The Control of Cell Division:A Review II. Special Mechanisms

The Control of Cell Division : A Review

II. Special Mechanisms

M. M. S WANN

(Department of Zoology, University of Edinburgh, Edinburgh, Scotland)

CONTENTS VIII. General discussion I. Introduction IX. The control of cell division and the cancer problem II. Differentiation and division 1. The metabolic pattern of cancer cells 1. The antagonism between differentiation and division 2. The fundamental basis of autonomy 2. The status of the growth and division mechanisms in differentiated cells I. INTRODUCTION a. Growth In the first part of this review (212), I attempted b. DNA c. Other division mechanisms to define a number of mechanisms that appear d. Discussion to be important in the initiation of division in 3. Division and growth as a form of differentiation a wide range of cell types. In this, the second 4. The differentiation problem part of the review, I shall consider to what extent 5. Further evidence on division and growth as a form these mechanisms are modified, and to what extent of differentiation 6. Conclusions further controls are imposed upon them in com munities of cells, particularly the tissues and or III. Vertebrate hormones 1. Bullough's in vitro experiments gans of higher animals. 2. Other in vitro work Within an organism, the majority of cells are 3. In vivoobservations specialized for various purposes and divide only 4. Long and short latent period responses to different infrequently. In Section II, I shall start by dis hormones cussing this antagonism between differentiation 5. The mode of action of the mitogenic hormones 6. Conclusions and division. There is some evidence that differ entiated cells have more or less completely sus IV. Control of growth in embryo and adult pended the various division mechanisms discussed 1. Compensatory hypertrophy and similar systems a. Liver in Part I; if so, the stimulation of such cells to b. Other organs and tissues divide must involve switching their synthetic ma c. The humoral feed-back type of control chinery away from making the products of differ 2. Skin wounds and limb regeneration entiation and over to making the various struc 3. Miscellaneous factors involved in the control of tural and enzymic proteins required for division. growth 4. The mode of action of regenerative and other growth One of the most important of these elements, stimuli and certainly the bulkiest, is the special protein 5. Conclusions required for the mitotic apparatus. It follows V. Plant hormones that division is best regarded as simply another 1. Auxins and kinins form of differentiation, there being no difference 2. Discussion and conclusions in principle between making proteins for some VI. Natural mitotic synchrony specialized function and making proteins for cell 1. Synchrony in syneytia division. 2. Meiotic synchrony Switches of synthetic pattern occur at intervals 3. Synchrony in cleaving eggs throughout development, being brought about 4. Mitotic rhythms in cultures of microorganisms by "induction." Having considered the nature 5. Mitotic rhythms in higher animals 6. Conclusions of this process, I shall turn to an examination of various lines of evidence which suggest that VII. Cell contact and cell division 1. Cell contact and the inhibition of proliferation the stimulation of differentiated cells to division 2. Conclusions may, in fact, be brought about in a similar way. 1118

In Section III, I shall review the problem of II. DIFFERENTIATION AND DIVISION those vertebrate hormones that stimulate cell di 1. THE ANTAGONISMBETWEENDIFFEREN vision. Although, no doubt, some of them exert TIATIONANDDIVISION effects on one or more of the division mechanisms It has long been realized that differentiated discussed in Part I, I believe that their most cells tend not to divide and that dividing cells important effect is more fundamental. It seems tend not to be differentiated. Waymouth (234) likely that they act inductively to switch the has assembled a large number of observations whole pattern of synthesis of differentiated cells and comments on this point; few people, however, over toward the making of protein for the mitotic seem to have considered why it should be so. apparatus and enzymes for a variety of other In spite of this antagonism, differentiated cells division mechanisms. are certainly capable of dividing. An embryo does In Section IV, I shall consider the control not consist of primordial undifferentiated cells, of growth in whole animals, particularly in con numbers of which, from time to time, differentiate nection with regeneration in its various forms. in various directions and then cease to proliferate. Here we have a situation where the recognized We can recognize in vertebrate tissue culture vari hormones are of minor importance, but where ous distinct cell types, all of which divide and substances, sometimes humoral, sometimes local, grow rapidly. Moreover, every tissue in the body are nevertheless involved. There is, moreover, of a higher animal shows at least occasional mi reason to think that, like hormones, these sub toses, with only two important exceptions: nerve stances act inductively to switch the pattern of cells1 and striated muscle. In the epidermis, in synthesis in a new direction. the gut lining, and in the blood-forming tissues, Sections II, III, and IV cover, I believe, the mitosis is very frequent, though the cells in ques most important aspects of the control of division tion might legitimately be regarded as not fully in higher animals, but there remain a number differentiated. However, even in obviously dif of other matters to be considered. In Section V, ferentiated tissues such as liver, kidney, and a I shall consider briefly to what extent plant growth hormones may act in the same way as the mito- whole range of glands, a few mitoses can always be found. Here again it is arguable that we are genie animal hormones. In Section VI, I shall dealing with undifferentiated reserve cells, though discuss natural mitotic synchrony; and in Section such evidence as there is suggests that these divid VII, I shall raise a few points which suggest ing cells are in various degrees differentiated (57). that cell division is in some way affected by However this may be, the differentiated cells, cell contact. I had originally intended to review except once again nerve and striated muscle, are also the questions of inhibition and stimulation certainly capable of dividing. Almost every cell inherent in gametogenesis, fertilization, diapause, in a regenerating liver, for instance, may undergo and dormancy. I believe, however, that the mech mitosis within a few days, and yet there seems anisms concerned are probably different from those to be little if any interruption of their normal employed elsewhere in higher organisms, and, since function (104). space is short, I have decided to leave out a dis The most that we can say about the antagonism cussion of these interesting, but rather specialized, of division and differentiation, therefore, is that problems. the more clearly differentiated a cell is, the less Summarizing the facts and arguments of this likely it is to divide, though, with the exception review presents certain difficulties, and rather of nerve and muscle, this capacity is never lost than adding a very long summary at the end irretrievably. I shall include a brief summary of conclusions at the end of each section. In Section VIII, 2. THE STATUSOFTHEGROWTHANDDIVISION however, I shall discuss the whole problem of MECHANISMSINDIFFERENTIATEDCELLS control in general terms. a. Growth.â€”In the first part of this review, Finally, by way of an appendix, I shall consider we saw that a certain minimum of growth was in Section IX the relevance of some of my con an essential prerequisite for division, though we clusions to the problems of cancer research. I did not inquire into the precise nature of this have deliberately restricted the discussion in this growth. However, there is no particular reason section to two matters, and I claim no particular to suppose that any type of increase in dry weight originality for my remarks. I hope, however, that will serve. Indeed, in the next section I shall they may add a little to some of the speculations 1Even differentiated neurons may in fact sometimes divide of others before me. (84, 158).

argue, following Mazia (150), that this essential is good reason to believe that this is owing to the minimum of growth may very well have to be presence of tetraploid and octaploid cells, as well synthesis of special protein for the mitotic figure as diploid ones, rather than to the presence of and other division mechanisms. Mere considera a large number of cells that have doubled their tion of the size of differentiated cells, therefore, DNA prior to mitosis. is not necessarily very illuminating and may give The evidence, then, indicates that most dif us no idea whether the essential type of growth ferentiated cells have not doubled their DNA has or has not taken place and whether, in conse and are held back or very much slowed down quence, differentiated cells are held in check by at some earlier point in interphase. The fact virtue of the growth mechanism or something else. that, as with growth, DNA in differentiated cells Some differentiated cells are certainly much does not double until a day or more after a hor larger than the undifferentiated cells that gave monal or regenerative stimulus suggests, moreover, rise to themâ€”in particular, nerve and striated that the DNA mechanism is held up at some muscle cells; but these are, in fact, the cells that very miwh earlier stage (Sections III, 5; IV, 4). never do divide. Most other differentiated cells c. Other division mechanisms.â€”In the first part are of the same order of size as the corresponding of this review, five further mechanisms were dis differentiating or undifferentiated cells; with the cussed as being probably an integral part of the exception of lymphocytes, they are certainly not division process: a nuclear PNA (pentose nucleic markedly smaller. If mere size is any criterion acid) cycle, a glutathione or sulfhydryl cycle, an for the minimum essential growth, it is possible, energy reservoir mechanism, a radiation-sensitive therefore, that differentiated cells have passed system, and a temperature-sensitive system. the critical point. The status of these mechanisms has not been in However, one line of evidence suggests that vestigated in differentiated cells. It is well known, size is not a valid criterion in this context. When of course, that PNA rises in actively growing ever fully differentiated cells are induced to divide, and dividing cells, but little is known specifically e.g., liver cells after partial hepatectomy (Section of nuclear PNA behavior. Sulfhydryl groups are IV, 1, a), none of them does so immediately, abundant in growing tissues (162), and this may or even after a short interval, as one might expect indicate that the -SH cycle in differentiated cells if the whole population were slowly growing (in is at a low ebb. On the other hand, a dose of the sense of essential growth). On the contrary, radiation that seems to be without obvious effect all the cells enlarge steadily for a day or more on the differentiated cells of amphibian tissues before they divide, suggesting that only under can nevertheless inhibit regeneration indefinite the regenerative stimulus do they start to make ly (30), suggesting perhaps that the radiation- the type of growth essential for division. Exactly sensitive division mechanism is still present. Noth comparable hypertrophy in differentiated or dif ing at all is known of the position of the remaining ferentiating cells has been noticed as a prelude two systems. for division in a wide range of other situations. d. Discussion.â€”The evidence discussed above It would seem indeed to be the rule (Tables 1 is capable of two interpretations. It may be that and 4). the growth mechanism (as defined in Part I of b. DNA.â€”Doubling of DNA, as we saw in this review) and the DNA mechanism are sus the first part of this review, is also a prerequisite pended at some point in the first hah*of interphase for division. It takes place usually in the latter (see in this connection Ref. 81). Alternatively, part of interphase, and this gives rise to a more if these mechanisms are not actually suspended, or less bimodal distribution for amounts of DNA they must be very much slowed down. In this per nucleus in the interphase cells of a dividing case, however, the later stages of interphase must population. The form of this bimodal distribution proceed much more rapidly to account for the can, in fact, be used to determine the point in essentially unimodal distribution of DNA values interphase at which DNA is synthesized (233). in differentiated tissues. DNA distributions for most differentiated tissues, On the evidence available, it is not possible however, even tissues showing some mitosis, are to make a firm decision between these two alter not markedly bimodal, but give a large peak natives. However, if we suppose that the growth around the normal diploid value, with only a and DNA mechanisms are slowed down, rather little or even no sign at all of a subsidiary peak than stopped, then we must suppose that they around the tetraploid value (138, 160, 180, 216, are slowed down to different extents in different 229). A few tissues, on the other hand, notably tissues and that, for instance, in those tissues liver, show a trimodal distribution, but there with mitotic indices of 0.1/1000 or less (Table

2), interphase is prolonged to as much as one of continuing, at any rate for a time, when at or even several years. This seems to me unlikely, least four of the other systems are blockedâ€” and it is, I believe, more reasonable to suppose namely, DNA, nuclear PNA, the temperature- that the growth and division mechanisms are sensitive system, and the radiation-sensitive sys actually suspended in differentiated cells. tem. Growth, then, is evidently not dependent To account for the few cells in a differentiated on the functioning of the majority of the division tissue that are found in division at any one time, mechanisms. Are these mechanisms, however, de there are then two possibilities. Either these divid pendent on the continuance of growth? The situa ing cells represent a definite germinal line, or tion in eggs, which divide but do not grow, strongly stem line, undergoing successive differential mi suggests that they are not, while a paper by Pardee toses2 (such cells might be regarded as the "em and Prestidge (168) suggests that, in bacteria, bryonic rests" or "reserve cells" that have been DNA doubling at least is not and that it can con postulated from time to time as being responsible tinue when growth is blocked. for adult growth, regeneration, and tumors), or It would seem, then, that no one mechanism they represent a shifting sample of the cell popu is capable of holding all the others in check. lation, selected in some more or less random If so, we must presumably look elsewhere for manner by hormonal or other stimuli. factors that can switch off the whole proliferative I believe, in fact, that the distinction between process. Moreover, if a differentiated cell is to these two possibilities is apparent rather than start dividing afresh, the same factors must pre real and that, although germinal lines of a sort sumably be responsible for switching it on again. certainly exist, they are not in any sense embryon In both cases, radical reorientations of synthetic ic cells with some special or inherent power of metabolism are likely to be involved. division. The evidence for "embryonic rests" or Such radical reorientations are, of course, com "reserve cells" is largely based on the somewhat monplace in embryology, in the process of differ equivocal experiments of irradiating various or entiation; and in the next section I shall consider ganisms and finding that they are no longer able evidence for the view that growth and division to regenerate, although their differentiated func are just as much processes of differentiation as tions are little if at all impaired. From this it the changes that ordinarily go by this name. has been argued that the embryonic cells with the power of division have all been killed or 3. DIVISIONANDGROWTHASA FORM inhibited. However, as we have mentioned earlier OFDIFFERENTIATION and as we shall see in Sections III and IV, nearly Where differentiation involves extreme physical all the cells in an animal can in fact divide, specialization, the loss of ability to divide is not following a suitable stimulus, within a day or perhaps very surprising. The mere size and shape two. It seems to me more reasonable to suppose, of a nerve or striated muscle cell, for instance, therefore, that all cells (except nerve and muscle) would seem to offer distinct objections to division. can potentially divide at any time and that wheth The case of less physically differentiated cells er or not they actually do so depends on the par is more puzzling, and there would seem to be ticular circumstances in the particular tissue. A no immediately obvious reason for the loss, albeit little thought will show that such a situation reversible, of ability to divide. It is possible, could readily give rise to "germinal lines" which however, that the reason is to be found simply are, however, not embryonic and which have in the sheer bulk of material required specifically no peculiar or unique capacity for division. Ac for mitosis and cleavage. cessibility of nutrients, for instance, might well The most obvious requirement here is, of course, account for the existence of germinal epithelia the mitotic spindle. Quite recently Mazia and in the skin and the gut lining, without the need Dan (150, 151) have shown that in the sea urchin for supposing these cells to be embryonic or in egg the spindle is formed from a distinct protein, any way different from other cells in the body. whose amino acid composition they have analyzed. Returning now to the idea that DNA synthesis, The electrophoretic diagram shows two quite fast- growth, and, presumably, the other division mech moving elements, one present in large and one anisms as well are held in check in differentiated in much smaller amounts, and ultracentrifugation cells, we must consider what might be responsible suggests a molecular weight of about 45,000. This for such a state of affairs. From the first part work makes it highly likely that mitotic apparatus of this review, we know that growth is capable protein is specialized for a single purpose only, 1References on this intriguing process are given by Ris and though this seems never to have been consid (182). See also an interesting paper by Danielli (5-t). ered before as a possibility, it is probable enough.

The spindle, after all, has a highly specialized for the greater part of the volume of a dividing contractile function to perform. cell. A part, moreover, will be taken up by systems, It remains to be seen whether mitotic figures e.g., of glycolysis and respiration, common to all from other cells prove to consist of one or two types of cells. The fraction of the cell mass then distinct proteins. If they do, as seems likely, there left for differentiated functions must be small. are some extremely interesting implications. In It follows from all this that a dividing cell the sea urchin egg, Mazia finds that the mitotic must, in fact, be making large amounts of special apparatus protein accounts for about 12 per cent ized components, just as much as a differentiating of the total cell protein. In ordinary cells, however, cell; the only difference indeed is in the particular it is clear that the spindle (and asters if they type of components being made. The growth and exist) must occupy a much larger relative volume division of proliferating cells can, in consequence, than this. I have found no published measurements be considered just as much a form of differentia on spindle volume, but I have made a number tion as any other form of cell specialization. of rough measurements myself on somatic cells Two tentative conclusions may be drawn at of various types in metaphase. These give relative this point. Firstly, as Mazia (150) has suggested, volumes ranging from 10 to 30 per cent, and it it would seem likely that it is growth of mi is evident that, in some tumor cells, the relative totic protein that constitutes the essential type spindle volume is considerably greater even than of growth that is the prerequisite for division. this (see, for instance, photographs in LettrÃ©[137]). Secondly, we can perhaps see why a dividing Volume is not, of course, a very satisfactory cell should not be differentiated and why a dif criterion for estimating dry mass, but there is ferentiated cell should not divide. The two forms evidence from interference microscopy that the of specialization will inevitably compete for raw density of the spindle is much the same as that materials and space within the same cell. If, of the rest of the cell (155), and it is well known for instance, liver cells were all moving through that the spindle is free from extraneous granules a division cycle, they would need to be perhaps (211). This, together with Mazia and Dan's find twice as big as they are, and the whole liver, ings on isolated spindles, makes it probable that similarly, would have to be twice as big. It is the volume of a spindle is a reasonable measure not difficult to see that such an arrangement would of its dry mass. The metaphase spindle, moreover, be inefficient for an organ that does not need to grows considerably in anaphase (211), while, in grow, and there is, no doubt, a selective advantage the sea urchin egg, where Mazia finds that mitotic for any animal in conserving its cellular energies protein accounts for 12 per cent of total protein, and in switching cells in the course of development the actual volume of the metaphase mitotic figure from a growth and division type of activity to is only some 2-3 per cent of the volume of the a differentiated activity and in switching back cell. It is possible, therefore, that the figures again only in the case of need. given earlier of 10-30 per cent for the relative volume of the spindle are underestimates for the 4. THE DIFFERENTIATIONPROBLEM relative dry mass of spindle material. It would From the arguments of the last section it will not be surprising, indeed, if mitotic protein ac be evident that cell division should probably be counted for one fourth or even one half of the regarded as a form of differentiation. We must, dry weight of somatic cells; and if it consists therefore, examine very briefly current notions of only one or two types of protein, these are of how differentiation is brought about, before likely to be very much the largest cell constituents. going on to consider the various stimuli to division Nor is this all. It is possible that the cell surface that operate within an organism. The term "differentiation" is normally used of dividing cells is similarly specialized, since it has peculiar growth and expansion functions. If for the more or less stable changes of cell type we follow Mitchison (154) in supposing the struc that occur during the development of an animal tural cell membrane to be as much as |-1 /* or plant. A distinction is usually drawn between thick (see also 213), then, in a cell of 15 p diameter, these changes and modulations, which are the the membrane will occupy an appreciable propor more readily reversible changes that can be elicited tion of the cell volume, between 15 and 30 per cent. by particular treatments. Differentiating embry If, further, we consider the special metabolic onic cells in tissue culture, for instance, lose much systems associated with division, it becomes clear of their specialized structureâ€”a change that used that proteins concerned in one way or another to be called de-differentiation. However, it is now with the division process may very well account known that such cells, if reimplanted in an or-

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1958 American Association for Cancer Research. SWANNâ€”ControlofCell Division: A Review 1123 ganism, can only revert to their original types. Two main types of theory have been elaborated. There is, therefore, no change in differentiation, The first of these postulates self-reproducing cyto but only a modulation. plasmic units, or plasmagenes, which mutate or The distinction between reversible and irre segregate in an orderly way, under the influence versible changes of cell typeâ€”between modulation of local cytoplasmic conditions. Different variants and differentiation as ordinarily definedâ€”is not of the theory suppose the plasmagenes to be altogether easy to preserve in the light of all generated initially by nuclear genes or to be de that is now known about cell behavior in regenera rived from other plasmagenes. They may again tion. It is often supposed that the fairly ob be replicated in a functional state, or in an inactive vious physical de-differentiation that takes place state, later to be activated by "prosthetic groups" in wound healing, or the regeneration of a lost derived from nuclear genes or from hormones. organ, is only a temporary affair and that dif The second type of theory does not postulate ferentiation follows sooner or later in the original self-reproducing cytoplasmic factors, though it usu direction. However, the evidence of Wolffian re ally allows cytoplasmic particles acting as gene generation of the lens (179) and of a variety intermediaries. It supposes differentiation to be of experiments with regenerative blastemata (re brought about by competitions and inhibitions viewed in Ref. 231) suggests quite strongly that at some metabolic level, leading to anabolic states de-differentiation may be genuine and may be of varying degrees of stability. These interactions followed by re-differentiation in a new direction. are supposed by some people to take place at There may, therefore, be a steady gradation from the level of enzyme synthesis. readily reversible changes of cell type to com Direct evidence for either type of mechanism pletely irreversible changes, and, if so, it may is scanty. There are a number of quite well authen be meaningless to ask whether the change of ticated cases of plasmagenes in microorganisms type involved in the resumption of proliferation and plants, but rather few in the cells of higher by, e.g., a liver cell in regeneration and the subse animals. There are also a few cases, in micro quent inhibition when regeneration is complete organisms, that point to competitive interactions are more like a modulation or a differentiation. and one piece of evidence, a rather significant If, on the other hand, there is a genuine distinction, one, in a higher animal. This is the work of Wilde we should presumably be inclined to regard the (244), who has shown that amphibian ectodermal change to proliferation as a modulation, since cells can be made to differentiate as melanocytes by it is certainly readily reversible.3 being treated with large amounts of phenylalanine The mechanisms underlying these changes of and other compounds closely related to tyrosine. cell type, and more particularly of the less readily Presumably, this must be a case of pushing syn reversible changes involved in differentiation as thesis in the direction of melanin formation by usually defined, are very little understood. The provision of an excess of raw materials, and it trend of recent thinking has been away from argues quite strongly in favor of the competitive the old ideas of nuclear differentiation, and it interaction type of theory of differentiation. is usually held that every nucleus in an organism On the theoretical level, both theories seem has its full quota of genes. The experiments of to provide as satisfactory a picture as one can King and Briggs (121) may perhaps mean some hope for at the present time, though the plas- revision of ideas on this score, but the possibilities magene theory has been criticized, rightly, per of genuine de-differentiation and re-differentiation haps, on the grounds that it is "uneconomical" must presumably mean that nuclear differentia and introduces additional complexities. tion, if it exists, cannot be a very far-reaching It is unnecessary for us to examine these prob affair. lems in any more detail or to attempt to make For these reasons, the basis of differentiation a decision between the different theories. The is assumed to be cytoplasmicâ€”that is to say, interested reader will in any case find no shortage it is generally supposed that the cytoplasm itself of discussions on the subject. An extremely thor determines in one way or another what genes ough and lucid survey is given by Waddington shall find expression at any particular time. (231). A brief but excellent statement of the prob lem has been made by Ephrussi (70). See also 3The cancerous transformation, on the other hand, while it Trinkaus (219). may at some early stage be reversible, is effectively irreversible in the later stages, and it seems to follow all the criteria of dif One particular point, however, needs some ferentiation in the accepted senseâ€”notwithstanding that thoughtâ€”namely, the extent to which the two tumor cells are often regarded as undifferentiated. types of theory can explain the relatively rapid

and reversible switches of synthesis that, as we discovered by Fell and Mellanby (78). Cells in shall see from later sections of this review, seem the Malpighian layer of skin normally differentiate to be involved in the control of proliferation. into a squamous, keratinized epithelium. However, As far as the competitive interaction theories if treated with excess vitamin A, they can be are concerned, rapid switches cause no difficulty. switched and made to differentiate into ciliated, All grades of stability of metabolic reactions would mucus-secreting cells. seem to be possible, and a change in available In all these cases, the detailed physico-chemical raw materials or in the activity of some particular mechanisms are largely obscure. Not much more reaction could well cause a switch to some new is known of the time relations of the inductive steady state and back again. process, although by definition the inductive stim The plasmagene theory does not so readily ulus produces effects which last much longer than account for these effects. It was indeed evolved the stimulus itself. Weiss and James (241), signifi largely in order to explain the relatively irre cantly, have re-examined Fell and Mellanby's ex versible nature of differentiation, and it accounts periment and shown that treatment with Vitamin satisfactorily enough for the steady progress of A for as little as 15 minutes is sufficient to produce cells from an undifferentiated to a fully differ the switch of differentiation. entiated state. No doubt it can be modified by supposing plasmagenes to be activated and inac 5. FURTHEREVIDENCEONDIVISIONANDGROWTH tivated in some way, but in so doing the theory ASA FORMOFDIFFERENTIATION becomes rather elaborate and cumbersome and We have already considered various reasons loses the attractive simplicity with which it began. for supposing that differentiated cells have ceased Differentiation in the course of development to make mitotic protein and that their various is brought about by a process generally known division mechanisms have been brought to a halt. as "induction." A wide variety of factors is known From this we have argued that the resumption to be involved (see [97, 231]). In the egg, particular of division must involve a major switch of syn substances may be localized in different regions thetic activity in every way comparable to the and may direct differentiation in various ways. switches involved in the process of induction, There may also exist gradients of substances, by which differentiation is brought about in the or of intensity of metabolism, controlling the course of development. Before considering the manner of differentiation. The nature of these sub various ways in which growth and division are stances and gradients is largely unknown, though controlled within an organism, I want to examine the numerical density of mitochondria seems to certain lines of evidence that would seem to sup be important. In later development, effects of port the idea of control of division by induction. particular regions (organizers) on adjacent regions There is, first of all, some evidence that the are widespread. Early work suggested that these proteins of rapidly dividing cells differ from those organizers liberated small molecular weight com of differentiated cells. Sorof and Cohen (202) pounds (evocators), possibly steroids. More recent have examined the soluble proteins of differen work, however, suggests that nucleic acids and tiated, embryonic, and cancerous liver cells elec- cellular contacts in some form or another are in trophoretically. They find that in the differen volved. There is evidence that transfer of particles tiated cells there is a variety of proteins, par between neighboring cells (infective transforma ticularly large, slow-moving ones, but that tumor tion) may occur in certain situations. The impor cells and embryonic cells show a quite different tance of mutual cellular inhibitions has also been pattern, with mainly smaller, fast-moving ele stressed (186). ments. It is at least possible that these are the A variety of simple chemical substances is mitotic proteins which, it will be remembered, known to affect the manner of differentiation. are small and fast-moving in the sea urchin egg Two of the best known of these are lithium and which will, of course, be much more prominent and thiocyanate ions, the former switching dif in embryonic and tumorous liver. It should be ferentiation toward endoderm (vegetalization), the pointed out, however, that these authors did not latter towards ectoderm (animalization). An in find the same marked change in regenerating teresting, and experimentally amenable, case of liver (201), though one would expect the change this phenomenon has recently been described by to be much less pronounced in this case. Willmer (245), who finds that the protozoan Nae- Secondly, there is the point, mentioned earlier, gleria can be caused to grow either in an amoeboid that differentiated cells hypertrophy for some time or a flagellate form by manipulating the ionic before they divide as a result of a hormonal or environment. Another interesting case has been regenerative stimulus. It is tempting to regard

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1958 American Association for Cancer Research. SWANNâ€”ControlofCell Division: A Review 1125 this as the period of manufacture of new mitotic Other explanations of course are possible but, protein and enzymes for the various division mech I think, less probable. It might be argued, for anisms. If we are right in supposing that differ instance, that one of the growth or division mech entiated cells have ceased to make these com anisms was lacking some key metabolite which ponents, then it is likely that, in cells that have was needed continuously, but only in small quan not divided for some time, these components will tities, and sufficient of which could be taken have been largely broken down and lost and that in rapidly. Against this we must remember that synthesis will have to start afresh, effectively from the whole growth and division system seems to zero. Synthesis would be expected, then, to take come to a halt in the absence of specific stimuli at least twice as long as in a normal interphase. and that some cells (e.g., wandering cells and In fact, interphase in tissue culture takes from some tumor cells) can, in fact, divide in serum 8 to 18 hours, and the latent period in regeneration, alone, without embryo juice. Serum, in short, etc., is always 24 hours or more (Tables 1 and 4). appears to contain all that is necessary to carry It is interesting also to find that the latent period out division, but not all that is necessary to of migration (which appears to be related to the la stimulate it, at least in most cells. tent period for mitosis) is always more for the adult One final point needs mentioning at this stage. than for embryonic or cancer cells in tissue culture If it is true that the cells in a higher organism (62, 110). This could well be because cells in are normally quiescent and need an inductive embryonic tissue will have divided recently and stimulus to activate their growth and division will be likely, therefore, to contain an appreciable mechanisms, then it would seem to follow, from amount of mitotic protein, whereas adult cells Jacoby, Trowell, and Willmer's experiments, that will probably not have divided for some time the stimulus is only effective for a single cell gen and may, therefore, contain much less, or even eration. Indeed, if this type of induction were no mitotic protein at all. to produce an effect that lasted for more than A further line of evidence of very great interest a few cell generations, we should not have a comes from the work of Jacoby, Trowell, and mechanism of growth control at all. There is, Willmer (118) on the mode of action of embryo therefore, a significant difference between induc juice in tissue culture (see Part I, Section IV, 1). tion of division and the usual types of induction, These authors isolated a tissue fragment in serum whose effects are more or less permanent. alone, so that it showed no mitosis. They then found that embryo juice needed only to be added 6. CONCLUSIONS for an hour or even less to produce a crop of Differentiated cells appear to be held back mitoses some 10-20 hours later. Further addition from division at some point well before the dou of embryo juice had no effect, until some 16 hours bling of their DNA. There is evidence that most later, when it would produce a fresh crop of if not all the other growth and division mech mitoses starting after a further 10 hours, by acting anisms are also held back, or more probably on cells that had just completed a mitosis. In suspended completely, in differentiated cells. other words, the essential factor or factors in One of the most important of these mechanisms embryo juice are required and taken up in only appears to be synthesis of protein for the mitotic a short period of time, long before mitosis itself. figure, and there is some evidence that in dividing The cells are then stimulated to complete one somatic cells this protein may account for a large mitosis, after which, however, they become qui fraction of the cell mass. If other elements re escent again, unless they receive a second dose quired for division are added to this, it seems of stimulant. Data from these experiments are probable that the materials required for division included in Table 4. may occupy as much as half the cell. It is suggested This work has often been quoted, mainly, it that this accounts for the well known antagonism seems, by way of pointing out the curious nature between division and differentiation, and that of stimulation by embryo juice. As far as I know, division, which involves the synthesis of a large however, its significance has not been appreciated. bulk of specialized material, should in fact be It seems to me that it may, in fact, provide regarded as a form of differentiation. the key to the control of growth and division Differentiation during development is brought within an organism, since this behavior is exactly about by a process known as induction, the es what one would expect of an inductive mechanism. sence of which is that a short-lived stimulus of It should operate for a short time, initiate a some sort produces a relatively long-lasting effect series of synthetic processes, and, thereafter, have on the pattern of synthesis. A number of lines no effect. of evidence suggest that the stimulation of differ-

entiated cells to divide is in fact an inductive of growth hormone in the body is to call forth process, though with one important difference the production of insulin, which is the real growth from embryological inductionâ€”namely, that the hormone. effect lasts for only one cell generation. c. Adrenalin.â€”Both adrenalin and adreno- chrome inhibit mitosis, with either glucose, fruc III. VERTEBRATE HORMONES tose, lactate, or pyruvate as substrates. Though A considerable number of the recognized verte adrenalin is reported to activate phosphoryla- brate hormones have effects on growth; but, except tion of glycogen and to inhibit glucose uptake, in the most general termsâ€”for instance, that Bullough argues that it must affect some post- anterior pituitary growth hormone leads to nitro pyruvate aspect of energy metabolism as well. gen retentionâ€”almost nothing is known of how It should be pointed out, however, that it might they stimulate cell growth and cell division. I well inhibit aspects of the division mechanisms do not propose to consider more than a very quite unconnected with energy metabolism. small fraction of the biochemical work that has d. Adrenocorticotrophic hormone.â€”ACTH pro been done on these hormones, nor shall I attempt duces a moderate stimulation of mitosis with to review even the main current hypotheses of glucose as substrate, but not with fructose or hormone action. These bear little relation as yet pyruvate. In view of this and its reported effects to the problems of the control of cell division, on stimulating respiratory rate, Bullough inter and there is in any case no shortage of expert prets the mitotic stimulation as the result of a surveys in the annual review journals, in the glucokinase activation. reports of conferences, and in books. e. Cortisone.â€”This hormone depresses mitosis 1. BULLOUGH'S in Vitro EXPERIMENTS with glucose, fructose, or pyruvate as substrates. Although there is considerable evidence that cor Very few people, it appears, have made a sus tisone and other glucocorticoid hormones depress tained attempt to examine the effect of hormones carbohydrate usage, it appears that their action on cell division itself. Bullough, however, has on mitosis cannot be at the glucokinase level, worked extensively in this field, and we may and Bullough assumes that they interfere at some usefully begin by considering his conclusions. point in energy metabolism after pyruvate; but, Bullough's work on the energy relations of as with adrenalin, it should be pointed out that mitosis has already been discussed at some length the effect might be on mechanisms other than in the first part of this review. It will be recalled energy metabolism. that he isolates small fragments of mouse ear f. Estrogens.â€”These hormones stimulate mi epidermis in saline and finds that only glucose, tosis in the presence of glucose, but not fructose JÃ¡ctate, or pyruvate, is required to bring a number or pyruvate. In view of a fair amount of reported of cells into mitosis. He concludes that a propor work that estrogens stimulate respiration and car tion of cells are in a stage of their cycle just prior bohydrate metabolism, Bullough interprets this to prophase (the "antephase") and need only as an action on glucokinase, resulting in more a supply of energy to complete the division energy for the cells in antephase. process. He also finds, testing a number of substances, Using the same technic, Bullough (33) has in that estrogenic and mitogenic potency are not vestigated the action of a number of hormones. closely correlated, so that the glucokinase effect, His conclusions may be summarized as follows: leading to increased mitosis, cannot presumably a. Insulin.-â€”Stimulates mitosis only in the pres be the essential feature of estrogenic action. ence of glucose, producing, at the optimum con Testosterone is found to be without effect on centration, a doubling of the number of cells ear epidermis in vitro. entering mitosis in a 4-hour period. Since insulin is now believed to facilitate the passage of sugar Before going on to examine other lines of evi dence, we should consider Bullough's general the through the cell membrane (139), its effect is likely to be one of making more energy available sis that the hormonal control of mitosis is mediated for cells in antephase. through the energy supply from sugar metabolism. b. Pituitary growth hormone.â€”With glucose as Can this be regarded as the primary mode of substrate, but not with fructose, massive doses action of all these hormones, or is it rather a of growth hormone depress mitotic activity. Bul secondary action that happens to be particularly lough suggests that this is owing to its reported important in the circumstances of the test? There effects in inhibiting glucose uptake and inclines are, I believe, a number of considerations that to the view of Young (247) that the function should make us cautious in supposing that energy

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1958 American Association for Cancer Research. SWANNâ€”ControlofCell Division: A Review 1127 supply is the universally important mechanism stimulation of "antephase" cells, can hardly be of control. more than a subsidiary effect. In the first place, Bullough is concerned only In conclusion, then, while we can accept the with the effect of hormones and metabolites on results of Bullough's experiments, we cannot ac mitosis in the first few hours after isolation of the cept his thesis that the various mitogenic hormones ear fragment. Ear epidermis, moreover, or rather act on interphase cells, solely via energy metab the Malpighian layer, is a population with a olism. We have to examine the effect of hormones fairly high proportion of dividing cells. Bullough on cells, not only at the very end of interphase, has already shown that some of these cells are but at other points in the cycle. Do hormones in "antephase" and need only a supply of energy speed up cells that are already moving through to divide. Any hormone, therefore, that stimulates a cycle, or do they switch differentiated non- metabolism, however secondary this effect may dividing cells into division? And are these two be, will, in the circumstances of the test, appear types of stimulation, if both exist, effected by to be mitogenic as the cells in antephase move different hormones? These and other problems into mitosis. are discussed in the sections that follow. What the hormone may do to the much greater number of cells that are not in antephase cannot 2. OTHERin Vitro WORK be discovered with Bullough's technic. To find Tissue culture methods have thus far given out, it would be necessary to bathe the cells disappointingly little useful information of the in a nutrient medium and follow mitosis over mode of action of hormones on growth and divi a much longer period. sion. It is indeed by no means easy to demonstrate The nature of the experiment, in short, deter in vitro hormonal effects at all. There are various mines that it shall pick out hormonal effects that possible reasons for this. In the first place, cells are due solely to energy metabolism and that normally lose their differentiated status in greater operate on cells very near to division. It is possible, or lesser degree when cultured in vitro and may, of course, that an increase of energy metabolism therefore, also lose some of their capacity for would similarly speed up cells at earlier points reacting to hormones. In the second place, to in the division cycle, though this has yet to obtain easily measurable growth in culture, it be proved. It is possible, as we shall see later, is usually necessary to add embryo extract, which that factors other than energy metabolites are almost certainly contains a mixture of hormones limiting at earlier stages. already. The net result, at any rate, is to achieve A further point is that even if energy me a population of cells dividing every 10 or 20 tabolism were to be established as the limiting hours, with a mitotic index of about 40/1000, factor in the earlier stages of division, it would whereas few tissues in the body have mitotic presumably lead to increased mitotic activity only indices of more than 1-2/1000, and many of in tissues where most or all the cells were moving the more static organs have indices of 0.01-0.1/ through a division cycle. As we have already seen 1000 (Table 2). The conditions of ordinary tissue (Section II, 2) there is no reason to think that culture in short may be quite inappropriate for in most tissues the bulk of cells are more or demonstrating hormone action. less differentiated and are not moving through The outlook for the future, however, is more a division cycle. In such cases, an increase in promising. It is now possible to grow a variety energy metabolism would be expected rather to of tissues in such a way as to preserve their lead to increased differentiated activity. If we differentiation (75, 82, 220). Technics for growing are to accept energy metabolism as the universal free cells in fluid media, and more particularly control, we should then have to postulate that in defined media, are also developing rapidly, some form of switching of energy from one channel so that the measurement of cell growth and cell to another was involved in such tissues. division in controlled conditions will become in Finally, anticipating Section III, 3, below, it creasingly easier (references in 25, 156, 235). should be mentioned that nearly all the critical experiments on the mitotic effects of hormones Existing work on the effects of hormones on in vivo show a clearly defined latent period of tissue cultures is reviewed by Waymouth (235), 24 hours or more before there is a burst of mitotic and a number of observations of hormone action activity. In most of these cases, the increase on organ cultures are considered by Fell (76). in activity is between tenfold and several 100-fold. The results are briefly summarized below: It would seem, therefore, that the twofold in a. Insulin.â€”There is fairly general agreement creases found by Bullough, resulting from the that insulin promotes proliferation, but the con-

centrations required and the effects produced are In the case of ear epidermis, the mitotic co very variable. With organ cultures of embryonic efficient of the Malpighian layer rises somewhat limb bones, it has been found to stimulate growth unevenly, after about 6 hours in one case and and retard differentiation. about 18 hours in the other, to a value about b. Pituitary growth hormone.â€”Rather few ex twice that in the control. In the acinar epithelial periments have been done with pituitary growth cells of the seminal vesicle and the coagulating hormone in vitro. In general, it has been found gland, however, where the resting mitotic rate to have either no effect, or an inhibitory one, is l/100th of that in the ear, there is a clearly though slight positive responses have occasionally defined latent period of 24-36 hours, and the been obtained. mitotic index then rises sharply by several hun c. Thyroid hormones.â€”These have been reported dredfold. In the ventral prostate the response to stimulate, to depress, and to be without effect to testosterone propionate is similar, but with on mitosis. In recent work with organ cultures estradiol benzoate the latent period is longer and they have been found to stimulate differentiation the response only about fourfold. and inhibit growth (the opposite of insulin). Allen inclines to an interpretation of these re d. Adrenalin.â€”General agreement that it de sults on much the same lines as Bullough, i.e., presses mitosis. that the hormones are raising energy metabolism; e. Corticosteroid hormones.â€”General agreement but, at least in so far as the curves for the genital that they are without effect on mitosis, or de tissues are concerned, this offers no explanation press it. for the long latent period and the enormous and f. Sex hormones.â€”Both male and female sex very sudden rise in mitotic index that follows it. hormones have been found to produce some stim Such a pattern is, however, very reminiscent of ulation of mitosis in vitro by a number of workers. the response of fully differentiated, nondividing Organ culture work has shown a complex response tissues to a regenerative stimulus (Section IV). that varies with the age of the donor animal. It is possible, then, that we have two rather different situations to consider. In one caseâ€”the 3. In Vivo OBSERVATIONS Malpighian layer of ear epidermisâ€”the mitotic The amount of work on the various hormones index is high, nearly 1/1000, so that a fairly affecting growth is enormous, but very little of large proportion of cells must be going through it is relevant to our present problem. The diffi a division cycle. In this case we may suppose, culties of the in vivo approach are of course con as Bullough does, that the hormone is merely siderable, since it is all but impossible to alter speeding up the division of those cells in antephase the level of one hormone -without calling forth by means of an effect on energy metabolism. secondary effects. It is difficult, nevertheless, not There is no reason why such an effect should to feel that much of the work has suffered from cause a latent period, and the observed latent being too strictly biochemical or physiological, period is indeed brief and ill defined. It may and insufficiently cellular in approach. Few work also be noted that the measure of the response ers, for instance, have even attempted to measure (twofold) is of the same order as Bullough finds mitotic index. Still fewer have measured cell size in vitro. or made any attempt to estimate cellular growth In the case of seminal vesicle, coagulating gland, rates. and ventral prostate, however, the mitotic index Such in vivo observations on the mitogenic is far lower, about 0.018/1000, and there is every effects of hormones as have been made, however, reason to suppose (see Section II) that only a seem for the most part to fall into a fairly clear very few cells are moving through the division pattern, indicating a definite latent period of 24 cycle at all. The latent period would then represent hours or more which is accompanied by cellular the time taken to switch a proportion of these hypertrophy; this is followed by a rather sharp cells over to division and would, in turn, account and often very large rise in mitotic index (up for the sudden appearance of mitosis. In the to several hundredfold). light of the discussion in Section II one would The sex hormones appear to have been investi expect this period to be associated with a con gated more extensively in this way than most siderable synthesis of mitotic protein and, there other hormones, and we may begin with two fore, of general increase in cell size. Allen gives of the more detailed studies of this sort, by Allen no information on this point, but cellular hyper (14, 15). This work concerns the response of trophy has been reported by Burkhart (35), who various tissues from castrate male mice to single investigated the action of testosterone on seminal injections of estradiol benzoate or testosterone vesicle and ventral prostate, and Drasher (64), propionate (see Table 1). who measured, among other things, cytoplasmic

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volume in rat uterus cells during the estrous cycle.4 portant; it appears, at any rate, to be less common, Allen's results are summarized in Table 1. In and the increase in mitotic activity is only slight. it I have also included the results of such other The response is in fact very much like that in adequately quantitative experiments as I can find Bullough's in vitro experiments, and it seems likely on the effects of hormones on mitosis.6 The in enough that a speeding-up of energy metabolism formation is all too scanty, but I believe we could cause the effect by bringing cells at the end can draw some tentative conclusions from it. of interphase into division. No significant latent The most typical response, it will be seen, is period would be expected, nor does there seem a very substantial increase in mitotic activity, any reason to expect cellular hypertrophy. It ranging from 12-14-fold in the case of large doses might be added that this effect is only likely of growth hormone acting on adrenal cortex, to to be detected in tissues where there is a fairly 870-fold in the case of testosterone propionate large proportion of cells actually moving through acting on coagulating gland (Table 1: 1, b, c; the cycle, i.e., in tissues with a fairly high mitotic 2, b, c, d; 3; 6, a, b; 7, b; 8). In all these cases, index. This is certainly true of epidermis and there is a clearly defined latent period ranging crop gland. from about 1 day to 3 days, and in several cases The first type of hormonal effect, the long cellular hypertrophy has been noticed as well. latent period response, is, I suggest, the important The response is generally greatest in the tissues one, and, on the rather inadequate data available, that have the lowest resting mitotic indices. There it seems to be the more common. It certainly are a few further cases in which the response includes the quantitatively greatest effects, and, is very much smaller but the latent period never- significantly, the greatest effects are noticed in

TABLE 2 TYPESOFMITOTICRESPONSESTOHORMONES Length of Magnitude Cellular Likely mode Type of response latent period of response hypertrophy of action Long latent period 1-3 days May be very large (up to Yes Complete reorientation of several hundredfold) synthetic metabolism Short latent period A few hours Small (2-3-fold) No Speed-up of energy me tabolism theless long, and where there is some indication the tissues with low mitotic indices, where the of cellular hypertrophy (Table 1: 1, d; 4; 5). majority of cells are almost certainly not moving It would seem that this type of response is es through a division cycle (Section II). This type sentially the same as the earlier type, but merely of response must, therefore, involve stimulating less in degree. these quiescent cells and switching their metab There remain four cases that seem to be rather olism over to proliferation. In the light of the different (Table 1: 1, a; 2, a; 7, a; 9). The response discussion in Section II, this is likely to involve is small, the latent period is shorter and less the synthesis of mitotic protein and enzymes in well defined, and cellular hypertrophy has only volved in other division mechanism, and, since been reported once. Two of these cases, estradiol these occupy a large part of any cell, it is only benzoate and testosterone propionate acting on to be expected that there will be substantial cel epidermis, have been discussed earlier. The other lular hypertrophy. The long latent period must cases are the small dose of growth hormone on then represent the minimum time required for adrenal cortex and the effect of prolactin on pigeon these metabolic reorientations to be completed. crop cells. Summarizing, we arrive at the tentative conclu I believe these two main types of hormonal sions shown in Table 2. effect on mitosis in vivo should be clearly dis 4. LONGANDSHORTLATENTPERIODRESPONSES tinguished. The second type of effect, the short latent period response, is perhaps the less im- TODIFFERENTHORMONES Since the long latent period response has not 4It is known that there is a considerable intake of water in been obtained with any certainty in vitro and the first few hours after treatment with sex hormones. Later, has not very often been demonstrated even in however, there is an increase in dry weight. 6Numerous other references to effects of hormones on vivo, it is not easy to be certain which hormones mitosis (using colchicine to make the effects more readily vis can act in this way. ible) are given by Eigsti and Dustin (68). Most of them are From the data of Table 1, it is clear that inadequately quantitative for our purpose. the long latent period response is typical of the

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1958 American Association for Cancer Research. SwA@â€”Control of Cell Division: A Review 1131 estrogenic and androgenic hormones when they in weight but very little mitosis (42). Presumably, act on the sexual tissues, and the response is therefore, there must be cellular hypertrophy, usually a large one; but when they act on epi but, since this is not followed by division, it is per dermis, at least, their effect seems to be of the haps due not to the synthesis of mitotic protein short latent period type. and enzymes for division but to increased dif The thyroid-stimulating hormone similarly pro ferentiated activity. It seems, in short, that ACTH duces a response (on the thyroid) of the long may be unconcerned with adrenal growth and latent period type. involved only in the control of differentiated ad The question of pituitary growth hormone is renal activity. Any effect it may have on division, more difficult. Its effect on the pancreatic islets therefore (and Bullough has reported that it pro (Table 1, 8) is of the typical, long latent period duces some stimulus in his in vitro ear epidermis type, but it is not entirely clear from Cater tests), is likely to be incidental and of the short and Stacke-Dunne's experiments on the adrenal latent period type. (Table 1, 7) whether it elicits a long or short The status of the thyroid hormones, like that latent period response, or perhaps both. It has, of pituitary growth hormone, is in some doubt. of course, long been known that very young ani The few cases quoted in Table 1 suggest a long mals and adults are relatively insensitive to growth latent period response, implying a stimulation hormone, and it is in the middle period of growth of inactive cells; but the magnitude of the effect that its effect is most marked. At this stage, is small. On the other hand, there is reason to growth hormone does not seem to produce the think that thyroid function has been modified sudden and very marked stimulation characteristic in the evolution of the vertebrates, and it seems of the sex hormones, and this might suggest that likely that in amphibian metamorphosis, thyroxin its effect is not to stimulate inactive cells to may be more powerfully mitogenic for certain growth and division so much as to speed up tissues. This effect, however, does not often seem cells that are already dividing. This is borne out to have been investigated in a suitably quantita by such observations as those of Leblond and tive manner. CarriÃ¨re (183) and Meinecke and Crafts (152), Against this must be set the various observa who find that hypophysectomy produces only tions that thyroid may be inhibitory for mitosis a slight drop in the mitotic activity of intestinal (65, 198), and in amphibian metamorphosis may mucosa or blood-forming cells, which can be re cause actual histolysis of some tissues. Thyroid versed in some degree by growth hormone. How hormones, in short, have multiple effects on mi ever, these are rapidly growing tissues, with a tosis, and it has been shown, moreover, in a num high proportion of active cells, and against such ber of cases that their effects are most marked evidence must be set the observations of the not on mitosis at all, but on differentiation (195, effect of growth hormone on bones. Becks et al. 209). (22), for instance, find that growth hormone will The mitogenic effects of insulin in vivo seem stimulate afresh the growth of bones in rats hy not to have been investigated in detail; but since, pophysectomized up to a year previously. These almost alone among hormones, it appears to have experiments were unfortunately not quantified a fairly clearly defined activity (in facilitating in such a way that they could be included in the passage of glucose and certain other sugars Table 1, but it seems likely that the response into the cell), one can be reasonably certain that was, in fact, of the long latent period type, with it would prove to have only a short latent period a sudden and marked rise in mitotic index. Green effect, of the type found by Bullough in vitro. span et cii. (94), moreover, found that a given It remains only to mention two hormones, dose of growth hormone produced a much greater or groups of hormones, that normally, if not effect on epiphysial thickness (measured a day always,depressmitosisâ€”adrenalinandthecorti after the injection) when it was given in three costeroid hormones (37). It is evident that a or four daily doses than when it was given in quantitative study into their effects on mitosis a singledose, which may perhaps indicate a latent in vivo is needed just as much as for the mitogenic period of more than 1 day. hormones. Do they, for instance, slow down those It might be expected that another pituitary cells that are moving through a division cycle hormone, ACTH, would exert a mitogenic action or do they inhibit the activation of quiescent at least on the adrenal cortex, but this does not cells? The antagonism of the sex and corticosteroid seem to be the case. Unlike growth hormone, which hormones (184), or cortisone and growth hormone produces mitosis but no great increase in adrenal (200), might suggest the latter effect. weight, ACTH produces a considerable increase The nearest approach to a fully quantitative

study here is the work of Robbins et al. (183), on the average, as a result of an increase in the based not on cell counts but on the incorporation number of cells that had doubled their DNA of phosphorus into DNA. It appears that ACTH prior to mitosis. Nuclear volume also increases, causes a substantial drop in cell numbers, in both with estrogen and growth hormone, appar rat bone marrow, spleen, and thymus. Total mi- ently because of an increase in nuclear PNA totic activity drops similarly, but it would seem and nonhistone protein (60, 85). Various other (though the authors do not say so) that the elements characteristically associated with nucleic percentage of cells in mitosis does not drop sig acids, e.g., phosphatases, may also increase (119, nificantly and may even rise. Such an effect is 172). perhaps most readily explained by an effect on All these effects are what would be expected length of cell life. in an outburst of mitosis and growthâ€”they can, The antimitotic effects of adrenalin seem not in fact, be paralleled exactly in regeneration, and to have been investigated in vivo in a fully quan they do not afford any grounds for or against titative fashion. supposing that hormones act primarily on either DNA or PNA. An extremely interesting paper 5. THE MODEOFACTIONOFTHE by Gelfant, Meyer, and Ris (86), however, pro MITOGENICHORMONES vides strong evidence that they do not act via We have already discussed the possible action DNA. These authors followed uterine growth after of the various mitogenic hormones on energy simultaneous stimulation by estrogen and inhibi metabolism and came to the conclusion that this tion by aminopterin or nitrogen mustard. These effect, though no doubt sometimes important, two inhibitors do not much affect the resting is not an adequate explanation of the character uterus, but they reduce the mitotic index of the istic, long latent period response that is to be hormone-stimulated organ very markedly indeed. found in vivo. On the other hand, they reduce the growth in An examination of the other metabolic effects weight of the stimulated organ only to about of these hormones is not much more illuminating. one half (having little effect on PNA synthesis They are, as a perusal of any hormone review or alkaline phosphatase). It is evident, in short, will show, multitudinous, and they do not, with that they are not seriously blocking the cellular the possible exception of insulin, yet make any growth initiated by estrogen. Weston (242) has coherent pattern. On any theory of hormone also argued that the action of cortisone in blocking action it must of course be assumed that the division is not due to an effect on nucleic acids. majority of effects are secondary, and it has long While it is desirable that we know more of been the hope of endocrinologists to show that the effects of mitogenic hormones on all the en one or two effects were the primary ones from zyme systems that impinge on growth and divi which all the others spread outwards. In the sion, I believe that such an approach may not case of cell growth and division, however, it is rapidly reach to the root of the problem. I want, very far from clear which are the master reactions. therefore, for reasons that will have become ap In consequence, the chances of deciding which parent, to consider the possibility that these hor are the primary effects of hormones in mitogenesis mones act (in the embryological sense of the word) are not good. As far as one can judge, indeed, by induction.6 Such an interpretation means that the effect of the mitogenic hormones, at least hormones are not stimulating mitosis by acting on the tissues most responsive to them, seems as some essential growth factor whose continued simply to be to induce a metabolic pattern charac presence is necessary to sustain some key enzyme teristic of growth in general, with no particular reaction, but that they are operating at a more compound or system behaving in any unusual fundamental level to determine a pattern of enzyme manner (119, and numerous references in 136, and protein synthesis, which in turn will determine 172). the cell's whole metabolic activity. Total PNA, for instance, increases, as does These questions have already been discussed PNA on a dry weight or per cell basis. Total in Section II, and it is only necessary to remind dry weight similarly increases, as well as dry the reader that switches of metabolic pattern weight per cell. Total DNA increases, though may be stable, and long-lasting, when they are usually less markedly, while DNA per unit of considered to be true differentiations, or less stable, dry weight often decreases, for obvious reasons. and temporaryÂ»when they are called modulations. DNA per cell has not been much investigated, Differentiation is characteristically brought about but seems not to change very much. At most, 6Suggestions that hormones act in some fundamental man of course, it would be expected to rise a little ner have of course been made before (see 107, 108, 171).

. 1 I 1,1 by the process of induction, the essence of \vluch a review on hormonal assay, mir in no PUSO was is that a stimulus of limited duration brings about mitosis the measured effect. The mitotic dose- a long-lasting change. Modulation, on the other response relationship for various androgens has, hand, is usually thought to depend on a sustained however, been investigated in some detail by stimulus, though the difference between differ Dirscherl et al. (59), using only two injections entiation and modulation may lie not so much of hormone and colchicine to magnify the effect in the necessary duration of the stimulus as in on mitotic index. These authors found a more the number of cell generations for which the or less linear relation between the effect and change of metabolic pattern persists. the log dose for testosterone propionate, but an The effect of a hormone is to produce a tempo upward curve for several other weaker androgens. rary outburst of mitosis in a tissue, so that the If hormones act as some sort of growth factor switch of metabolic pattern involved must clearly that is required continuously, then we should be of the temporary, modulatory kind. What expect, on the simplest assumptions, a logarithmic evidence is there that this hormonal switch is dose-response relationship. If, on the other hand, of a truly inductive nature? Does the hormone they act inductively, then, assuming a normal trigger off the changes leading to mitosis, after scatter of sensitivity of cells to the inductive which its presence becomes superfluous? And does stimulus,7 we might expect to find a relationship the triggering lead (cf. Section II, 5) only to of the cumulative probability type, i.e., a sigmoid a single cell division, after which a fresh triggering curve. Furthermore, if the data given by Dirscherl by hormone becomes necessary? et al. (59), as well as by Martin and Claringbold There seems to be no definite evidence on the (148), are replotted on a linear scale, they do time for which a hormone must act to stimulate give perceptibly sigmoid curves. Clearly, how mitosis, but there is at least some reason to think ever, the whole question requires detailed inves that the in vivo response is not dependent on the tigation. continuous presence of hormone. A single dose It is also important to know more of individual of estrogen, as numerous workers have found, cell behavior under the influence of hormones. produces mitotic activity that lasts for several Is the rising mitotic index with increasing doses days. In contrast to this is the rate at which of hormone an indication that a given proportion steroid hormones are broken down and excreted. of cells are moving more rapidly through the cell The bulk of steroid hormone given in a single cycle, as might be expected of a hormone that acted dose is evidently removed within a day (63), continuously on some particular system essential or even much less; half-lives of less than 1 hour for proliferation? Or does it mean that an increas have been reported (83). Growth hormone, too, ing proportion of cells are moving through the has been estimated to have a physiological life cell cycle at a more or less fixed rate, as might of only 9 hours (227), with a half-life for large be expected of an inductive mechanism? It should doses of only 26 minutes (140). Closer examination be possible to answer these questions by means of the necessary duration of action of all the of autoradiographs. It may be remarked, in pass hormones and the duration of the response is, ing, that if hormones merely speeded up the I believe, crucial. rate at which a given proportion of cells moved Detailed knowledge of the dose-response rela through the cell cycle, these cells would be left tionship might also be helpful. The most detailed scattered at various points in the cycle when work here is that of Greenspan et al. (94), who the stimulus died away, whereas the inductive found a linear relation, over a very wide range, mechanism might be expected to bring the reacting between the effect of pituitary growth hormone cells to a halt after completing one mitosisâ€”which (measured by the tibia test) and the logarithm is probably the normal situation in a differentiated of the dose. Unfortunately for our purpose, how tissue (Section II, 2). ever, the measure of effect was only cartilage thickness, the dose was given in four successive 7That there is a considerable scatter of sensitivity to the inductive stimulus is strongly suggested by Jacoby, Trowell, daily installments, and for the higher doses, at and Willmer's (118) experiments on the effect of applications least, several cell generations must have been for limited periods of embryo juice to cells in tissue culture. involved. All these factors, and in particular the Five per cent of juice, for instance, activates a few cells but last two, make any interpretation difficult. Similar leaves the majority unaffected ; 15 per cent activates apprecia objections apply to work by Chai and Mendelsohn bly more, and 40 per cent more again. A second application of juice at the same strength, after the first crop of mitoses is over, (48) on response to androgens. produces a similar effect, but it is the original cells, or rather their A variety of other dose-response curves for progeny, that respond. The cells that were originally unaffected various hormones are given by Emmens (69) in by the concentration in question seem to remain unaffected.

Before leaving the question of whether or not These subsidiary or additional effects have not the various growth hormones act inductively, it been much investigated and have been quantified should be mentioned that minute quantities of even less than the mitotic response to hormones. steroids have, in fact, been shown to be capable A start has, however, been made by Ebling (67) of induction, though it has also been argued that in an interesting series of papers dealing with this effect is a secondary one. It was shown the effects of sex hormones on the cells of the many years ago by Waddington and others that hair follicles. they could cause the formation of neural tubes 6. CONCLUSIONS in amphibian embryos (231). More recently it In vitro experiments with hormones have tended has been shown that estradici tends to suppress to give variable results, though Bullough has endoderm formation in sea urchin egg development advanced the theory that certain of them increase and to act, therefore, in somewhat the same ani- mitotic activity by stimulating energy metabolism. malizing way as thiocyanate (187, and see Section Bullough's experiments are discussed in some de II, 4). tail, and it is concluded that the effect can only The evidence, in short, though mostly circum be a secondary one. stantial, does afford some indication that the In vivo experiments seem to show two main mitogenic hormones operate by induction. How types of response to mitogenic hormones. The they do so is, of course, entirely obscure, but, more important one involves a long latent period if one were to follow current thought about protein (1-2 days), cellular hypertrophy, and usually a synthesis, one might suppose that they must act very large increase in mitotic index (up to many in some way or another at the level of microsomal hundredfold). It is suggested that this effect is particles. It may be significant that KÃ¶rner(127) an inductive one, the latent period and cellular finds that microsomes in a cell-free system in hypertrophy representing the period of switch corporate amino acids appreciably faster if the over and synthesis of mitotic protein and other animal from which they are taken has been pre materials for division. The second type of response viously treated with growth hormone. It would involves only a short and ill defined latent period, seem desirable, at any rate, to look for labeled no obvious cell hypertrophy, and only a modest hormones in the different fractions of cells in increase of mitotic index (two- to threefold). It the latent period of mitogenesis. is suggested that this may be explained by a We must also consider the possibility raised general stimulation of energy metabolism along by Hechter (107, 108; see also Mueller, 157) the lines of Bullough's theory. that hormones affect the pattern of metabolism There is a certain amount of evidence in favor by controlling the entry of substances into the of the idea that the important long latent period cell. response is an inductive effect; in particular, hor In conclusion, it should be pointed out that mones have a very short half-life in the body. mitosis is by no means the only end-result of the various hormones that affect proliferation.8 IV. CONTROL OF GROWTH IN Characteristically, cell movement is also stimu EMBRYO AND ADULT lated, and this effect becomes apparent before In the last section, we discussed a number mitosis and continues long after mitosis has died of hormones with important effects on growth. down (1). Collagen deposition may follow mitosis Some of these, such as the pituitary growth hor (1), and length of cell life may be affected (67). mone and the thyroid hormones, appear to exert A wide range of effect is of course not surprising rather widespread effects, and, though they may as a result of a stimulus of the inductive type be important for particular tissues at particular and presents no conceptual difficulty, even though times, they are not essential for regeneration or the mechanism involved may be obscure. the control of growth in the adult. Other hormones, in particular various pituitary and gonadal hor 8Two minor effects of steroids or related compounds on cell mones, are trophic for certain glands and tissues. division may also be mentioned. They have, somewhat sur prisingly, a colchicine-like effect on mitosis itself, though prob In these cases, the level of hormone appears to ably only in concentrations well above what is required for exert the main, and perhaps the only, control their normal stimulatory effect (146, 218). Agrell (8, 9) has of growth in the relevant organ. Reduction of shown that they are without effect on the first few divisions hormone causes the target organ to regress; ad of sea urchin eggs but that they become inhibitory at about ministration of excess causes it to hypertrophy. the time the store of preformed DNA runs out and the egg be gins its own synthesis. These two effects are not easy to corre In the case of the pituitary hormones that late with the normal effects of steroids, though the colchicine are trophic for various glands, feed-back systems effect may perhaps be a simple narcotic action. are well known to exist between the pituitary

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1958 American Association for Cancer Research. SWANNâ€”Control of Cell Division: A Review 1135 und llie gluiid in tjucslioii. Tliyroxiu, for iiiatuiice, erate, however, puts tliem outside the scope of depresses the secretion of thyroid-stimulating hor this review. mone; absence of thyroxin stimulates it. The sys Many of the earliest experiments on regenera tem is, therefore, self-regulating in large degree. tion were concerned with the healing of skin Similar, if somewhat more complex, systems op wounds or the growth of new limbs after amputa erate between the pituitary, on the one hand, tion. This work is well summarized by A. E. and the gonads and the adrenal cortex on the Needham (161), and we shall return to it later other. in this section. The problem in such situations, The various recognized hormones do not, how however, is complicated, particularly in the case ever, seem to provide any such regulatory systems of limbs, by the need for extensive morphogenesis. for all the other tissues and organs of the body, There has, therefore, been a trend in recent years although there is, as we shall see, clear evidence toward studying the problem on systems that of rather precise and definite mechanisms of con seem at least to be simpler; for example, liver trol. In this section, I shall attempt to review regeneration after partial hepatectomy, compen what is known of these other, more elusive sys satory hypertrophy of one kidney after removal tems. of the other, or renewal of blood after hemorrhage. There are a number of situations to consider. Of these, liver regeneration has been the most In the first place, several tissues in the body intensively examined. are continuously renewing themselves, even in the adult. These are the skin, the gut lining, the TABLE 3 epithelia of the lung and bladder, the testis, and APPROXIMATEMITOTICINDICESFORVARIOUS the hemopoietic tissues. Cells in these tissues RATANDMOUSETISSUES divide fairly frequently, with mitotic indices (in Cells in tissue culture; variable, up to about 100. the rat and mouse) ranging from about 0.5/1000 Data mainly from References 124, 134, and 243. to 30/1000â€”see Table 3. In addition to steady Mitotic indices Group Tissue (mitoses/1000nuclei) renewal, however, all these tissues can rapidly I Gut lining 3.0-30 make good more serious and sudden loss, either Lung 0.7- 2.5 Epidermis 0.5- 1.5 from wounding or, in the case of blood, from Bladder 0.3- 0.6 hemorrhage. The mechanisms involved may be Hemopoietic tissues 1.4-14 the same as those concerned with normal renewal, Testis 0.5- 1.2 n Adrenal 0.2 but, in the case of epithelia at least, they may Liver 0.1 be different. Thyroid 0.05 A second group of tissues shows much lower Seminal vesicle Coagulating gland > 0.02 mitotic activity: adrenal cortex, liver, thyroid, Prostate J kidney, and connective tissue for instance, with m Striated muscle 0 mitotic indices ranging from 0.02/1000 up to 0.2/ Nerve cells 0 1000â€”seeTable 3. These figures are not sufficient even to keep up with the normal growth rate 1. COMPENSATORYHYPERTROPHYAND of an adult rat of 1 per cent per day (134), SIMILARSYSTEMS and the tissues in question are presumably only a. Liver.â€”The problems of liver regeneration growing slowly, and not to any significant extent after partial hepatectomy have been reviewed renewing themselves. Nevertheless, most if not recently by Harkness (104). About two thirds of all of them can react very dramatically to a the rat liver can be easily and neatly removed. The partial loss of tissue or organ, and can repair remaining one third then restores itself, roughly exponentially, to its normal size in a matter of the damage, sometimes very accurately, within 10-20 days. a matter of days or weeks. The mechanisms that No mitosis is evident for 24 hours, though control this regeneration are usually supposed in the second half of this period there is an to be the same as those that limit growth in em increase in DNA and in cytoplasmic PNA, as bryonic and later development. well as an appreciable growth in weight. In con Finally, there are a few cells, in particular sequence, there is marked cellular hypertrophy. nerve and striated muscle, that never normally Shortly after the first day, there is a burst of mi divide at all in the adult animal, though the cells tosis in the parenchymal cells (which make up can regenerate lengthwise, and cellular hyper the bulk of the liver), followed about a day trophy, particularly under stimulation, may be later by lesser bursts of mitosis in the other important. The fact that such cells do not prolif cell types. The rise in mitotic index is very large,

about 300-fold in the parenchymal cells, and it adults inhibits regeneration, whereas serum from is evident that most if not all of the cells in hepatectomized animals does not. the tissue that remains divide in the course of The concentration of plasma proteins does of a few days. These details are incorporated in course fall after hepatectomy, but scarcely rapidly Table 4. enough, it would seem, for it to act as the stimulus It has become fairly clear that neither the to regeneration. Against this objection Glinos (87) recognized endocrine glands, nor excretory prod has produced some evidence that it may fall in ucts, nor diet is crucially involved in the regenera the vicinity of the liver cells before it falls in tive stimulus.9 It has been held that the blood the blood. It might further be argued that the supply to the liver is fundamentally important, important change is not simply one in total plasma particularly the portal flow; but more recent work albumin concentration but in the concentration TABLE4 THEMITOTICRESPONSETOREGENERATIVEANDOTHERSTIMULI

larhyper Natureofstimulus1. typeLiver trophyYesYesYes?Yesence(2)(109)(117)(145)(165)(246)(166) Partial hepa paren tectomy2. chymalcells LiverlittoralcellsBile I/18,500)0.1Not duct epi theliumKidney Ligature of given given oneureter3. Contralater-al NotgivenNot NotgivenApprox. X4X30X25XllXÂ»X200Latentperiod(hours)24484824-4824-482415 ?YesN.I.Yes?YesNX?YesRefer kidneyGall Ligatureofcommon bladderepitheliumEpidermisTailgiven10.51-20.1Maximummitoticindexnuclei)28.79.35.9Not60255.510-1120IncreaseofmitoticindexX287>X200X59X70 bileduct4.

Regeneratinglimb5. days24-4824-486

Subcutaneousgraft fin epi ofspinalcord dermisSpleen material6. Antigen7. redpulpTail

Maximalfeed-ins fin epi daysCellu after star-AnimalRatRatGuineapigNewtAxolotlRatAxolotlTissueandcelldermisRestingmitoticindex(mitoses/10000.1Nil(< vation for 10 days 8. Short appli- Chick Periosteal fi- 1 X20 10-12 N.I. (118) cation of em- broblasts bryo juice N.I. = No information. ? = Definite information not given by author. makes this unlikely. The liver of the parabiotic of some specific type of albumin, making up twin of a hepatectomized rat, for instance, shows perhaps a small fraction of the whole. increased mitosis. It now seems that the critical Though much more remains to be discovered, factor is in some way connected with the con it is clear that we are not dealing with a stimulus centration of plasma proteins. due to any of the recognized hormones. Indeed Glinos and Gey (88) found that mere dilution we are, strictly speaking, not dealing with a stimu of the plasma caused mitosis in the liver of normal lus at all, but with the release of an inhibition. rats, and more recently Glinos (87) has shown It may be noted in passing that an inhibitory that regeneration can be inhibited by increasing system of this sort implies that the essential al the plasma concentration and that the active bumin fraction must be continuously elaborated fraction is in the plasma albumin. Stich and Flo by some or all of the liver cells and rapidly rian (208) have also shown that serum from normal broken down, presumably outside the liver. 9Cater (41), however, finds that growth hormone intensi Whether the control of liver growth is effected fies the regenerative response. solely by the formation of an inhibitory substance

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1958 American Association for Cancer Research. SWANNâ€”Control of Cell Division: A. Review 1137 isnotclear,ItmightkrxpeetcdjbyanalogÃ¯Ã¯hatfrtrthetruesituation!itlooksatfirst with the pituitary feed-back systems, that the sight as though the regulation of kidney growth inhibition worked on some other tissue which is rather different from the regulation of liver accordingly produced more or less stimulant for growth and is not due in any way to the release liver mitosis. No such stimulant has been dis of an inhibition, but to the excretion, or failure covered, however, and Kohn (125) has argued of excretion, of definite stimulatory substances. against its existence. This question is discussed The problems of red cell productionâ€”erythro- further in Section VIII. poiesisâ€”have been reviewed by Grant and Root A variety of chemicals do, of course, produce (93) and by Erslev (71) (for more recent refer mitosis in the liver, but it is generally assumed ences see 115). It now seems doubtful if degrada that this is simply a case of regeneration following tion products of hemoglobin act as a stimulus; damage. Thiourea, however, seems to cause no and, though anoxia undoubtedly causes increased damage but nevertheless produces the characteris red cell production, it does not do so by any tic burst of mitosis, with a long latent period direct effect on the hemopoietic tissues (see also (61). Whether thiourea bears any relation to a 215). Hormones are certainly involved in eryth- naturally occurring stimulant, or alternatively af ropoiesis, and van Dyke et al. (226) have par fects the inhibitor, remains to be established. tially isolated a pituitary erythropoietic hormone b. Other organs and tissues.â€”When one kidney rather similar to ACTH. Nevertheless, hormones is removed, the other rapidly hypertrophies. This seem to exert a modifying rather than a funda situation has not been investigated in nearly mental action, and cells are produced, though as much detail as liver regeneration, but from less rapidly than otherwise, in the hypophysec- what is known (see references in 68) it seems tomized animal. to follow a very similar course. There is a cor The fact that homologous anemic plasma in responding latent period, followed by a sharp jected into an animal causes marked erythropoiesis burst of mitosis in the convoluted tubules. Other seems to point to some blood-borne factor, and cell types show bursts of mitosis somewhat later. a number of workers now seem near to identifying It is not known whether compensatory hyper it. Erslev and Lavietes (73) find a factor in either trophy of kidney, like that of liver, is subject the albumin or the a- or 0-globulin fraction of to any form of inhibition from plasma proteins; plasma. More recently Rambach et al. (178) have but whether or not this is so, it would seem that identified it as a heat-stable mucoprotein in the 02- other factors must be involved, since mitosis can globulin fraction. A rather different factor, though also be produced in one kidney by ligature of probably a protein, has been found by van Dyke the corresponding ureter, though it is then most et al. (225) in the urine of anemic patients. Non- pronounced in the connective tissue cells (see protein factors have been found by Linman and Table 4).10 The unligatured kidney also shows Bethell (143). some rise in mitotic activity (109). Even more A further point of some interest is that Erslev surprising is the finding that a kidney rendered and Lavietes (73), using nitrogen mustards, and ischemie by the ligation of the renal artery also Linman and Bethell (144), using radiations, have shows mitosis, but in this case mainly in the come to the conclusion that the erythropoietic excretory tubules and the pelvis. factor is not produced by the rapidly dividing These observations strongly suggest the exist and sensitive lymph or hemopoietic tissues. ence of a stimulatory factor in the form of some The nature of the stimulus for the secretion waste product. In a sense, therefore, they point of erythropoietin itself is uncertain, but it is to the traditional explanation of hypertrophy as probably not the removal of an inhibition, since being due to functional overload, though it does simple plasma dilution, at least within limits, not, of course, follow that the stimulus is due does not lead to red cell formation, although to an excess of all waste products but, rather, it does lower the oxygen-carrying capacity of to an excess of one particular substance. WTiat the blood. Up to a point, however, this diminution substance or substances are involved is not known. can be made good by more rapid circulation of Herlant (109) suggests that it may be products of the blood, and Erslev (72) has suggested that the protein catabolism. Haddow (references in 99), on critical factor is the oxygen content of the tissues. the other hand, has suggested that it may be xan- A control system that accounts for most if not thopterin or related compounds (see, however, 27). all the facts would therefore seem to consist of 10Ligature of the bile duct similarly produces a burst of some differentiated tissue that responds to anoxia mitosis in the gall bladder epithelium, after the usual latent by secretion of erythropoietin. What this tissue period (117). See Table 4. may be is not yet certain, but there is evidence,

somewhat surprisingly, that it may be kidney figuration, after the antigen-antibody scheme."11 (116, 159). It is also clear that cobalt is involved One of these compounds is an intracellular spe in the production of erythropoietin (89). cific catalyst, the template. The other is an anti- The control of white cell production has not template, released continuously from the cell into been examined in so much detail, but it is evident the blood stream and rapidly broken down. that hormones are important (91) and that other Removal of part of an organ thus reduces the leukopoietic substances (references in 130) are rate of production of anti-templates, so that tem probably involved. Osgood (163, 164) has sug plates for a time predominant, and growth con gested that mature leukocytes produce relatively tinues until the balance is restored. Addition of unstable inhibitors of mitosisâ€”a system analogous a part, on the other hand, will inhibit growth, to that controlling liver growth. unless a proportion of cells disintegrate and die, c. The humoral feed-back type of control.â€”In the in which case templates will be released and a previous two sections we have discussed in some burst of growth will ensue. Necrosis or wounding detail the regenerative and growth-controlling will have the same effect. mechanisms in a number of tissues. These systems It will be evident at once that the facts of have a certain amount in common: all involve liver regeneration fit Weiss's theory very well. one or more blood-borne factors, and all show Indeed, the inhibiting albumin fraction of plasma some sort of feed-back mechanism. In detail, how might well be equated with the anti-templates. ever, they differ widely. The evidence from kidney is less certain, pointing How many more such systems can we expect as it does to mitotic stimulation by a waste to find? Nerve and striated muscle do not, of product that is normally excreted. Weiss does course, grow, in the adult, by cell multiplication, not deny that "functional overload" of this sort though the hypertrophy of muscle with continued may be important. He suggests, however, that activity, and its wastage with disuse, suggest the template-anti-template mechanism may oper that it may nevertheless have its own special ate as well and quotes experiments by Wayne- feed-back mechanism, and it is possible that this Ferris on embryonic nonfunctional metanephros is concerned in regulating embryonic growth. How which showed a measure of the characteristic ever, a number of other organs are known or sus compensatory hypertrophy. The evidence from pected to show some form of compensatory hyper erythropoiesis cannot so easily be fitted in, since trophy, working, it may be supposed, in similar it shows, fairly clearly, a stimulatory substance ways. We have already mentioned briefly the in the circulation. The thyroid and probably other various glands with pituitary feed-back: the ad glands are also controlled by stimulatory humoral renal cortex, the thyroid, and the gonads. Com factors which are, of course, the appropriate pi pensatory hypertrophy is also found in the lungs, tuitary trophic hormones. the pancreas, orbital glands, salivary glands, and With the exception of liver, therefore, the evi the pituitary itself (references in 1, 36, 239). dence is rather against circulating inhibitory fac It is clear, however, that this sort of mechanism tors and in favor of circulating stimulatory factors. would be inappropriate for the more widely dif The evidence for intracellular stimulators (the fused tissues and could not in fact operate without templates), on the other hand, is somewhat more extreme regional specificities. The loss of one limb, convincing. Weiss (239) found that tissue cultures for instance, does not result in hypertrophy of of kidney and heart cells were stimulated to greater the others but, if it leads to anything, causes growth if the homologous organ was left in the regeneration of the limb itself. Skin wounds simi embryo from which embryo juice was prepared larly do not lead to hypertrophy of skin remote than if the homologous organ had been removed. from the injury but to healing of the wound. Andres (17) injected kidney or liver cell debris, It would seem more likely that other mechanisms either fresh, or frozen and thawed, into the em must operate in these cases, and we shall discuss bryonic chick blood stream. He then showed that them in the next section. Meanwhile, we may the ratio of mitotic rate in the kidney and in consider the various humoral feed-back mecha the liver was raised by kidney debris and depressed nisms in more general terms. by liver debris, each organ debris thus exerting A number of attempts have been made to a homologous effect. Teir and co-workers (214) unify these mechanisms and produce a generalized similarly found that liver suspensions increased scheme of growth control. The best known of these the mitotic activity of rat liver. Comparable ef- is the template-anti-template theory of Weiss 11These compounds should no doubt be considered as one (239). This supposes cells of any given type to pro example of a much wider class of complementary compounds, duce "paired compounds of complementary con called by Tyler (222) "natural auto-antibodies."

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1958 American Association for Cancer Research. SWANNâ€”Control of Cell Division: A Review 1139 fects were also obtained with orbital gland and the blood, it is only to be expected that a failure stomach epithelium. Saetren (189) has found a to do so on a sufficient scale should lead to somewhat more complex situation. Liver tissue growth. This will be achieved if absence or par injected intraperitoneally reduced the normal mi tial absence of some metabolites stimulates liver to tic wave that follows hepatectomy, when the growth either directly or indirectly. amount of injected tissue was comparable with If, then, we examine the control mechanisms the amount of extirpated tissue (cf. 208). The without attempting to force them into too tight injection of lesser amounts, on the other hand, a common mold we see that they all have a par was markedly stimulatory. A corresponding effect ticular appropriateness, and we arrive at some was found with kidney, and the active substances sort of a justification for the classical idea of appeared to be heat-labile and nondialyzable. control by functional load.12 It is evident, too, In some well known work Ebert (66) made that in every case, except liver, a circulating chorioallantoic transplants of adult chicken spleen stimulatory substance has been identified, at least and found specific stimulation of chick spleen in a tentative way. In general, this seems to growth, but this type of experiment has been be a protein, and in the case of the various criticized by Simonsen (194) on the grounds that endocrine glands, it is a recognized protein hor adult lymphocytes may have colonized the chick mone. The feed-back system may be completed spleen and reacted against the chick antigens. in various ways and may or may not involve The evidence on intracellular factors is admit circulating substances, produced by the organ tedly confused, but as far as we can see at present itself, that are inhibitory for the tissue producing it suggests the existence of both specific stimu the stimulatory substance. In the case of the latory and specific inhibitory substances. The evi thyroid, the adrenal, and the gonads, these cir dence on extracellular, humoral factors is more culating inhibitory substances are the relevant solid and, with the exception of the liver in hormones. hibitory albumin fraction, indicates the existence A final point of some importance is the mode of stimulatory rather than inhibitory substances. of action of the various stimulatory (or inhibitory) It is difficult to reconcile all this evidence with substances. Some of these we have already dis the idea of a universal template-anti-template cussed in Section III, along with the hormones. mechanism of control. Can we, however, make However, it will be evident that the others exert out any other underlying pattern? In detail, the an effect that is extremely like, if not identical control mechanisms probably vary a good deal, with, the hormonal stimulus. In both liver and and I suggest that, beyond the fact of their kidney regeneration there is the same latent pe involving feed-back mechanisms and blood-borne riod, the same cellular hypertrophy, the same factors, the only common feature we can recognize sharp rise in mitotic activity, and the same changes at present is their appropriateness to the function in DNA and PNA. There would seem, therefore, of the particular organ in question. to be good reason to think of these agents as The job of the kidney, for instance, is to excrete, exerting the same kind of inductive effect as and it is not surprising, therefore, that continued the recognized hormones (Section III). failure to do so on an adequate scale should lead to enlargement in order to meet the demands 2. SKIN WOUNDSANDLIMBREGENERATION put upon it; and what is more appropriate than To what extent can the types of control dis that the stimulating factor should be a substance cussed in the previous sections be extended to normally excreted? Again, it is the job of the the problem of wound healing and limb regenera red blood cells to carry oxygen. If they fail to tion? If we regard the skin as one tissue, or paired do so in sufficient quantity we should therefore limbs as equivalent to paired kidneys, clearly expect that some tissue, presumably one of those the humoral, feed-back type of control will not most sensitive to oxygen lack, would set in motion be suitable. We have already said that it would the production of a stimulatory substance, and be inappropriate if one limb were to hypertrophy this, it would seem, is just what happens. Or when the other is amputated, or if the whole again, it is presumably the job of endocrine glands skin grew when one region was wounded, and it such as the thyroid or the adrenal cortex to does not in fact happen. maintain a certain level of hormones in circulation, These difficulties could be overcome if we were and that their secretions should inhibit the secre to suppose extreme regional specificity, such that tion of a further trophic hormone is just what we 12It is significant that most organs do not regenerate to might expect. Lastly, since the liver is concerned their original size, and Addis and Lew (6) suggest that they with releasing a wide range of metabolites into grow only to the size at which they function adequately.

each limb, and each small area of skin and under pletion of limb regeneration. Nor, it would seem, lying tissue had its own feed-back mechanism. can they account for the accurate cessation of This is not inconceivable, but, I think, hardly growth when sufficient proliferation has occurred likely. We have, moreover, to account for the to make good the lost tissues. It is possible that fact that even tissues with a well developed feed cell contact (Section VII) may be important at back mechanism, such as liver, can respond to this stage. It is also possible that leukocytes may a wound in the same localized sort of way as be involved. It has long been known that they skin (1). It is also significant that one wound exert some sort of trophic effect on epidermal appears to have no effect on the rate of healing and fibroblastic cells in tissue culture and will, of another (26). in fact, enable these cells to grow in serum without The nature of the localized stimulus to mitosis the addition of embryo extract (38). There is that must be involved in these situations is by now good histological evidence that leukocytes no means clear, but there seems little doubt that secrete (181), and it is not unlikely that they some form of "wound hormone" or "regeneration should have a trophic effect in vivo (141). promoting factor" is released by the damaged The localized response to wound hormones is, cells. Such substances have been postulated many in the nature of things, not easy to examine times, but never fully isolated and still less chem quantitatively, but a preliminary attempt has ically characterized. There is a large, but rather been made by Abercrombie and Santler (5) in confused amount of literature on this subject, the case of Wallerian degeneration of peripheral which is reviewed by Davidson (56) and A. E. nerve. It is not altogether clear that this is in Needham (161). Reference may also be made to fact a situation of the same type as skin or limb papers by Menkin (153). regeneration, though it seems likely. The burst Although almost nothing is known about the of mitosis in the various accessory cells of a chemistry of wound hormones, we can deduce crushed, damaged, or severed nerve is certainly a little about their general mode of action. It reminiscent of other types of wound healing and would seem, for instance, that they cannot be may be owing to a wound hormone released by specific for a given cell type, since in any wound the degenerating nerve fibers. a wide variety of cell types undergo mitosis more The method used was to crush peripheral nerves or less simultaneously. The mitotic response in and then crush them again after various intervals wound healing or limb regeneration is, moreover, of time, so varying the amount of new fiber very reminiscent of the response to the other capable of releasing wound hormone. It was found hormonal and regenerative stimuli of a more spe that the amount of proliferation in the surrounding cific kind. There is, for instance, a well defined cell population depended on the amount of de latent period, during which no mitosis occurs,13 generating nerve. On the other hand, the rate and there is marked cellular hypertrophy (18, of proliferation varied inversely with the density 39, 161). The onset of mitosis is then relatively of the population; a given amount of stimulus sudden, and the increase of mitotic index may be (provided by a given amount of nerve degenera considerable, at least in limb regeneration (Table tion), in fact, seemed to result in a more or less 4). The increase of DNA and PNA also follows fixed number of new cells. Abercrombie and Sant the pattern for hormone action and liver regenera ler conclude that the proportion of cells undergoing tion (161, see also 106). We can, therefore, pro division is probably determined by the average visionally interpret the wound hormone stimulus amount of stimulant per cell. in the same way as the hormonal and organ If we suppose, as we have discussed earlier regeneration stimuli, as switching synthesis and in Section III, 5, that there is a normal distribu metabolism toward the making of mitotic protein tion of sensitivity of cells to hormonal and other and materials required for the other division mech division stimuli, then we shall expect to find, anisms. over a considerable range, a rough linear cor Whether wound hormones are a complete ex respondence between average dose per cell and planation of these types of repair and regeneration percentage response, and Abercrombie and Sant- ler's results would therefore seem to be consistent is doubtful. They can, for instance, hardly explain a prolonged response, particularly in the com- with our general hypothesis.

Â»This latent period is rather longer than is the case with 3. MISCELLANEOUSFACTORSINVOLVEDIN hormones, perhaps because of the variety of cell activities that THECONTROLOFGROWTH seem necessarily to precede mitosisâ€”e.g., the vascular reac tion, demolition, differentiation, epidermal closure, cell migra The various systems we have so far discussed tion, and blastema formation (161). cannot, by any means, represent all the factors

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1958 American Association for Cancer Research. SWANNâ€”Control of Cell Division: A Review 1141 involved in the control of growth in a whole function and as such must fall into the same animal, and it is in fact possible to recognize general category of control mechanisms as we quite a number more. None of these has been have discussed in previous sections. No doubt subjected to a detailed analysis, and all of them, many more such cases remain to be discovered there is no doubt, would merit further investi (see for instance [53]). gation. A rather different case concerns the general Cell growth and division are undoubtedly af nutritional level. In a general way it is, of course, fected in some degree by nerves (discussion and well known that growth is dependent on the level references in Ref. 161). Intense stimulation of of feeding, and it might be assumed that starva functional activity may, for instance, lead to mi tion slows down the process by reducing the tosis in exocrine glands (128). Innervation is also level of nutrients circulating in the blood and important in limb regeneration, and some tissues hence slows down individual cell growth. Some ex will, in fact, not regenerate without a nerve supply tremely interesting observations of Overton (166) (20, 196). The effect seems to be a fairly direct suggest, however, that the situation is not so one on mitosis, presumably mediated by some simple. Starvation reduces the mitotic index of sort of neurohumor. Overton (165) has investi amphibian epidermis to a very low level, but gated this action by grafting central nervous tissue on refeeding there is a well defined latent period, under amphibian epidermis. She found the usual followed by an enormous and very sudden burst effects of a sharp rise in mitotic index, with a of mitosis, which later settles down to a more well defined latent period (Table 4). The chemical normal level (Table 4). It would seem, therefore, nature of this stimulus has not been investigated. that starvation has not slowed cell growth down Antigens can also induce mitosis in the anti uniformly, but rather that it has resulted in a body-forming tissues, and it is even possible that higher proportion than normal of cells halted this is a normal part of the response to antigens. in early interphase. In other words, cells in a Coons and co-workers (135), using fluorescent starved animal complete mitosis fairly readily antigen on the lymph nodes of animals previously but are not so readily or so frequently stimulated immunized, found that it adheres (initially in into the next division. The effect of starvation the nucleus) to small clusters of cells, which get on mitosis, in short, seems to be indirect, and larger with time, suggesting that a few individual perhaps Immorally mediated. It is, of course, well cells originally took up the antigen and then known that starvation inhibits the secretion of proliferated. Wissler et al. (246) have investigated endocrine glands (references in 95, p. 243). It mitotic indices in spleen red pulp, following in may be remarked in passing that the length of jection of antigen, and find a sharp rise, following the latent period appears to depend on the length the usual latent period of 1-2 days (Table 4). of starvation, being, in the case quoted above, Mechanical tension may similarly lead to pro 5-6 days after 10 days of starvation and 3-4 liferation, especially in the uterine wall, in skin, days after 5 days of starvation. in the gall bladder, and in the bile duct (references Bound up with the question of level of nutrition in [1]). The mechanisms involved are unknown. is that of tissue competition. This problem has Loss of cells by mechanical abrasion also leads been discussed by Bullough (32), but very little to growth and mitosis in epithelia, particularly is known about it. A number of cases of local in the skin and gut linings (reviewed by Leblond competition are knownâ€”for instance, the regres and Walker [134]). Pinkus (173), for instance, sion of warts when the surrounding hair is growing finds that the amount of mitosis in the Malpighian actively or the decrease of hair growth when layer of skin relates closely to the number of the underlying mammary glands are active. Re superficial dead cells removed experimentally. Le mote competition is also evident between a mam blond and Walker point out that a variety of malian embryo, or a tumor, and the rest of the environmental effects may influence this sort of body. The pattern of bodily change in starvation regeneration but that it seems necessary to postu late some "inherent" control mechanism. There is further evidence of competition between one is no indication of what this may be. It may tissue and another. It is not known, however, be remarked that the digestive epithelium renews whether these are direct or indirect effects. Are itself even in a fasting animal (134), so that certain cells and tissues, and especially, perhaps, mechanical removal of cells is certainly not the those cells moving through a mitotic cycle, more only factor to be considered. efficient at picking up certain key food substances All the preceding cases, it will be noticed, in short supply? Or is the competition mediated involve growth in relation to some differentiated in some way by the growth control mechanisms

discussed earlier in this review? There appears the nature of the situation makes any experiment to be no evidence on these points. ing difficult. Nevertheless, it should not be im possible to devise an experiment, say with eryth- The dozen or so growth situations discussed ropoietin, to test the idea. so far can represent but a tiny fraction of the The locus of action of an inductive stimulus different mechanisms involved, since the growth is quite unknown, but, as discussed in Section. of every tissue in the adult and the embryo must Ill, one would be inclined to look at the level be under the control of its own particular system. of microsomal particles. It is, therefore, interesting: Embryonic growth, indeed, consists of a constantly that van der Decken and HuitÃn (224), investi shifting pattern of mitosis in both space and time gating the microsomal fraction of regenerating (e.g., 101, 240), but the controlling mechanisms rat liver, find various changes, and in particular have scarcely been examined at all. The genetic an increase in amino acid incorporation, between control of growth is another field that has received 12 and 24 hours after hepatectomy, i.e., in the very scant attention at the cellular level (see 58). Yet another intriguing problem is "cutely," latent period. In conclusion, it should be pointed out, again the situation in various invertebrates such as as in the case of hormones, that proliferation is rotifers in which cell number is constant. Such not the only end-result of regenerative and other animals appear to switch over irreversibly at a growth stimuli. Cell movement and very often particular point from proliferation to differentia collagen deposition are also involved. The reader tion and are subsequently unable to regenerate is referred to Abercrombie's (1) valuable paper (23, 223). Investigation of any of these problems in which this aspect of growth, among others. might lead to valuable advances in the study is discussed at some length. of control mechanisms. 5. CONCLUSIONS 4. THE MODEOFACTIONOFREGENERATIVE If various internal organs or tissues are partially ANDOTHERGROWTHSTIMULI removed, the loss is rapidly made good by cell The biochemical changes associated with re growth and division. The mitotic response is al generation have been investigated in some detail, most identical with the long latent period type but, as with hormones, they seem to fall simply of hormone response; that is to say, there is a into a pattern characteristic of growth in general. latent period of 1 or more days, cellular hyper DNA increases, as would be expected from the trophy, and a sudden and very large increase timing and intensity of mitosis.14 PNA increases of mitotic index. It is suggested once again that markedly. Phosphatases and thiols also increase, this is an inductive effect. as does respiration. These changes are tabulated Some of the agents responsible have been in in some detail by A. E. Needham (161). A variety vestigated. In the case of kidney and red cells of references are also given by Leslie (136) and there are definite stimulatory substances; in the Harkness (104). case of liver there is an inhibitory substance, In Section III, 5, it was argued that such the lack of which brings about mitosis. Feed-back results give no reason for assuming that DNA, systems of one sort and another control the levels PNA, or any other metabolite is the immediate of these humoral agents. target of hormone action. The same arguments, The growth of other tissues and organs such I believe, hold for the regenerative stimuli, and, as skin, gut lining, and limbs is not controlled following the reasoning of Sections II and III, in the same way, and humoral substances seem I suggest once again that we are dealing with to be less important. There is evidence, however, an inductive mechanism, which initiates the syn for local stimulatory agents. The mitotic response thesis of mitotic protein and the other biochemical to these agents seems to be of the same long changes mentioned above. latent period type, and is presumably also induc One of the essential features of induction, it will be remembered, is that a short-lived stimulus tive in nature. produces a longer-lasting response. In the case A variety of other factors are also concerned of hormone action, there is at least some sugges in stimulating or limiting growth in particular tis tion of such an effect. In the case of regenerative sues. Nerves, antigens, mechanical tension, abra stimuli, however, there is, as far as I am aware, sion, as well as feeding after starvation all appear no information one way or the other, and indeed to produce effects of a similar type, and it is "Unexpected results have, however, been obtained in suggested that in most, if not all, of these cases kidney regeneration (74, 174). inductive stimuli are at work.

V. PLANT HORMONES cell growth rate but that not all increase division Research in the last 2 or 3 decades has revealed rate. This is, of course, what might be expected, that plant growth, like animal growth, is controlled since division presupposes growth, whereas the by a number of hormonal mechanisms. Not sur reverse is not necessarily the case (Part I of prisingly, the actual hormones are different, but this review). On the whole, substitution in the the complexity of their pattern of interactions 4-position is required for division, and in the is nevertheless very reminiscent of the situation 2-position for growth; 2,4-dichlorophenoxyacetic in animals. The precise mode of action of the plant acid has, therefore, a strong effect on both growth hormones is similarly elusive, and, as with most and division. animal hormones, no plant hormone has yet been As one might expect, the auxins show a wide shown to operate primarily by affecting a particu range of metabolic effects: respiration, water up lar enzyme reaction. There is even a parallel be take, salt uptake, and sulfhydryl groups are all tween animal and plant hormones in the striking involved (see reviews in Ref. 232). This very effects of making substitutions in the ring struc multiplicity of effect rather points to a more ture of the auxin and steroid molecules. fundamental mode of action (228), and it is there In at least one respect, the investigation of fore of considerable interest that Skoog and his hormones in plants is more troublesome than co-workers should have found a connection be in animals: the hormone-producing cells are not tween auxins and nucleic acids (197). localized in easily removable glands or organs. It appears that a rather delicate balance be On the other hand, the hormones themselves are tween small amounts of indolyl acetic acid and more readily isolated, identified, and assayed, and much larger amounts of adenine will determine in vitro experiments are easier. In consequence, the type of growth and organ formation in tobacco the study of plant hormones has developed along tissues, and that other naturally occurring purines rather different lines from the study of animal and pyrimidines can enhance or modify the effect hormones, but in various respects the two fields of adenine. In a search for a complex of IAA are complementary, and it is possible that some and adenine, a substance of considerable poten of the findings about plant hormones may have cy in causing cell division was isolated and has important implications for the study of animal now been identified and synthesized. It is 6- tumors (see Section IX). (furfurylamino)purine, usually known as kinetin. Various analogs are also active. 1. AUXINS AND KlNINS In tobacco pith tissue grown on a suitable The best known natural plant hormones are the medium, kinetin brings about division, and, since so-called auxins, the most important of which ap the cells in question were found to have the pears to be indolyl acetic acid (IAA). A large num tetraploid value of DNA, it was first thought ber of synthetic auxins (and auxin inhibitors) are that the block overcome by kinetin occurred after also known, most of them being substituted ar- DNA duplication. It appears, however, that the yloxy-alkyl acids (e.g., 2,4-dichlorophenoxyacetic cells are genuine tetraploids and that kinetin must acid). operate at an earlier stage. It seems, in fact, It has become fairly clear that the primary that comparable but small amounts of both IAA effect of most auxins is on cell size, or more and kinetin are required at some point to bring precisely cell extension. The action is at least about DNA synthesis and the ensuing mitosis. twofold and involves a softening of the cell wall Moreover, the interaction of the two substances as well as true cell growth. Cell division may at different concentrations produces a wide range also be stimulated (but, as will become apparent of effects on organ formation and differentiation later, interactions with other nonauxin hormones (197).16 are certainly involved here). The over-all response K,inetin, however, is not the only factor that to auxins, however, is distinctly complex, since seems to be involved in this way. Steward and the various tissues and regions of the plant may Shantz (207) have isolated a variety of other respond differently, some being stimulated and unrelated substances from coconut milk that have some inhibited; there may also be formation of similar effects. One of these is diphenylurea. The new structures by differentiation. others have not yet been identified. These hor Steward and Shantz (207) have studied the mones and kinetin are collectively known as kin- effects of different substituted phenoxyacetic acids ins, but their mode of action is unknown. on cell growth rate and cell division rate in isolated A third group of hormones has recently been iso- potato tuber cells, grown in a fluid medium. They 15Kinetin has also been tested on animal cells, with con find that a wide range of substituents increase flicting results (references in 98).

lated from the fungus Gibberella. They are known All these effects are important in a study of as the gibberellins (210). In many respects they control mechanisms, though they have received, behave like auxins, but there are important dif in most cases, very scant attention. I do not ferences. Their action seems to be primarily on propose, therefore, to consider them in detail, cell growth and little, if at all, on cell division. especially since they are being reviewed at length by Harnden (103). Mitotic synchrony, however, 2. DISCUSSIONANDCONCLUSIONS does raise a number of points of interest that We have argued, in earlier sections, that animal I want briefly to consider. growth hormones operate inductively and switch the metabolism of quiescent cells from the syn 1. SYNCHRONYINSYNCYTIA thesis of differentiated proteins over to the syn Mitotic synchronism of nuclei within one mass thesis of proteins for the mitotic figure and the of cytoplasm appears to be an almost invariable other mechanisms concerned with cell division. rule, no matter how many nuclei are involved. They operate, that is to say, at a very fundamental It has been described in multinucleate protozoa, level, to determine a whole pattern of synthetic in fungi, insect eggs, multinucleate vertebrate metabolism. Do plant growth hormones work in cells, fish embryos, slime molds, and plant endo the same way, or are they required continuously sperm (references in [103]). as essential factors in some particular aspect of A particularly intriguing case has been described the growth and division mechanisms? by Howard (112) in the slime mold Physarum There is, as far as I am aware, no evidence polycephalum. This organism consists of one huge of the type collected in Table 1, about the existence syncytium, many centimeters across, containing or otherwise of a latent period with cell hypertro enormous numbers of nuclei. Within any one phy.16 Nor has anyone determined whether plant region, the nuclei divide, apparently synchronous hormones need to be applied throughout the cell ly, but the mitoses actually spread across the syn cycle or merely for a short time at the beginning. cytium as a wave, which takes about 10 minutes On the other hand, the multiplicity of effects, to go from one side to the other. and the way in which a balance of auxins and As far as I know, only one investigation has kinins can determine not only division, but various ever been made that throws any light on the mech types of differentiation, both suggest rather strong anisms underlying syncytial synchrony. Quite re ly that the plant hormones operate at a level cently Rusch (private communication) has suc deeper than that of particular enzyme systems. ceeded in growing Physarum under sterile culture Although there is no direct evidence, it would conditions. If these sterile cultures are shaken not be surprising, therefore, if plant hormones, continuously, the plasmodium breaks into numer like animal hormones, proved to work, at least ous pieces. The mitoses in each piece are syn in part, by the induction of particular patterns chronous (the intermitotic period being about of synthesis. 12 hours), but there is no correlation between the divisions of the different pieces. If, however, VI. NATURAL MITOTIC SYNCHRONY all these separate pieces are centrifuged down, The nuclei of syncytia, or of multinucleate they coalesce, and the resulting large syncytium cells, almost invariably divide in perfect unison. divides synchronously after 6 hours. In other Ordinary cells seldom show such an effect, but words, a fragment that went through mitosis, the mitoses and mÃ©iosesofanimal testes and plant e.g., 1 hour previously and a fragment that went anthers nevertheless often exhibit a very high through 11 hours previously, in some way produce degree of synchrony,17 as do the early cleavage an average effect of having been through mitosis stages of many eggs. Outside these rather special 6 hours previously (cf. Part I, VIII, 3). cases, it is usually assumed that mitoses occur It is clear from Rusch's work that DNA dou only at random. In fact, however, partial syn bling is not a limiting factor for mitosis in this chrony, amounting to a more or less clearly defined organism, since it evidently takes place shortly mitotic rhythm, has been found quite often, both after mitosis. Presumably, then, it is not impli in the tissues of higher animals and plants and cated in the phenomenon of syncytial synchro in cultures of micro-organisms. nism. However, even if the DNA had doubled le In any case, the structure of the plant cell and the exist much later, Rusch's second experiment would ence of cell extension dissociated from cell division would make have suggested that it was not concerned. This any direct comparison with animal cells difficult. experiment indicates, in fact, that the limiting 17Female gametogenic divisions, however, do not appear to mechanism must be something that involves a be synchronous, though there may be waves of mitosis in continuous rather than a discontinuous build-up estrus.

of some substance. It is difficult to see how the The substance involved has not been identified. times after one division and before the next could There is some evidence that it may be DNA be made to average out in any other way. DNA or a DNA precursor, and, if the protoplasmic is, of course, the one division mechanism that bridge explanation be ruled out, this is rather is a discontinuous affair; but, since growth, as likely. The inductive type of control discussed well as all the other division mechanisms postu in earlier sections, which appears to operate in lated in Part I of this review, is definitely, or most cells of a higher organism, is clearly ill very probably, of a continuous nature, more work fitted to give a high degree of synchrony, because is needed to find out whether one of these, or yet of the long and potentially variable interphase. some other process, is involved. Control by the supply of some metabolite required Lastly, it is of considerable interest that the lim for any of the continuous division mechanisms iting mechanism in syncytial synchronism should discussed in Part I of this review will not give be "miscible" in the way Rusch's experiments great synchrony for the same reason. Control indicate. This might seem to suggest that the by the supply of some metabolite required for mechanism is cytoplasmic, though, strictly speak a discontinuous mechanism, particularly if it op ing, it need mean only that whatever substance erates shortly before mitosis, might, however, give is involved can diffuse freely around the organism. such an effect, and DNA doubling, at least in It could very well be elaborated in the nucleus, most cells, is just such a mechanism. though not presumably held there. These consid erations would seem to rule out the nuclear PNA 3. SYNCHBONTINCLEAVINGEGGS cycle, but the choice still remains among a con Eggs vary widely in their cleavage behavior. siderable number of other mechanisms. Some cleave perfectly synchronously for many Growth, in the sense defined in Part I of this divisions; others do not cleave synchronously at review (in the light of Section II of this review, all (103). It has usually been assumed that syn probably synthesis of mitotic apparatus protein), chrony, where it exists, is simply the result of is a continuous process. So also is the energy development at equal rates in the different blasto- reservoir, the -SH cycle, the temperature-sensitive meres. However, some experiments by Harnden cycle, and probably the radiosensitive mechanism. (102) suggest that in the sea urchin egg, at least, The synchronizing mechanism in Physarum might a measure of cell interaction is involved. be any of these or some other yet undiscovered Elastomeres separated at the two-cell stage system. will continue to cleave normally, but Harnden 2. MEIOTICSYNCHRONY found that, whereas the two half eggs showed Meiosis and mitosis are very commonly syn synchrony within themselves, they differed con chronous in the spermatogenesis of animals. This siderably between themselves in the time of cleav synchronism may be limited to small groups of age. It has been claimed that there are proto cells that are the descendants of one spermato- plasmic bridges between the different cells of the gonium, or it may be more widespread. A similar sea urchin egg, and, if so, we are presumably situation is found in many plant anthers. Tetrads concerned once again with a special case of syn of pollen grains may be synchronous; in some cytial synchrony; but, in the absence of more cases all the tetrads in a whole pollen sac may definite evidence on this point, we must also divide in perfect unison (references in [103]). consider the possibility that cells may interact Where only small groups of cells divide syn within the same egg, or between one egg and chronously, it may be enough of an explanation another, through the liberation of diffusible sub that they are all recently descended from a com stances. mon ancestor. However, in the more extensive Allee and Evans (11) claimed that sea urchin cases, this is clearly not so. It is possible that eggs divided more rapidly in dense suspensions. protoplasmic bridges are involved, in which case Harnden (102) has investigated this effect in more we may be concerned with a special form of detail and finds that there is, in fact, a definite syncytial synchrony, but definite information on optimum density of suspension for rate of develop this point is lacking, ment but that only the first cleavage is affected. There is, however, some evidence that materials He also finds a slight but not very consistent can pass from one pollen cell to another (19, increase in the degree of synchrony of the whole 55, 191). There is also evidence that pollen cells population of eggs. These effects are probably may absorb material from the tape tal cells (167) not the result of pH or other simple environmental and that Drosphila eggs may absorb material changes. It may also be significant that various from the giant nurse cells (126). workers have claimed to show that extracts of

sea urchin embryos can speed up their division however, been extensively criticized (see 103). (12). Nothing is known of the substances involved It is clear, for instance, that small temperature in these effects. variations, pH, redox potential, COa concentra In an interesting series of papers (references tion, food supply, and a number of other factors in [9]) Agrell has investigated the early synchrony must be very carefully controlled before any re and subsequent loss of synchrony in the sea urchin sults can be accepted without reservation. Never egg. He points out that synchrony is lost at theless, it does seem that the effect is a genuine about the same time as the cytoplasmic reserves one in bacteria and very possibly in other cells also. of DNA run out, and the embryo has to synthesize It may be mentioned that a similar effect has fresh DNA for itself. It would seem possible, been reported in tissue cells. Early attempts to therefore, that DNA doubling may be involved set up pure lines from single cells all failed, and both in eggs and in meiotic divisions (Section Likely, Sandford, and Earle (142) succeeded in VI, 2) in the maintenance of a high degree of doing so only by growing cells in a small capillary synchrony. tubeâ€”presumably the very small volume made it possible for the single cell to build up the 4. MITOTIC RHYTHMS IN CULTURES necessary concentration of substance in measur OF MICROORGANISMS able time. More recently, Puck and Marcus (175) It is usually supposed that division in cultures found that clones of tissue cells could be estab of microorganisms occurs at random. However, lished if single cells were grown in a medium it has been noticed by various workers that suscep containing large numbers of cells that had been tibility to transformation in Pneumococcus, sus prevented from dividing by irradiation. In a later ceptibility to cold shock in Escherichia coli, and paper, however (176), it was shown that single oxygen consumption in Salmonella all show cyclic cells could be made to grow without these "feeder" waves, presumably due to an element of natural cells, provided that they were isolated more care mitotic synchronism. Other workers have shown fully. The importance of size of inoculum in estab directly that cultures of Bacillus megatherium and lishing ascites tumors has also been examined Chlorella exhibit waves of division (references in some detail (122). in [103]). The chemical substances (if any) that are re It has usually been assumed that these rhythms sponsible for all these reported effects have not are the result of all the cells starting to grow been identified. Malmgren and Heden (147), work simultaneously on inoculation into a fresh medium, ing with bacteria, suggest that they may be DNA or of starting a culture from a single organism. or DNA precursors. Mast and Pace (149), us Adolph and Bayne Jones (7), however, raised ing Chilomonas, believe that there is a specific Bacillus megatherium cultures from single cells "growth-promoting substance" released. and noted that the synchrony seemed to improve with time. 5. MITOTIC RHYTHMS IN HIGHER ANIMALS Harnden (102) has investigated cultures of the As we have seen earlier in this review, waves fission yeast Schizosaccharomyces pombe in some of mitosis amounting to partial mitotic synchrony detail and finds that, whereas some cultures show are commonplace and indeed almost characteristic a definite mitotic rhythm, others do not. He in a number of situations. They have been noticed also finds, significantly, that there is no initial in healing wounds, regenerating organs, following burst of mitosis after inoculation. It seems rather hormone treatment, and in the normal course likely, therefore, that the cells are liberating some of development. They are also to be found after substance into the medium that is responsible the initial setting up of a tissue culture. As we for the effect. have argued earlier, they are presumably due to Further evidence that microorganisms can in inductive stimuli of one sort or another acting some way affect the division of other individuals on a cell population that is predominantly qui comes from the numerous observations that cells escent, and we need not consider them further. in a small inoculum will not grow at all or will There are, however, some observations on tissue show a much longer lag phase than those in a cultures that are not so readily explained. It large inoculum. This is a well known phenomenon has been noticed (e.g., 79) that small groups in bacteria (see Part I, IV, 2). It has also been of cells within a tissue culture are likely to divide reported in protozoa and unicellular algae. The with a fair degree of synchrony. As far as I effect forms the basis of Robertson's (185) well know, such observations have not been subjected known theory of "allelocatalysis." This theory, to any rigorous statistical treatment to make and the evidence on which it is based, have, sure that the grouping of the dividing cells is

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1958 American Association for Cancer Research. SWANNâ€”Control of Cell Division: A Review 1147 not simply due to chance. However, if we accept 6. CONCLUSIONS the observations at their face value, we must Three grades of mitotic synchrony can be recog suppose, once again, that there are fine proto nized within organisms: the perfect synchrony plasmic bridges from cell to cell, or, as would of syncytia or multinucleate organisms, the high seem to be the case in microorganisms, that cells degree of synchrony of testes and plant anthers, exert some influence on each other. This might and the partial synchrony or mitotic rhythms be the release of chemical substances or perhaps found in many plant and animal tissues. an effect mediated by cell contact. It is suggested that the perfect synchrony of It remains to consider a specialized and recur syncytia is due to some "miscible" cytoplasmic rent form of mitotic wave, namely the diurnal division mechanism, as yet unidentified. The syn mitotic rhythms that have been found in a large chrony of mitotic and meiotic divisions, on the number of tissues of higher plants and higher other hand, is more likely to be owing to diffusible animals, particularly mammals.18 This question metabolites concerned in DNA synthesis. Mitotic has been extensively investigated and reviewed rhythms may be due to variations in the level by Bullough (32). of energy metabolism, though the possibility of In the light of his work on epidermal mitosis hormonal effects cannot be excluded. in vitro, in which he showed that an energy supply alone is sufficient to make "antephase" cells divide VII. CELL CONTACT AND (Part I, Section II; see also Part II, Section III, CELL DIVISION 1), Bullough attributes the diurnal rhythm to It has long been realized that cells may show variations in carybohydrate metabolism. He finds, specific adhesiveness (114), but it is to Holtfreter for instance, that muscular exercise and cold both (111) more than anyone else that we owe our reduce very markedly the amount of mitosis, present realization of the importance of such ef and both factors do, of course, cause a heavy fects in development. It is now clear that shifting drain on carbohydrate reserves. Similarly, he has patterns of adhesiveness are essential in controlling shown that insulin and starvation reduce mitotic cell movements and in the blocking out of organs activity and, conversely, that the mitotic rate and tissues. They are important in the innervation can be raised by injection of carbohydrate. of organs (240) and probably also in sperm egg However, a study of the diurnal cycle of blood interaction (221). sugar concentration in normal mice has shown Recently Abercrombie, Ambrose, and co- that it is high when mitosis is low, and vice workers have examined these problems in vitro versa (see also 131). Bullough, therefore, suggests and have attempted to quantify some of the that the critical factor is the carbohydrate level effects. They have shown, in particular, that cell within the cell, and he has shown that in skin, at movement is inhibited in proportion to the number least, the intracellular glycogen content runs paral of cell contacts in cultures of fibroblasts (3).19 lel with the epidermal mitotic rate. The factors They have also shown that cell contact depends controlling intracellular glycogen content, how on a specific adhesiveness which is lacking in ever, are unknown. tumor cells (4). This dependence is evidently as In the light of Bullough's work and our earlier sociated with a change in surface electrical charge discussions, we may suppose that a steady stream (16). It has, in fact, been known for some time of cells starts moving through interphase, but, that tumor cells within a tissue do not adhere because of varying levels of energy metabolism, to one another so tightly as normal cells (50), a greater or lesser number will be speeded up and there can now be little doubt that the loss from "antephase" into division. Since the diurnal of specific adhesiveness is closely connected with invasiveness and metastasizing power. variations of mitotic index are only about twofold, The physical basis of specific adhesion is by this may well be a sufficient explanation. On no means entirely clear, but it is generally as the other hand, we cannot exclude the possibility sumed to be a phenomenon of the same general that the initial activation, whether hormonal or type as enzyme-substrate and antibody-antigen local, is also a rhythmic process. The observations reactions. Presumably the surfaces of any one of Halberg et al. (100), indeed, rather suggest type of cell bear charges suitably distributed to some such effect, since the adrenal cortex appears enable them to attract one another, or are suitably to show a mitotic rhythm which is the exact sculptured to allow close contact and adhesion opposite of other tissues. 19A further finding of considerable interest is that cells in 18Including tumors (230). mitosis do not adhere to their neighbors.

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1958 American Association for Cancer Research. 1148 Cancer Research Vol. 18, November, 1958 by hydrogen bonds and van der Waal's forces. there is a marked temporary rise in respiration The various possibilities have been discussed by rate. Weiss (238), Schmitt (192), and Steinberg (205). Evidence of quite a different sort, and more Not surprisingly, specific adhesive effects can be specifically concerned with cell contact, comes modified in various ways by antisera (96, 170, from a study by Harrison (105) on the mode 203, 222). of growth of small, free-living amebae (Hartmanel- la spp.) maintained in sterile culture on peptone. 1. CELL CONTACTAND THE INHIBITION Harrison found, first of all, that cultures which OF PROLIFERATION were permanently agitated, so that the amebae Although most investigations of cell adhesion remained in suspension, grew like any other cul have been concerned with its importance in the ture of microorganisms, i.e., exponentially at first, movement and disposition of cells, there are a and then slowing down to a maximum. Identical number of grounds for thinking that it may also cultures that were not agitated, and in which be involved in the control of proliferation. In the amebae rested on the bottom of the vessel, the first place, it has long been realized that grew exponentially at first, but only until the cell division and cell movement are in some way amebae reached a density of about 106/sq cm. associated (references in [1]). It would not be At this point they made a fairly continuous sheet surprising, therefore, if contact, which certainly of cells all over the bottom. Beyond this point inhibits movement, were also to inhibit division. they grew not exponentially, but accurately linear Such an effect may explain the fact that, both ly, until they reached a density of about 5 X IO6/ in embryos and in adults, mitoses are very fre sq cm, when they slowed down and very gradually quently to be found along the free edges of organs reached a maximum. and tissues, giving rise to germinal epithelia. It The amount of nutrient fluid above the amebae might be argued that this is more likely to be made, within wide limits, no difference to the owing simply to the accessibility of oxygen and pattern of growth, and it was found, if cultures nutrients. On the other hand, as we shall see were grown on a fine sintered-glass surface through later, it may be not so much a question of the which nutrient solution was gently perfused, that proximity of nutrients as that cells which are the growth curve was still identical with that not making close all-around contact are mechani in a normal culture. From these and other experi cally in a better position to take up nutrients ments it is evident that neither nutrients, oxygen, and particularly to take up large molecular weight pH, nor indeed any effect other than cell contact substances by means of pinocytosis or vacuole can be involved. formation. Then again, it is possible that contact The contact effect is not apparently species- plays an important part in wound healing, if not specific, since two species of amebae grown to in the initiation of division after wounding, at gether did not reach a higher joint maximum least in the cessation of division when epithelial of cells. Some extremely interesting results were, continuity is restored (see Section IV, 2). however, obtained with antisera. It was found Turning to more precise information about the that culture growth was somewhat retarded in relation of the cell surface to the control of divi dilutions of 1 in 10 to 1 in 80 of ameba antiserum sion, there are two lines of evidence to be con in peptone, but the exponential growth typical sidered. Recently, Tyler (222) has shown that of the early part of the growth curve was extended antisera prepared against fertilizin can block mi to a higher density than in controls grown in tosis in the sea urchin egg. Since fertilizin is a comparable dilutions of normal serum in peptone, polysaccharide, present in the surface of the fer while the linear phase was considerably reduced. tilized and unfertilized sea urchin egg, which can Antiserum, in short, seems to oppose the contact be prepared in a pure state from the jelly that inhibition effect, presumably by preventing proper surrounds the unfertilized egg, this is an experi contact. ment of considerable interest. It is, in fact, the It seems likely that contact inhibition involves first time that an antiserum prepared against the processes of cell growth rather than of cell a known surface constituent of a cell has been shown division, for the simple reason that there are to have an inhibitory effect on division. no clear cell-size variations at different points Nuclear as well as cytoplasmic division is in in the cycle of culture growth. In what way hibited by this treatment, and the block does cell contact affects growth, however, remains to not appear to be specific for any particular stage be discovered. In the absence of evidence to the of division. Its mode of action is unknown, though contrary, it is perhaps best to think in terms of

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1958 American Association for Cancer Research. SWANNâ€”ControlofCell Division: A Review 1149 a direct effect of nutrients on intake. Extensive anisms are in varying degrees dissociable: there contact inevitably reduces the area of cell surface can be no certainty, therefore, that the same to which nutrients have access, furthermore, it one is always the final one. appears to impede the ingestion of food vacuoles If the idea of a single key mechanism triggering by pseudopodial activity, though the proportion division is doubtfully valid for isolated cells, it of peptone taken in by these means is not known. becomes completely untenable when we consider The significance of the linear growth curve is the cells of an organism. Within an animal or also uncertain. It could be explained by supposing plant, every type of differentiated cell, in a huge that only cells on the glass substratum were divid range of different organs and tissues, is capable ing, but this seems not to be the case. It is more of dividing in its own time and at its own rate. It probably due to a steady decrease in growth rate is inevitable, therefore, that there will be (in ad as the number of cells, and hence the amount of cell dition to the general mechanisms discussed in contact, increases. Part I) as many special mechanisms as there are It remains to be seen whether simple mechanical cell types, i.e., presumably some hundreds. considerations are a sufficient answer to this prob There are various ways in which this multi lem, but there are at least some grounds, from plicity of control mechanisms might be achieved. other work, for thinking so. When a piece of It would not be impossible, for instance, to con tissue is cultured in the ordinary way on a plasma ceive of each differentiated cell type as being clot, or in a fluid medium on glass, it gives rise, deficient in a different substance involved in one of course, to rapid unorganized growth. If, on of the various division mechanisms. A liver cell the other hand, contact with a solid surface and might lack something required for DNA synthesis, all-around accessibility of nutrients are both pre a kidney cell might lack something required for vented by floating the tissue on a "raft" on the -SH cycle, and so on. These cells could then the nutrient medium, growth is more usually divide only if they were supplied from elsewhere slow and organized. We get, in fact, not tissue with the appropriate compound. There is some culture, but organ culture. It would seem, there evidence (Section VI) that mitotic synchrony may fore, that simple mechanical considerations are be brought about by such means, but there is important and might be sufficient to account, little evidence to suggest that it is a universal at least in part, for the inhibitory effect of cell pattern of control (see, however, 28, p. 71). contact on cell growth. Alternatively, we might suppose that every cell was lacking in something concerned in one 2. CONCLUSIONS particular division mechanism and that the control Specificity of cell contact is known to be im systems consisted of a variety of methods for portant in morphogenesis, and the lack of it to making good the deficiency. This, I believe, is be concerned in the invasiveness of tumor cells. essentially the pattern of control that is most There is some evidence also that cell contact commonly envisaged, the division mechanism in tends to inhibit growth and division. It is not question supposedly being DNA. yet clear whether this is simply a case of mechani The trouble, however, is that DNA doubles cal hindrance of pinocytosis or some more complex during each cell cycle and can fairly readily be phenomenon. estimated. It is, therefore, only too easy to suppose that, because some stimulus leads to the doubling VIII. GENERAL DISCUSSION of DNA, it is bringing this about directly and There is a common tendency to think of the that the cell in question was not dividing because control of cell division in terms of a single key it was blocked at the point of DNA synthesis. mechanism, but I doubt whether this is a very While it seems to be true that differentiated useful concept, even in isolated cells. It is true cells are held back at some point prior to the that some process must be the last to reach doubling of DNA, we have examined at some completion, and to that extent it might be re length the reasons for supposing that all the garded as the key mechanism for triggering mi other division mechanisms are similarly suspended tosis. However, as we saw in Part I of this review, (Section II, 2). It may also be mentioned that if there are a considerable number of these mecha DNA synthesis is artificially blocked (by radia nisms, all of which must reach completion before tions or alkylating agents) it does not lead to mitosis begins, and it is surely meaningless to a blockage of the other division processes, and regard any one of them as being more important growth in particular continues, giving rise to giant than the others. In any case, the different mech cells (Part I, Section VII).

In Section II, 3, it was argued that there is to division, we have two contrasting situations a good reason for the apparent suspension in to consider. On the one hand there are the various differentiated cells of all the processes of division, recognized mitogenic hormones, the local wound since mitotic protein in particular, and the other hormones, the humoral substances concerned with elements concerned with division to a lesser degree, kidney growth, red cell production and so on, occupy a large fraction of the dividing cell. A and a number of other ill defined systems of differentiated cell that kept these mechanisms one sort and another. All these appear to act going would, therefore, have relatively little space by means of definite stimulatory substances whose left for differentiated functions. The situation then presence induces cell growth and cell division. seems to be that a differentiated cell suspends On the other hand there is, in the case of liver, all its division mechanisms and resumes the ap a definite inhibitory substance, only in whose propriate syntheses only in case of need, as a absence do growth and division take place. The result of some mitogenic stimulus. case of the pituitary may be similar, the inhibitory A switch of synthetic pattern of this type is substances being in this case the various hormones comparable in every way to a differentiation, secreted by the glands under pituitary control. and from this I have argued that the antagonism In their absence the pituitary certainly secretes between differentiation and division is only to more trophic hormone, and probably grows to be expected. It is, indeed, no more surprising some extent as well. than that a liver cell, for example, should not Are we to accept this evidence at its face secrete pituitary hormones. value and suppose that division is controlled in In the course of embryological development, some cases by stimulatory substances, and in switches of differentiation are brought about by other cases by inhibitory substances? Or are we means of "induction," the essence of which is to assume that in one or the other group we have that a stimulus of short duration should produce discovered only hah* the control mechanism? By a much longer lasting effect. Very little is known inventing suitable feed-back systems, it would of the chemistry of this process, though, following no doubt be possible to force every situation current lines of thought about protein synthesis, into a common mold, of either the stimulatory we might expect it to operate at the level of the or the inhibitory kind. microsomal particles. In a stimulating discussion of this general prob Is there then any direct evidence that the lem Abercrombie (1) argues strongly in favor of stimuli to mitosis within an organism are of the accepting controls that depend ultimately "ei inductive type? These stimuli have been consid ther on a specific 'regulatory substance' that the ered at some length in Sections III and IV. organ normally adds to the blood, or on one They consist of certain recognized hormones, hu that it normally subtracts from the blood." I moral factors of one sort and another concerned would add a further argument in favor of the in regulating the size of organs, and a variety of same point of view. I believe that it is basically local factors concerned in maintaining continuity wrong to think in terms of stimuli or inhibitions of tissues, and in the healing of wounds. where division is concerned. I have argued earlier In the case of various hormones, there is quite that what is normally called a mitogenic stimulus good reason to think in terms of an inductive involves the start up of an entirely new pattern action, since the hormones in question are broken of synthesis, and very probably the cessation down so rapidly in the body that the stimulus or partial cessation of the previous pattern of can only be present for a relatively short time synthesis concerned with differentiated function. (Section III, 5). There is also some evidence, both Similarly, when the mitogenic stimulus is over, from hormones and from regeneration, that micro- there is a reversal of these effects. From the somes are affected at a very early stage in mito- point of view of synthesis, therefore, there is no genesis (Section III, 5; IV, 4); and, finally, there switch on, or of, but rather a switch from one is the apparently universal phenomenon in mito- direction to another. It might indeed be better genesis of a long latent period with cellular hyper to use the word steering, instead of switching, trophy which, as we have seen in Section III, which tends to carry the implication of on or off. is only readily explained in terms of induction. If then, we think not in terms of switching There is, however, one important point of differ division on or off, but of steering synthesis in ence from normal embryological induction, namely, one direction or another, there is no particular that the effect appears to last only for a single reason to expect that the division pattern will cell generation (Section II, 5). be determined by the presence of some substance, Turning to the nature of the inductive stimulus and the differentiation pattern by its absence,

rather than vice versa. We might well expect together "with oasic systems sxich as those of to find both situations, and the present evidence, energy metabolism, must, therefore, take up so as far as it goes, suggests that in fact we do. much of the space of a dividing cell as to leave A final point of some importance is the locus little room for other differentiated functions. This, and mode of action of these steering substances. we argued, must be the ultimate cause of the In earlier sections I have suggested that they are antagonism between differentiation and division likely to act on the microsomal particles. Of course, and must be responsible for the state of affairs this is very speculative, and the mode of action whereby differentiated cells do not apparently may well be indirect and mediated perhaps via carry a useless load of mitotic protein and asso intra-cellular membranes or via the cell membrane ciated mechanisms, but switch over to making itself (cf. Section VII). It may also be that we these things only in case of need and under the have to think in terms not only of primary steering influence of some hormonal or other stimulus. substances but of secondary modifying agencies It is likely that the mere fact of switching that provide a fine adjustment to the steering over to this type of synthesis will lead to a process, and of yet other substances that determine decreased synthesis of differentiated elements as the rate of synthesis in various directions. These a result of intracellular competition; but even are all problems for the future, and it would if this is not so, the effect of extra synthesis be idle to speculate further on them at the present. in the new direction will result in a relative The complexity of differentiation, of division pat decrease of differentiated elements. This is pre terns, and of hormonal action all suggest, however, sumably the situation in regeneration and hyper- that this is the direction in which we must look plasia. for an understanding of the control and coordina Once a cell has become neoplastic, however, tion of growth in higher organisms. this change is likely to be accentuated by selection, since any cell synthesizing less of the differentiated IX.THE CONTROL OF CELL DIVISION elements, and concentrating its energies on the AND THE CANCER PROBLEM very considerable task of synthesizing the elements The subject matter of both parts of this review required for division, will be at an advantage. impinges at a number of points on the practical And this, of course, will lead to the gradual problems of cancer research. I propose, however, loss of differentiation, both biochemical and struc to consider only two matters that I believe to tural, so characteristic of the tumor cell. be of fundamental importance. The first concerns This same process should also lead to a loss the possible significance of the characteristic met of cytoplasmic volume, and tumor cells are in abolic pattern of tumor cells and arises from the fact usually smaller than their normal counter arguments of Section II of the present review parts. The situation may be somewhat compli about the large proportion of a cell's mass occupied cated by increase of ploidy, since this invariably with cell division.20 The second concerns the pos leads to a corresponding increase in cell size (Part sible nature of tumor cell autonomy in view of I, Section VIII, 1); but, in any case, the volume the arguments advanced earlier for the control of cytoplasm relative to nucleus will fall (51). of proliferation by inductive stimuli operating In a typical case (liver cell hepatoma) this ratio at the level of protein synthesis. was found to shift from about 6:1 to 3:1 (193). It was shown that nuclear volume did not change, 1. THE METABOLICPATTERNOFCANCERCELLS so that the amount of cytoplasm must roughly The great wealth of work on the biochemistry have halved. If we suppose that the hepatoma of cancer, so ably summarized by Greenstein (95), cell has lost most of its differentiated functions has shown incontrovertibly that all cancers, what and consists largely of mitotic protein, the various ever their origin, tend to converge gradually on division mechanisms, and the basic mechanisms a common metabolic pattern. I want to consider of energy metabolism and protein synthesis, we the possible significance of this pattern, in the are then led to the conclusion that these elements light of the arguments in Section II of this review. make up a mass equal to about half the cytoplasm There, it will be remembered, we argued that of a normal, differentiated liver cell. This figure, specialized mitotic protein, together with the vari it will be recalled, agrees well enough with the ous division mechanisms, accounts for a consider estimate of Section II, derived from direct meas able proportion of the total mass of a dividing urements of spindle volume in normal cells. cell, perhaps as much as a half. These elements, The case of energy metabolism in tumor cells 20My views are essentially an extension of those of Husch is a particularly interesting one in this connection. (188). A succession of workers, starting with Warburg,

have felt that the high glycolysis, and more par to be regarded as in some way causal; but if ticularly the high aerobic glycolysis of tumors, it is accepted that the process of proliferation was in some way an integral part if not the requires very extensive synthesis of specialized actual cause of neoplasia. The relevant facts are protein for the mitotic figure, and lesser syntheses set out in some detail by Greenstein (95) and of special systems concerned with initiating and Weinhouse (237). It is now apparent that the aero controlling the process of division, the whole ac bic respiration of tumors, though low, is not counting for the bulk of the cell mass, then the lower than in some normal tissues. It is also changes associated with cancer take on a new apparent, however, that the various enzymes, aspect. They represent in fact "streamlining" of particularly in the cytochrome system, are work cells that do nothing but proliferate. The common ing to the limit of their capacity in tumor cells, pattern of tumor metabolism is likely, therefore, whereas in normal cells there is a considerable to be simply the basic minimum of structural reserve in hand. The glycolytic enzymes in tumors and metabolic requirements for proliferation. The also appear to be working at the limit of their fully autonomous, highly malignant cell, in short, capacity, whereas in normal cells they, too, have is probably the nearest we can get to a pure "proliferating machine," shorn, it would seem, large reserves in hand. Returning to our earlier argument that pro of virtually everything that does not contribute liferation absorbs a large part of a cell's resources, to this end. we can expect that selection for cell growth rate within a tumor will lead to a steady reduction 2. THE FUNDAMENTALBASISOFAUTONOMY in these costly and seemingly unnecessary reserves It is now generally agreed that two processes of respiratory and glycolytic enzymes. It is not are crucially involved in the cancerous transforma surprising, indeed, that they should be reduced tion. Cells must lose, partially or completely, effectively to nil, since, unlike the normal cells their normal control mechanisms and their normal of many organs, the tumor cell has no unexpected contact relations. Loss of the first, but not of calls on its energies. It is simply growing steadily. the second, results in a benign tumor. Loss of If, moreover, the cells are growing in somewhat the second, but not of the first, is less readily anaerobic surroundings, which, because of inade recognized, but it may be that metastatic prostate quate blood supply, may be the case, we can carcinoma and various other hormone-dependent further expect that selection will favor the syn tumors fall within this category. thesis of glycolytic rather than aerobic respiratory As far as we can judge, these two changesâ€”loss mechanisms. This, together with selection in favor of normal control mechanisms, and loss of normal of economy, might well lead to a situation in contact relationsâ€”are in themselves sufficient to which the respiratory mechanisms could no longer make a cell neoplastic. It does not follow, however, oxidize lactic acid as fast as the glycolytic mech that cancer is necessarily the result of two single anisms produced it. In short, selection for econ and sudden steps. Both processes may be gradual omy in synthesis, conducted in slightly anaerobic and ins^olve several steps. The loss of growth surroundings, may be enough to account for the control, indeed, is known to be gradual in the tumor cell's characteristic energy metabolism. If case of hormone-dependent tumors,21 and there so, the well known high aerobic glycolysis may is a certain amount of statistical evidence, from merely be a more or less inevitable result, and the pattern of incidence of tumors with age, which not in any sense the cause of the neoplastic state. suggests that at least three steps are involved Similar considerations ought, perhaps, to be in the neoplatic transformation, and very possibly applied to other changes that characterize cancer more (references in [80]). cells, before assuming that they may be funda Contact relations fall largely outside the scope mental causes of neoplasia. Many of these changes, of the present discussion, and, apart from empha I believe, may turn out in due course to be sizing the importance of discovering more about secondary effects. the changes of surface structure that must be The essence of these arguments is that the involved (see, for example [177]), I do not propose process of proliferation, from the point of view to consider them further. We can, however, use- of the cellular economy, is an expensive one, with 21There are also hints that other tumors may retain some the synthesis of mitotic protein accounting for degree of dependence. Paschkis et al. (169), for instance, re most of the budget. As long as it was possible ported that the growth of a hepatoma was stimulated during liver regeneration. In the case of multiple tumors it has also to doubt this, and I think it generally has been been reported on occasion that removal of one speeds up the doubted or at any rate overlooked, then the nu growth of the others (90). This is a situation exactly com merous changes that occur in neoplasia are likely parable with normal compensatory hypertrophy.

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1958 American Association for Cancer Research. SWANNâ€”ControlofCell Division: A Review 1153 fully examine the loss of growth control in the to react, since we know nothing of how or where light of the evidence and of arguments brought the inhibitor works. However, if we assume that forward in earlier sections of this review. the inhibitor normally attaches to synthetic cen Within an organism we find, it would seem, ters in such a way as to prevent access of raw a situation in which the cells are normally qui materials, then failure to react presumably in escent, as far as proliferation is concerned. Par volves failure to attach or, in other words, some ticular stimuli,22 inductive in nature, are then progressive loss or change in the surface properties needed to activate the growth and division mech of the synthetic centers themselves. anisms. Most stimuli seem to be fairly specific, On a priori grounds it is perhaps more likely but some, e.g., the wound hormones, appear to that loss of control will be associated with a activate a wide range of cell types. How many loss of specific substances, rather than with the different stimuli are required to activate a given acquiring of an ability to synthesize some new type of cell is uncertain. In the various situations substance. On the other hand, every cell pre we have considered in earlier sections, we have sumably contains the genes for synthesizing any been able to identify in most cases only one of the growth stimulators, so that we can hardly stimulatory factor. It is, nevertheless, perfectly rule out this possibility. possible that several stimuli are really involved. It is clear that we need to know much more The fact that, in many types of response, hormones of both the locus and the mode of action of the seem to exert at least a modifying influence makes inductive agents before we can hope to understand this, indeed, rather likely. the nature of tumor cell autonomy. Nevertheless, How, in view of these mechanisms, can we there are a number of possible leads to follow, account for the loss of growth control in tumor of immediate relevance to the cancer problem. cells, and, a further point of some importance, It should not be out of the question, for in how can we account for the gradual nature of stance, to find out directly whether any tumor this loss? There are a number of situations to cells are synthesizing their own stimulatory sub consider. In the case of cell types in which control stances. Are tumors derived from sexual tissues is by means of a definite stimulant, it would seem such as uterus, mammary gland, or prostate mak likely that a tumor cell must make progressively ing small quantities of androgens or estrogens, more of the appropriate stimulus substance itself, barely sufficient, perhaps, for their own purposes? or that it must gradually bypass the systems that Unlikely as this may seem, it might provide a require the stimulus. Since we know effectively simple explanation of the facts discussed by Klein nothing of what these systems are, we can hardly and Klein (123) in an extremely interesting paper say much about the nature of a possible bypass on the evolution of independence from stimulatory mechanism; but if, say, the synthetic centers and inhibitory effects in tumors. A number of for growth and division were normally isolated ascites tumors, originally hormone-dependent, but from the supply of raw materials by substances transplanted often enough to have achieved ap specific for each differentiated cell type and the parently complete autonomy, will nevertheless appropriate stimulus factor then attached itself show signs of hormone dependence if administered in such a way as to allow nutrients access to the in small inocula. In general, moreover, larger in synthetic centers, the bypass would presumably ocula are needed for establishing an ascitic growth consist in the disappearance or alteration of the than for a solid growth. The authors suggest original specific blocking substances. that the cells "gradually acquire the ability, either The case of cell types such as liver, where by way of adaptation or by selection of variant control is by means of a circulating inhibitor, types arising de novo,to produce and utilize endog is rather different. Failure to produce the inhibitor enous substances capable of acting as substitutes may be involved, but it is difficult to see that for the exogenous stimuli previously needed. With this would make any significant difference until a small cell number, such cell products may be the tumor was well established, since all the other come too diluted and fall below an effective level, normal cells would in fact be producing it. We thereby making the cells once more dependent must, I suggest, think rather in terms of failure on the previously needed exogenous stimulus." to react to the inhibitor, at least in the earlier Furthermore, "the fact that a critical inoculum stages of the growth of the tumor. As with cells size is necessary to initiate free-cell growth, and that normally react to a division stimulus, we cell numbers below this critical limit cannot grow cannot say much about the nature of this failure in the ascites, but only in the solid form, can 221 use the word stimulus here to include activation by also be explained by this assumption. The better release from inhibition. growth of small inocula in the solid tissues might

be due to the less severe dilution and slower In the first place, if tumor cells have acquired diffusion of the active cell product(s). A relatively something of critical importance, it is most likely small number of tumor cells might be sufficient to be the ability to synthesize their own inductive to build up the critical concentration necessary stimulatory substancesâ€”either hormones, or or for cell division in solid tissues, whereas in the gan-specific controlling substances, or "wound hor peritoneal fluid the products of few cells become mones," depending on the type of cell in question. rapidly diluted by the relatively large amount It should be added, however, that cells which of continuously circulating liquid." operate through an inhibitory mechanism, e.g., Further evidence in favor of the idea that some liver cells, are presumably not capable of following tumors may in fact synthesize the substances this road to autonomy. that their normal counterparts require for growth If, on the other hand, tumor cells have lost and division comes from work on the crown gall something, it is likely to be substances or struc tumor of plants (29). Tobacco pith tissue normally tures that react with the various inductive agents, requires both auxins and kinins to proliferate, whether stimulatory or inhibitory. Of the chemical but the same tissue rendered cancerous by crown nature of these substances we know nothing at gall bacteria can apparently synthesize these sub all, but we can at least guess that they will stances, though to an extent that depends on be functionally related to microsomal particles, the length of the induction period. Varying degrees and may perhaps be physically attached to them. of tumor autonomy can therefore be produced In our present state of ignorance about these at will. A point of some interest is that the ultimate controlling processes of growth and divi inductive stimulus (crown gall bacteria) is only sion, it would be idle to attempt to decide among effective when the cells to which it is applied these various alternatives.23 As we have seen earli are at or near division. er, it seems that crown gall tumor cells in plants The possibility that some tumors may become fall into the first category, of synthesizing their autonomous because they begin to synthesize the own stimulatory substances, but it does not follow necessary stimulators themselves is obviously that the same situation prevails in animal cells. much the easier problem to investigate, since In any case, as we have seen earlier, not all quite a range of stimulators have been identified, animal cells are likely to be capable of this aber either partially or completely. The alternative ration. It would not be surprising, indeed, if possibility, that tumor cells are failing to react autonomy were to arise in all the ways we have in the usual way to the appropriate stimulatory discussed. or inhibitory substances, is much more difficult to approach, since we know little or nothing of The fact that we can perhaps begin to see the normal situations. As a start, however, it what may have happened to the mechanisms of would be desirable that hormone dependence, or control in tumor cells does not get us any nearer dependence on any other form of stimulus or to an understanding of why these changes should inhibition, should be quantified in some way. have come about, and I have in fact deliberately How, for instance, does the mitotic dose-response excluded the problems of the etiology of cancer relationship compare, during carcinogenesis and from this discussion. It should, however, be said the progression of a tumor, with that of the in conclusion that the two main theories of cancer, normal tissue? Further advances are likely to the viral and the epigenetic (113), can both ac depend on new discoveries about the fundamental count quite readily either for the loss or the mechanisms concerned. gain of specific synthetic abilities. They could indeed lead to identical situations, as far as damage The argument of the preceding pages amounts to the mechanisms controlling proliferation is con simply to saying that, amid the various physical cerned. and chemical changes associated with neoplasia, ACKNOWLEDGMENTS cancer cells have either acquired something, or In attempting to review fields with which I am not very lost something, of critical importance in the control familiar, I have been helped inestimably by a large number of of proliferation. The alternatives of what we may colleagues. To all those who have given me the benefit of call positive or negative loss of control, have, 23If indeed they are alternatives. In an interesting paper, of course, been put forward often enough before, Weiler (286) has shown that cytoplasmic particles (microsomes and indeed one or other (or both) points of view and mitochondria) from normal kidney possess an antigen that corresponding particles from kidney tumor cells do not. And, must inevitably be true. However, in the light conversely, that the tumor particles possess an antigen that of our earlier discussions, we can perhaps give the kidney particles do not, but which is to be found either in a little more precision to these speculations. red cells or in particles from lung tissue.

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