in regulating cyclic nucleotide levels [176]. In fibroblastic cells, there is at present no evidence for or against such calcium ion changes, although flbroblast growth factor can stimulate slowly growing 3T3 cells in low (50/~M or 100/AVl) CaC12 concentrations to enter S phase at faster rates [177]. The possibility remains that changes in cyclic nucleotide levels in fibroblasts could be accomplished in part by this mechanism. (2) Changes which are controlled by alterations in cyclic AMP concentrations e.g., the uptakes of uridine and glucose. (3) Changes which are probably coupled with changes in activity of the Na÷/K ÷ membrane ATPase e.g., transport of phosphate ions and certain amino acids (A or alanine group) [178,179]. (4) Changes which involve RNA and protein synthesis e.g., the late (after 1-2 h) increases in uptake of glucose and phosphate. Thus, there is no single control mechanism or pleiotypic modulator [167,180] like cyclic AMP [171] for regulating the overall activity of the diverse changes in membrane permeabilities. However, whether these changes are causally related to changes in r [181] is discussed in the next section. VIIB. Use of growth factors and hormones to establish possible correlations with rates of cellular entry into S phase Before pure growth factors became available, preliminary experiments had suggested that some of the increases in uptakes (aminoisobutyric acid uptake [182], phosphate uptake [163] and glucose uptake [183]), which occurred after the addition of impure mitogens or glucocorticoids to quiescent fibroblastic cells could be partially uncoupled from changes in rates of DNA synthesis. However, since no pure mitogens were available it was impossible to discriminate between changes in the early and late increases in uptake rates

116 and to determine which o f them correlated with changes in rates o f DNA synthesis. Experimental results obtained by adding different growth factors, such as fibroblast growth factor and prostaglandin F2a, and hormones, such as insulin and hydrocortisone, to quiescent 3T3 cells have provided evidence on possible correlations between specific components o f uptake processes and the percentage o f cells synthesising DNA in a given time (labelling index). This is summarized in Table III [161,184]. In detail, when prostaglandin Fza was added at saturating (300 ng/ml) concentrations it stimulated 20% o f the cells to enter S phase in 28 h, but it failed to stimulate uridine uptake or the e a r l y protein synthesis independent glucose uptake normally seen upon serum addition [96,184]. Prostaglandin F2a, however, stimulated the rapid increase of rubidium-86 (a radio-tracer for potassium), and phosphate uptake and also the protein synthesis dependent increase in the later or second phase glucose and phosphate uptake [161]. Conversely, insulin, which failed to increase the labelling index stimulated the rapid increases in glucose [96], uridine [164], rubidium uptake [160,161] and the protein synthesis dependent increase in phosphate uptake [161], but failed to increase the late protein synthesis-dzpendent increase in glucose uptake [96]. The synergy observed upon the value of the labelling index between prostaglandin F2a and insulin is elsewhere observed only for the protein synthesis dependent increase in glucose uptake. These results show that no single, early uptake change so far measured is strongly correlated with increasing the labelling index and hence r. Furthermore, that the triggering of the early phase of glucose, uridine, and the rubidium uptake alone are not sufficient to

TABLE III EFFECTS OF GROWTH FACTORS AND HORMONES ON THE STIMULATION OF SOME UPTAKE PROCESSES AT THE START AND OF DNA SYNTHESIS AT THE END OF THE LAG PHASE OF 3T3 CELLS Uptake rates are cxpressed as early (E) (within 1 h) and late (L) (4 h) "after additions. Saturating concentrations of prostaglandin F2c~, insulin, hydrocortisone, and fibroblast growth factor were added at 300 ng/ml, 50 ng/ml, 40 ng/ml, and 50 ng/ml respectively. The rates of uptake are expressed relative to those for no additions (100%) [96,161,184] and DNA synthesis by the percentage of cells synthesising DNA after 26 h in parallel experiments [150]. 86Rb is a radiochemical tracer for potassium ions. n.d.: not determined. Additions

None PGF2a Insulin PGF2~ + insulin Insulin + hydrocortisone PGF2a + hydrocortisone PGF2~ + insulin + hydrocortisone FGF + insulin FGF + insulin + hydrocortisone Serum (10%)

Glucose

Phosphate

E

L

E

L

100 110 310 350 350 110 * 360 * n.d. n.d. 400

100 600 350 1900 n.d. 260 * 460 * n.d. n.d. 3800

100 330 130 350 n.d. n.d. n.d. n.d. n.d. 700

100 500 370 600 n.d. n.d. n.d. n.d. n.d. 1200

* Unpublished results of Jimenez de Asua.

86Rb E

Uridine E

DNA synthesis (% labeUed nuclei)

100 196 160 176 n.d. 159 164 158 153 340

100 100 260 270 260 100 n.d. n.d. n.d. 400

0.2 20.0 1.0 55.0 1.0 2.0 22.0 50.0 80.0 100.0

117 cause alteration in r. These results also show that prostaglandin F2a and insulin can stimulate separate effects (as suggested in Section IVC), possibly at the level of the plasma membrane, but some of these effects interact, not at this level, but at a later, protein synthesis-dependent step, such as the synthesis of the glucose carrier or a membrane component which presumably facilitates the insertion of the carrier in the membrane [96]. Similar conclusions were obtained by using hydrocortisone with fibroblast growth factor or prostaglandin F2~ in Swiss mouse 3T3 cells or by using polyoma or SV40 viruses as mitogens to stimulate DNA synthesis in permissive cells. Hydrocortisone failed to change the early increase in glucose, uridine or rubidium uptake triggered by fibroblast growth factor or prostaglandin F2a with insulin despite the fact that it had opposite effects on the labelling index with these growth factors. However, there was a partial correlation between the changes in the protein synthesis-dependent glucose uptake and the eventual changes in the labelling index [184]. Similarly, infection of quiescent mouse embryo cultures with polyoma virus or BALB/c 3T3 cells with simian virus 40 caused only the late (protein synthesis) dependent increase in glucose [ 185,186] and phosphate [ 181 ] uptake. Like the early uptake changes, the role of the cyclic nucleotide changes observed after addition of serum or growth factors and hormones to quiescent cultures of fibroblastic cells is uncertain [187]. After addition of serum [168-172] or growth factors such as prostaglandin F2a [54], cyclic AMP levels fall within minutes, although under certain conditions with fibroblast growth factor and BALB/c 3T3 cells no decreases have been reported [ 173]. Reduced cellular proliferation rates accomplished by allowing the cells to exhaust serum growth factors in the medium or by the removal of serum usually [168, 170,171,188,189], but not always [188,190,191], leads to an elevation of cyclic AMP levels. Addition of high concentrations of cyclic AMP analogues or prostaglandin El, which elevate intracellular levels of cyclic AMP, cause a reduction in proliferation rates similar to that observed after removal of serum [141]. Maintaining high intracellular levels of cyclic AMP during the first half of the lag phase prevents the later increase in r induced by prostaglandin F2~ [150]. The exact contribution of changes in cyclic AMP levels to changes in proliferation rates are unknown. Indeed, results with cyclic AMPdependent protein kinase mutants in $29 mouse lymphoma cells have suggested that cyclic AMP is a nonessential regulator of the cell cycle [192]. Still less is known about the role of cyclic GMP in cell proliferation and two opposing views have arisen from experiments performed in different conditions. On the one hand cyclic GMP has been postulated to be a positive regulator of cell proliferation [193] based on the findings that addition of serum [172,174], fibroblast growth factor [173], to quiescent 3T3 cells or epidermal growth factor to quiescent rabbit lens epithelial cells [194] caused transient, early increases in cyclic GMP. Furthermore, high concentrations (10 -s M) of analogues of this nucleotide caused small increases in DNA synthesis in quiescent 3T3 cells [ 172] and in proliferation of mouse L cells [ 195]. On the other hand, cyclic GMP may have a negative role since, after, addition of serum to 3T3 cells slowly growing in heat-inactivated plasma rather than low levels of serum, cyclic GMP levels fell [175] and addition of even higher concentrations of cyclic GMP analogues (10 -3 M and above) caused reduced proliferation rates [175]. The fall in cyclic GMP in this latter system was not a consequence of different methodologies or differences in cell type [196]. In summary, there is no single initial plasma membrane-linked event observed to date after the addition of serum to quiescent fibroblastic cells which is uniquely correlated with altering r. This is not too surprising since commercial serum contains a mixture of growth factors and synergising hormones (e.g., hydrocortisone and insulin) which we have

118 suggested initially trigger separate pathways, before these pathways converge to alter r. Moreover, in the previous section, we had shown that even for a single pure mitogen, prostaglandin F2a, two signals, signal C) and signal (~)have to be generated to explain the observed kinetics for cellular entry into S. This clearly rules out the possibility of a single initial event causing these alterations in rates. Since the delivery of signal (~) and signal (~) can also be separated in time, then this suggests that triggering all the early plasma-membrane-linked events will insufficient to cause increases in r. The implications of this new type of model will be discussed in Section IXA.

VIII. Specific and general changes in protein synthesis and degradation rates and their relationship to altered rates of cellular proliferation VIIIA. Changes in general rates o f protein accumulation during transition from one proliferative state to another The proliferation of a continuously replicating population of cells must involve two distinct physiological processes, the generation of increased mass, mainly protein, and new daughter cells. To maintain a population of cells of approximately the same average mass per cell under constant conditions or during transition from one proliferative state to another, the controls on cell proliferation rates and on protein generation need to be co-ordinated in some way. If this co-ordination is to be achieved within one cell cycle in transition from one proliferative state to another, then changes in the rate of cellular entry into S should be accompanied in the same cycle by parallel changes in intracellular rates of accumulation of protein, i.e., the sum of protein synthesis and degradation. The experimental evidence for this supposition is as follows: (1) Addition of growth factors, fibroblast growth factor [28], epidermal growth factor [197], prostaglandin F2~ [96] and synergising hormones as well as serum [121,198,199] cause increases in specific rates of de novo protein synthesis before changes in r are observed. Indeed, adding varying concentrations of fibroblast growth factor or serum to quiescent 3T3 cells cause concomitant increases in protein synthetic rates measured when these attained maximum rates after 6 h, and in the fraction of cells in S phase after 30 h [281. (2) Addition of fibroblast growth factor, hydrocortisone and insulin to quiescent rat fibroblasts [200] as well as serum in this and other systems [201-203] also cause a reduction in the rates of protein breakdown, mainly of the long-lived but not the shortlived proteins. The former rate is linearly correlated with reductions in rates of DNA synthesis [200]. (3) Increased rates of protein synthesis and accumulation, and increased rates of DNA synthesis are both observed even when Vero cells fail to multiply due to a block in the G2 phase of the cell cycle, which then leads to cells with high protein and DNA content [1341. (4) The rate of protein synthesis and the value o f r rise together after adding insulin at the end of the lag phase established by an cartier addition ofprostaglandin F2~ to quiescent 3T3 cells [204]. (5) When transition from a relatively high to a low proliferation rate in human WI-38 fibroblasts is produced by reducing the concentration of serum in the medium, then within 3 h, the rate of protein synthesis is decreased and the rate of degradation increases sufficiently to cause cessation of protein accumulation. The rate of DNA synthesis is reduced after 6 h [147].

119 Thus the extracellular control of changes in the rate of general protein accumulation depends on adjustments in both the rate of protein synthesis and rate of protein degradation and these adjustments are usually observed before alterations in r occur. These observations do not prove any causal relationship between changes in rate of protein accumulation and changes in r. However the requirement for protein synthesis for increasing r is well established since antibiotic inhibitors at concentrations which are sufficient to block protein and/or RNA synthesis block increases in r [205]. In particular in HeLa cells collected at the point of entry into S by thymidine starvation and then released, both protein-synthetic and RNA-synthetic inhibitors cause a rapid decay in DNA replicase activity. A critical protein with a short half-life is postulated to accumulate close to the onset of DNA synthesis and be responsible for triggering DNA replication [206]. Whether this 'protein' is also responsible for controlling r depends on whether the HeLa cells are collected before or after the rate-limiting step, which was not determined in these experiments. However, similar conclusions for the control of cellular entry into S have been reported after addition of a protein-synthetic inhibitor to serum-stimulated 3T3 cells [143]. However, whether changes in overall rate of protein accumulation merely accompany or cause changes in r is uncertain. Nevertheless, the fact that there is a requirement for the synthesis of protein(s) for increasing r suggests that any process which affects the overall rate of protein synthesis during the lag phase may also change r. Thus the elucidation of the important biosynthetic steps in changing r may resolve into two areas, the elucidation of the mechanism which changes general protein accumulation rate, and the mechanism which changes the relative concentration of specific proteins required for controlling the step which determines the value of r. We shall return to possible mechanisms that could exert co-ordinated control over both processes at the end of this section.

VIIIB. Intracellular mechanisms which cause increased rates o f protein accumulation during the lag phase As stated in the previous section increases in the rate of protein accumulation during the lag phase are mainly produced by increases in protein synthetic rates rather than decreases in protein breakdown rates [207]. The mechanism of altering rates of breakdown of proteins is poorly understood. Indeed, whether changes are wrought in the proteins as substrates, in the activity of the degradative enzymes, or in the increased accessibility of substrate to the degradative enzymes is unknown [208]. Much greater effort has been exerted in the elucidation of the mechanism of the control of the rates of protein synthesis. In the case of BALB/c 3T3 or Swiss mouse 3T3 cells after addition of saturating concentrations of fibroblast growth factor, hydrocortisone [28], prostagiandin F2c~ and insulin [96], or 20% serum there is a 2-3-fold increase in rate of protein synthesis per mg of cell protein 6 - 9 h after the additions (also 10% or 20% serum in other systems) [ 121,209 ]. The rate is then relatively constant for the remainder of the lag phase. This represents a 4- to 6-fold increase in rates of protein synthesis per cell, since the protein content has nearly doubled by this time. The increased rate of protein synthesis during this time arises by production of more ribosomes and cytoplasmic messenger RNA per cell (2- to 3-fold) (but little increase in messenger RNA per ribosome) and an increased probability of attachment of messenger RNA to ribosomes [209, 210], since 2 - 3 times more poly(A)-containing RNA (presumptive messenger RNA) preferentially accumulates as free ribonucleoprotein particles in the cytoplasm of quiescent cells [210-213] compared with serum and fibroblast growth factor and hydrocortisone stimulated 3T3 cells. Protein-chain-elongation rates are relatively unchanged in this and

120 other systems tested [209]. Thus, the increased rate of initiation of translation of messenger RNA and the rate of the combined production of more ribosomes and messenger RNA contribute about equally to the overall increase in protein synthetic rate per cell. In other cell systems, the relative contributions may differ. Thus, after addition of serum to quiescent 3T6 cells, the net rate of protein synthesis seems to be only dependent on the rate or production of messenger RNA and ribosomes [214]. In various fibroblastic cells the generation of ribosomal RNA occurs through both an increased rate of synthesis of nuclear 45 S precursor RNA [211,215] and a cessation of 18 S and 28 S RNA breakdown [210,211,216,217]. Increased production of cytoplasmic poly(A)-containing messenger RNA, however, arises mainly from an increased processing rate of nuclear poly(A)-messenger-containing RNA [218] rather than changes in transcription or breakdown rates of cytoplasmic messenger RNA [211,219-221]; the exact contribution to the rate of production of new ribosomal RNA or messenger RNA may vary in different systems. The elucidation of changes which occur at the molecular level is only just starting. Thus, RNA polymerase I, the polymerase for synthesising 45 S precursor ribosomal RNA seems to be more active from amino acid stimulated- than from amino acid-starved Ascites tumour cells, which suggest perhaps that a permanent modification of the enzyme has occurred [222]. The most detailed changes at the molecular level have been observed in the translational apparatus, since well-defined in vitro systems which correctly initiate protein synthesis are available, whereas the study of the initiation of transcription and its control is in its infancy in animal cells. In detail, protein synthesising-extracts from quiescent cultures of 3T3 cells have lower activity than those from serum-stimulated or fibroblast growth factor- and hydrocortisone-stimulated cultures, the change in activity residing mainly in the salt-wash proteins released from the ribosome [223] and in total cytoplasmic poly(A)-containing messenger RNA [224]. Two changes in the ribosomal wash proteins from quiescent cultures have been identified so far, the reduced activity of both the initiation factor 'IF2' which binds the initiator tRNA, Met-tRNA~ et and a factor which binds capped ends (m7G(5')ppp(5')Nmp) or poly(A)-containing messenger RNA. The preferential undercapping of poly(A) + messenger RNA in messenger ribonucleoprotein particles compared with that in polyribosomes could account for the changed efficiencies of translation of messenger RNA [224]. Whether messenger RNAs are capped immediately upon entry into the cytoplasm, and then caps are removed in quiescent cultures is unknown. Since the activity of 'IF2' is increased in the brine shrimp Artemia salina, in its transition from a cyst to a growing organism [225] and since cyclic nucleotide changes can affect this activity in reticulocyte lysates in vitro [226], then for the first time a possible site of action may be established for one class of intracellular messengers, the cyclic nucleotides, which control an aspect of the growth cycle. Much more data, however, is required to clarify this issue. Other possible changes at the translational level such as destabilisation of messenger RNA after loss of cap [227], and changes in messenger RNA-discriminatory factors [228] have not yet been investigated.

VIIIC. The production of specific proteins during the lag phase prior to increases in rates o f cellular entry into S phase Before turning to the synthesis of specific proteins after addition of serum or fibroblast growth factor and hydrocortisone to quiescent fibroblasts, it should be noted that the changes in cytoplasmic poly(A)-containing messenger RNA complexity during the lag phase are negligible [211], and the majority of the sequences are the same in quiescent

121 and growing 3T6 cells, only a 3% difference or less being detected [229]. Thus, high resolution techniques on different cell fractions will be required to identify the few anticipated changes in the net synthesis of specific proteins. Studies in several laboratories have shown that there is an increase in the rate of synthesis of nonhistone-chromosomal proteins occurring soon after quiescent cells are stimulated, and before increased values of r are observed [230-233]. Using high resolution two-dimensional gel separation systems to separate the nonhistone-chromosomal proteins [234] of 3T3 cells radioactively labelled for periods of 2.5 or 5 h, O'Farrell [235] has shown that, of over 350 radioactively labelled spots, only two show large increases in intensity during the lag phase after addition of either serum or prostaglandin F2~ and insulin. The overall rate of synthesis of nonhistone proteins is increased by 2-3-fold per mg of protein by the end of the lag phase, like the overall prorein synthetic rate [96]. The first change, which occurs after about 3 h, results in an increase and then a decrease (over 5 h) in synthesis of a particular component, while towards the end of the lag phase a component (mol. wt. 32 000) is observed whose relative rate of synthesis appears to rise in parallel with the later increased rate of cellular entry into S, both changes occur after addition of either serum or prostaglandin F2~ and insulin to quiescent 3T3 cells. Hydrocortisone inhibited the synthesis of the second but not the first component with prostaglandin F2a and insulin. Changes in non-nuclear proteins have also been observed. In particular, the activity of protein(s) responsible for the protein synthesis dependent increase in 2-deoxyglucose uptake (specific protein accumulation) shows a high correlation with k [96]. However, mere correlation is not sufficient to show a causal relationship. Increases in the production of specific proteins can arise in at least four ways: (1) At the transcriptional level by specific gene derepression of a particular messenger RNA. (2) At a precursor messenger RNA processing level by selection of nuclear RNAs to be processed and dispatched as messenger RNAs into the cytoplasm [236]. (3) At the translational level by allowing the translation of previously untranslated messenger RNA [210], as seen in the early stages of differentiation of Dictyostelium [237]. (4) By altering general rates of protein synthesis or degradation which may cause preferential accumulation or destruction of proteins with different stabilities [238]. The following facts suggest that 'mechanism 1', the selective control of transcription of messenger RNA for proteins which are required for progress through the lag phase and/or for controlling the step which determines the value of k may be the most likely possibility: (1) A temperature sensitive cell-cycle mutant of Chinese hamster cells [239] causes the overproduction of a protein by overproduction of its messenger RNA and arrest of the cells half-way through the lag period at the nonpermissive temperature [240]. (2) There was a very specific requirement for messenger RNA synthesis at the same time as the synthesis of an unstable protein hypothesised to be required for the initiation of DNA synthesis [4].

VIIID. Possible relationships of specific and general protein production Finally, how can co-ordinated control of the production of specific proteins and of protein accumulation be maintained when cells are proliferating under both constant and changing conditions? Two basic schemes can be envisaged for either situation, namely (1) direct or (2) pleiotypic control, probably at the genome level. Thus, either (1) the rate of protein accumulation governs the rates of production of proteins required for controlling the step which determines k or (2) a control protein or related complex both governs the rate of protein accumulation, through the processes outlined, and specifically increases

122 the production of messenger RNAs coding for proteins required for controlling the step which determines k. Similar considerations apply in conditions where growth rates are reduced. The same mechanism need not necessarily be followed when cells are growing under constant or changing conditions. If conditions can be found either in the steady growth state or in transition between growth states where alterations in the net rate of protein accumulation occur without affecting k, then 'model 1' can be eliminated.

IX. Speculations and conclusions

IXA. Possible mechanisms for a growth factor to generate two independent intracellular pathways Our simple model envisages two independent processes going on in the lag phase after addition of the growth factor, prostaglandin F2a to cultured 3T3 cells, both processes converging before the change in the rate of the limiting step for entry into S. Since the timings of some hormonal interactions suggest that a concurrent linear sequence of events is occurring in quiescent 3T3 cells during the lag phase, then at least one of the growth factor-induced processes is probably organised in a linear sequence of intracellular steps which eventually leads to changes in r. How can two independent processes be generated by one growth factor? The growth factor could interact with (1) the same receptors but the interaction could be separated in time while the receptors became vacated for the second binding step, (2) the same receptors and generate two or more intracellular signals, (3) two independent sets of receptors in the plasma membrane and (4) one set of receptors in the plasma membrane and one set elsewhere in the cell. The fact that different operational signals in our system can be generated by different prostaglandin Fza concentrations may tend to support a two receptor hypothesis [3,4], however, surface binding studies with epidermal and nerve growth factors reveal only one class of receptor [87,148,241]. Hypothesis (4)cannot be completely excluded since there are suggestions of nuclear receptors for nerve growth factor and insulin [241,242]. Since there is little evidence at present for two functionally different sets of receptors, however, we shall only consider those models based on one set of receptors. To explain the ability to separate the delivery of the growth factors' mitogenic response in time (signal (T) and signal (~), Section VIB), the growth factor may be required either (1) at two discrete times or (2) during an extended period of time. In either case the initial interaction of the growth factor with the cell could generate a linear sequence of events, but in the second case continuous binding could be required for their maintenance. The time when the second interacting pathway became functionally operative would then be determined either (1) by the second binding of the growth factor to the cell Or (2) by becoming competent to interact only at a later specific step or steps in the primary pathway. These may not be entirely mutually exclusive. The formal biochemical distinction could be made by removing a growth factor from the cell after delivery of signal (~) which can now be easily tested. An example of the former process is the concanavalin A stimulation of lymphocyte DNA synthesis where concanavalin A is required to be present for only the first 1"or 2 h during the lag phase to deliver signal C) [243]. An apparent example of the second case is a continuous requirement for serum during the majority of the lag phase before fibroblastic cells can alter r [128,145]. However, since serum contains both growth factors and synergising hormones which may require a spe-

123 cific time period to operate [150], the interpretation of these latter experiments is ambiguous. The above two hypotheses for the one receptor model imply, respectively, that the duration of the lag phase is determined either (1) by a membrane controlled phenomenon, the reappearance of the growth factor receptors or ( 2 ) b y intracellular phenomena. These intracellular phenomena possibly operate at the level of the DNA, since it is difficult to conceive of other types of intracellular pathways that could achieve the same constancy over the extended times required [101 ]. Biochemical evidence in support of'hypothesis 1' has been obtained from binding studies of epidermal growth factor to cultured human diploid fibroblasts [148]. The epidermal growth factor binds to the cell surface and is 'internalised' at 37°C. The human fibroblasts were then capable of rebinding only a very small quantity of fresh growth factor, but the full binding capacity returned after about 10 h at the end of the lag phase [244]. The return of the binding capacity required serum and could be prevented or delayed by antibiotic inhibitors of RNA and protein synthesis [148]. Agents which disrupted microtubule assembly (colchicine) and lectin receptor mobility (concavalin A) can, under certain conditions, also block the effect of serum in increasing rates of DNA synthesis in fibroblasts [245], while a colchicineresistant clone of hamster ovary cells is also cold-sensitive for increasing rates of DNA synthesis, the lesion occurring during the lag phase [246]. These changes could possibly block the return of the epidermal growth factor receptor and thereby inhibit the stimulation of cellular DNA synthesis. Biochemical evidence in support of the second alternative is scanty. However, changes in chromatin structure are triggered during the lag phase as measured by increased dye binding to DNA [247,248,248a] and increased synthesis of RNA during [ 142] and histone messenger RNA at the end [249] of the lag phase ofWI-38 fibroblasts are observed. However, an analogous situation is encountered in the estrogen-primed-induction of ovalbumin synthesis in the chick oviduct. A constant lag of three hours is observed in this system before an abrupt change to the new rates of ovalbumin messenger RNA production occurs, although nuclear receptors are saturated with estradiol within 15 rain [250]. Palmiter et al. [250] have proposed a translocation or delayed switch model, and a similar model could apply in the case of growth factor-induced increases in r, the lag phase corresponding to the time taken to translocate the initial growth factor-induced 'signal' along the chromatin. Transcription of different RNAs during the lag phase could then be triggered at fixed times prior to the triggering of the messenger RNA which codes for the hypothetical 'protein' required to increase r. The fact that saturating amounts of prostaglandin F2a or insulin added at the end of the lag phase initially established by low concentrations of prostaglandin F2a, cause almost immediate effects on rates of protein synthesis [204] and r suggests that in this type of model, once the initial 'signal' has been translocated along the chromatin, then the chromatin is in a state such that it is immediately responsive to further incoming 'signals'. All models will have to explain the abrupt change from the old to the new rate for cellular entry into S observed at the end of the lag phase. This implies that a quick, synchronous, all-or-none event has to occur such as (1) the abrupt return of receptors to the cell surface for the eventual binding of the growth factor and delivery of signal (~) or (2) an abrupt change of state of the chromatin as a prerequisite before transcribing messenger RNA species essential for changing rates of cellular entry into S. Similarly all models will have to explain the progression from a hydrocortisone-sensitive to a hydrocortisoneinsensitive stage of the lag phase triggered by prostaglandin F2a. Both types of model

124 could possibly explain this observation. The glucocorticoid in 'model 1' would only interact to enhance the first signal at the level of the surface receptor; when this has been removed from the cell surface it can no longer interact. In 'model 2', when the initial signal from the growth factor has changed the structure of chromatin sufficiently, then the eventual interaction of the glucocorticoid with the DNA will be ineffective. Finally, either of these two models could explain the differential effect of hydrocortisone with fibroblast growth factor or prostaglandin F2~, either by causing a differential effect on these growth factors' respective receptors or by the glucocorticoid interacting in a differential manner with chromatin after prostaglandin F2~- or fibroblast growth factor-addition. More experimental evidence is required to differentiate between these and other type of models.

IXB. The nature o f the step which eventually governs the rate o f initiation o f DNA synthesis

Although a few cell cycle mutants have been isolated from mammalian cells, these have been obtained in diverse cell systems and hence an overall comparison of the mutants which affect regulation of the cell cycle is difficult. Rao and Johnson ]251 ] have applied the technique of fusing HeLa cells synchronised in one phase of the cell cycle with those in another using inactivated Sendal virus to study the regulation of DNA synthesis and mitosis. HeLa cells can be synchronised by collection of mitotic cells and when plated in fresh medium and serum there is a constant 'prereplicative' or 'lag' phase of about 8 h before appreciably increased rates of cellular entry into S are observed. These rates also seem to be those of a first order process (Fig. 8). This is ostensibly similar to the kinetics observed when k changes after addition of serum or growth factors to quiescent fibroblasts. The following differences, however, may exist: (1) It is uncertain if a low rate of cellular entry into S is occurring during the 'lag' phase of the HeLa cells, (2) the 'lag' phase of the HeLa cells may contain an additional period of cellular recovery and attachment after the mitotic selection and plating and (3) there may be possible traversal of part of the determinate phase of the cell cycle after mitosis and before S phase (the remainder of G1 outside 'Go' or 'A' state [112,113]). Nevertheless, the fact that similar, although not such detailed conclusions were obtained with mono- and bi-nucleate hamster fibroblasts after cytochalasin treatment and addition of serum [252], and by fusion of synchronised populations of mouse, hamster fibroblasts and HeLa cells [253], suggests that much of this 'lag' phase of HeLa ceils is equivalent to the lag described previously for fibroblasts. The following conclusions are drawn: (1) the initiation of cellular DNA synthesis is under positive control, it can be induced in the nucleus of a cell at the start of the 'lag' by fusion with an S phase ceil, thereby abolishing the 'lag'. Similar findings have been reported in nuclear transplantation studies in other systems [254,255]. (2) The rate of entry into mitosis is influenced by negative as well as positive controls; in a binucleate cell incomplete DNA replication in one nucleus prevents the more advanced G2 nucleus from entering mitosis. Fusion between late and early G2 cells reduces the time for the lagging nucleus to enter mitosis, but G2 cells have no stimulatory or inhibitory effects on the processes occurring during G1 0r S. Thus, cytoplasmic factors are postulated to exist both for increasing and for independently inhibiting or stimulating progress of cells to mitosis. In fibroblastic cells, however, the main growth factor- and hormone-regulatable phase occurs in G1 and not at subsequent steps in the cell cycle. Hence, the regulatable elements which are subject to external growth factor and hormone control, the putative factors

125 ~oo

ii

80 ._ 6 0 ®

\

~= 40 (o E 20

i

5

10

115

20

Hours

Fig. 8. Kinetics of cellular exit from G 1 after fusion of HeLa cells at different stages in the 'lag' phase. Data from Rao et al. [256] with permission. HeLa cells from the early or late part of the 'lag' phase were fused with Sendal virus to yield binucleated cells. Key: early 'lag' phase, mononucleates (o), binucleates from fusion of two early 'lag' phase cells (o), binucleates formed from fusion of early 'lag' phase with late 'lag' phase cells (•). The cumulative labelling index in the early 'lag' phase nuclei was followed as a function of time after fusion and release from mitosis, and the data is plotted as the logarithml 0 of the percentage of unlabelled cells (% in G1) against time.

which increase r, are of more immediate concern in these systems. Fusion of HeLa cells at different stages in the 'lag' phase has shown that (1) there are no differences between mono- and bi-nucleates of early 'lag' phase cells for the value o f k [256] (Fig. 8) and both nuclei in a cell initiate DNA synthesis at the same time [251]. Similar results were obtained with mouse [253] and hamster fibroblasts [252,253]. (2) The cells co-operate by pooling their cytoplasmic resources for the purposes of determining the length of the 'lag' phase. Thus, an early 'lag' phase cell has its 'lag' time reduced by fusion with a cell near the end of the 'lag' without affecting k [256] (Fig. 8). Thus, the inducer molecules required for the rate-limiting step for the initiation of DNA synthesis are common to both nuclei in binucleate cells and hence are probably extranuclear in origin, and the total number of inducer molecules present within a cell is important for the initiation of DNA synthesis rather than their concentration. These results support the earlier findings in Stentor [257]. The initiator for DNA synthesis probably operates in a non-concentration dependent manner, a critical threshold per cell has to be achieved within the cytoplasm before the rate-limiting step for the initiation o f DNA synthesis is triggered, rather than the concentration of the initiator per se determining the firing [251,256]. This step presumably governs the first order kinetics for cellular entry into S. This initiator activity is gradually lost during S and disappears by G2 phase. The time taken to increase the effective number of inducer molecules or lower the threshold for initiating DNA synthesis represents the 'lag' phase. This time can be shortened for cells in the early part o f the 'lag' phase by fusing them with cells near the end of the 'lag', once again suggesting the temporally linear array of events within the 'lag' phase as discussed earlier. The fact that the degree (hours) of advancement of a late 'lag' phase cell towards the end o f the 'lag' was related to the number [252] and age in the 'lag' of other cells added to it [256] indicates that progression through the 'lag' phase is unlikely to be dependent on the concentration

126 of intracellular components, but rather more complex models have to be envisaged in accord with those discussed in Section IXA. Some support for the idea of cytoplasmic control of the initiation of DNA synthesis has come from the isolation of cytoplasmic factors from early embryos or proliferating cultured cells which, when added to quiescent cells from animal tissues, increase the rate of incorporation of [3H]thymidine triphosphate into DNA [258,259]. The increased appearance of replication 'eyes' was taken as evidence of increased rates of initiation of DNA replication. Furthermore, cytoplasmic extracts of the temperature sensitive mutants of the cell division cycle of yeast which are deficient in events of the dependent pathway leading to S phase" are all capable of stimulating DNA synthesis in the nuclei of frog spleen cells at the permissive, but not the nonpermissive temperature, whereas mutants in cytokinesis exhibit the same effect at either temperature [260]. However, a yeast mutant which has an impairment in DNA replication after the initiation of DNA synthesis also shows lower cytoplasmic activity for stimulating DNA synthesis at the nonpermissive temperature. This suggests that the yeast cytoplasmic extract is acting to stimulate DNA replication processes following the cellular initiation step. In the future the study of possible cellular components which either trigger the initiation of cellular DNA synthesis or cause alterations in its rate will be of paramount importance in understanding how the intracellular pathways triggered by growth factors and hormones integrate their effects before altering rates of cellular entry into S. Their identification and isolation, however, will depend on obtaining a satisfactory test for the initiation of cellular DNA synthesis. In this respect, the complications and difficulties encountered with proving that correct initiation of DNA synthesis occurs in nuclear or cell-free DNA synthesising systems may be circumvented by introduction of extracts of growth factor- and hormonestimulated cells directly into quiescent cells, either by direct micro-injection [261] or fusion techniques [262] and observing their effects on both r and progression through the remainder of the cell cycle.

IXC. Relationship of growth factor and hormonal control of fibroblast proliferation rates with the control of proliferation rates in other cell systems In this section, we shall compare results obtained with microorganisms and animal cells in vivo with those obtained by the method of growth factor and hormonal control of events in the fibroblastic cell cycle. In a budding yeast, different proliferation rates of cells grown with different glucose concentrations are reflected in different time spent in the unbudded (G1) phase of the cell cycle, the length of time spent in the remainder is constant [263]. Transition to a higher growth rate can be obtained by transferring 'stationary' cells to fresh medium and the kinetics for the change are qualitatively identical to those in cultured fibroblastic cells: a constant lag phase is followed by a first order process for cellular exit from G1 [264]. Similar results for the increase in rates of DNA synthesis were originally observed when a bacterium was shifted from a poor to a rich growth medium [265]. In yeast [14,127] and possibly Chlamydomonas [100] DNA synthesis, nuclear and cell division cycle can be partially uncoupled by antibiotic inhibitors [14] or temperature sensitive mutations [100,127] from the growth cycle which includes the main processes of protein and RNA synthesis and the discrete synthesis of certain enzymes, a possibility discussed earlier for mammalian cells. However, a particular cell mass may be required for an early event in the yeast lag phase since small cells took longer to bud than large cells after transfer to a fresh medium [266,267]. This is possibly similar to the effects of prolonged quiescence of WI-38 cells, namely a low mean cell size

127 and a longer lag phase after addition of fresh serum [268]. Observations on the relative sizes of mother and daughter yeast cells, especially under nutrient starved conditions, have suggested that, although the step known as 'start' is dependent on cell size, subsequent stages in the cell cycle are not [267]. Thus, the relation between these two cycles requires some interlocking controls but they may be loose and can be uncoupled. Comparison of the action of growth factors and hormones in the fibroblastic cell cycle with that obtained with the equivalent cells growing in the animal has not yet been attempted. Little is also known about the control of cell proliferation in other animal tissues since many of the agents which control this process are unknown. As an example, the process of liver regeneration is under hormonal control and a variety of agents including insulin, glucagon, hydrocortisone, thyroid hormone, polypeptides and metabolites have been implicated as regulators of proliferation rates of the hepatocytes [269,270]. Insulin- or glucagon-supply could be delayed as long as 6 h after partial hepatectomy, but maximum DNA synthetic rates were still observed at the same time as if they had been continuously present [271]. This suggested that insulin and glucagon interacted in vivo in a synergistic way with an unknown blood-born mitogen during a 'lag' phase prior to an increase in DNA synthetic rates. These results were confirmed and extended in studies with cultured rat hepatocytes in that insulin, hydrocortisone and thyroid hormones have to be added during a specific time interval of 6 - 1 2 h after addition of fresh serum-free medium and inosine to attain maximum increased rates of cellular entry into S, after a constant 'lag' phase of 10-12 h [30]. The identity of the major mitogen in vivo is unknown, but epidermal growth factor can synergise with insulin and glucocorticoids in increasing rates of cellular entry into S in cultured hepatocytes [272]. Despite the stimulation of DNA synthesis, cell division occurs infrequently in these cells suggesting either that the dividing cells detach or that extra components are required to allow the cells to divide [131,272]. The original stochastic two parameter model for cycling cells was postulated for proliferation of hamster cheek-pouch epithelium in vivo [112]. However, although the control of cellular proliferation rates by positive growth factors has not been extensively investigated in different tissues of the animals the evidence suggests that more complicated regulatory processes are at work. Thus, in the mouse mammary gland, ovarian steroids seem to influence the duration of S phase [273]. The ear and skin epidermis show variations from the above model [274,275] and the fact that some chalones cannot only slow proliferation rates in the G1 phase but also in the G2 phase [68] suggests that the regulatory controlling elements and the way they affect the cell cycle in vivo may be more complicated than those established in vitro on fibroblastic-like cell lines. Similarly, additional complications may arise in renewing and differentiating cell populations in vivo where choices between self-replication and differentiation have to be made [276]. To test these ideas defined cell lines which can both differentiate and respond to interacting growth factors and hormones will have to be established in vitro so that accurate measurements of the cell cycle parameters can be made.

Acknowledgements We thank members of the Imperial Cancer Research Fund Laboratories and the Friedrich Miescher-Institut for many fruitful discussions. In particular, the authors are deeply indebted to Miss Dorothy Bennett, Drs. R. Biirk, R. Brooks, P. King, Angela Otto, Veronica Richmond, J. Smith and G. Thomas for constructive criticisms of the manu-

128 script. We also t h a n k Miss C y n t h i a D i x o n a n d Mrs. J a r o m i r a L o h m a n for h e l p f u l assist a n c e in p r e p a r a t i o n o f t h e figures.

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