Reflections About Hormone Action and Implications for the Cancer Problem*

Reflections about Hormone Action and Implications for the Cancer Problem*

O. HECHTER

(Worcester Foundation for Experimental Biology, Shrewsbury, Mass., and Department of Physiology, Boston University, Boston, Mass.)

In his theoretical consideration of hormone ac strates available to integrated mult Â¡enzymesys tion, Dr. Mueller has given us the views of a mod tems within semi-isolated compartments of the ern biochemist, conscious of cell organization, cell by regulating the transfer of substrate, or faced with a fundamental problem in cell physiol products, through cytostructural barriers. One as ogy. As an endocrinologist, trained in physiology, pect of this "new view," in essence, amounts to the I find myself in agreement with so much of what rediscovery of the potential importance of perme Dr. Mueller has said that the differences between ability relationships in regulating metabolic se us seem minimal. But differences there areâ€”not so quences, a concept postulated by the pioneers in much in terms of philosophy, but in the delinea pharmacology and cell physiology, with particular tion of profitable areas for future investigation. In reference to the cell membrane. Dr. Mueller's view, the enzyme is the unit of struc For certain reasons, Dr. Mueller has indicated ture and function in the cell, but I cannot help that hormone action upon cytostructural barriers, thinking that this view would not necessarily be while theoretically possible, seems unlikely as a upheld by cytologists, genetists, physiologists, mode of hormone action. I have previously stated pharmacologists, or even by all biochemists. This (12) and still feel that it is precisely in this area, in is a problem in semantics with philosophic over the control of permeability relationships, that we tones, which I do not propose to discuss. I think have the most likely mechanism of hormone ac all would agree that enzymes are important for tion. I readily admit, however, that a host of al cell function, that hormones in one way or another ternative possibilities exist. I should like to discuss modify cellular enzymatic reactions, and that, by a the possible action of hormones at the cell mem study of the manner whereby metabolic sequences brane, particularly with respect to the regulation are regulated in the living cell, hormone action will of growth. From this will arise certain ideas which eventually be elucidated in fundamental terms. may have implications for the cancer problem. I In discussing how and where hormones might shall not restrict this discussion to the steroid hor act at the cellular level, Dr. Mueller listed the old mones exclusively; in my view, the aspect of hor possibilities and some of relatively recent vintage mone action which is of prime importance to the in biochemistry. The old ways, in their essence, are problem of cancer is that which is concerned with those previously discussed by Green in 1940 (8). the regulation of growth of specifically differenti New ideas of hormone action have come into bio ated cells. This trophic action of hormones is not chemistry from a consideration of the enzymology restricted to steroids but is characteristic of the of intracellular structures coupled with the knowl pituitary trophic hormones, of protein or poly- edge from recent advances in cytology (made pos peptid nature as well. If I have broadened the area sible by new technics in electron microscopy), to of discussion by emphasizing that trophic hor gether with an old concept in biologyâ€”organiza mones of diverse chemical structure serve a similar tion. From this has emerged the view that hor biological function, I do not wish to imply that a mone action may be concerned with making sub- single mode of action need necessarily be opera * The work described in this paper was supported by The tive. Indeed, as I have previously indicated (12), it Commonwealth Fund, U.S. Army Contract No. DA-49-007- seems more reasonable to expect to find differences MD-546, U.S.P.H. Grant No. RG-4749, and National Science in mechanisms of action where such profound dif Foundation Grant NFS-G-2966. ferences in the chemical structure of the activating Presented at the second meeting of the Scientific Review Committee of the American Cancer Society, held at the West- agents exist. Keeping this reservation in mind, let ehester Country Club, Rye, New York, December 13 and 14, us proceed. 1056. Can hormones act to control permeability relation- 512

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1957 American Association for Cancer Research. HECHTERâ€”Reflections about Hormone Action and Cancer Problem 513 ships at the cell membrane?â€”inthe case of insulin mine whether this is actually the case in diaphragm the answer is almost certainly "yes." There is now muscle in vitro (where an insulin effect upon glu a growing body of evidence that one of the primary cose uptake is well documented), Dr. Resnick and mechanisms of insulin action is to increase the I estimated the rate of sugar penetration (inde transfer of sugar into the cells of certain but not all pendent of secondary metabolic effects) by study tissues (17). Muscle is a key example of an insulin- ing the entry of galactose into the cells of rat dia responsive tissue; brain and erythrocytes are ex phragm muscle, and these studies will soon be pub amples of tissues and cells which do not respond to lished (21). In confirmation of previous reports insulin. According to the hypothesis of Levine and that galactose is not utilized by muscle (17), we Goldstein (17), a "barrier" (probably the cell have established definitively (by analysis of the membrane) normally limits the free entry of sugar radioactivity of COz formed during incubation of into the cell; in responsive tissues insulin acts upon tissue with galactose-C14, as well as by paper a sugar-transfer mechanism to facilitate the pene chromatography of the radioactivity present in the tration of sugar through this barrier where it is tissue and medium) that galactose-1-C14 was not detectably transformed in our system. By deter TABLE 1 mining the content of galactose in the cells at vari INTRACELLULARENTRYOFGALACTOSEIN ous periods of incubation (correcting for tissue DIAPHRAGMMUSCLE extracellular sugar) and employing kinetic equa N*o insulin Insulin Diff. tions derived for us by Dr. A. K. Solomon at (mg/gm/hr) Harvard, it was possible for us to calculate the Resnick and Hechter (al) rate of entry of galactose into the cells in the units No preincubation 1.3 1.4 60 min. preincubation 2.4 4.4 2.0 of mg sugar/gm diaphragm (wet wt.)/hour of in-

TABLE 2 GLUCOSEUPTAKEBYDIAPHRAGMMUSCLE CONDITIONS Init. glucose Incubation concentration period No INSULIN1.81.91.61.83.12.5*3.62.71.62.72.21.5iNÃ¤CLIX(mg/gm/hr)3.43.52.8S.45.44.3*6.64.52.34.33.82.2Dirr.1.61.61.21.62.31.83.01.80.71.61.60.7 STUDY (mg per cent) (hr.) Gemmili and Hamman (7) 200 3 Stadie (23) 200 2 Villee and Hastings (24) 200 2 Haft et al. (IO) 200 1 \Yalaas and Walaas (25) 140 0.5 U tÃ U U 140 1.0 Brown et al. (1) 140 0.5 Krahl and Park (16) 140 1 Krahl and Cori (15) 100 2 Perlmutter and Greep (20) 100 2 Li et al. (18) 100 2 Demis and Rothstein (6) 100 1 * The mean of two sets of values presented in the same paper. made available to the intracellular enzymatic ma cubation.The entry rates obtained in these studies chinery. are shown in Table 1; they are compared to rep The nature of this sugar-transfer mechanism resentative studies reported in the literature on upon which insulin acts is still obscure; however, glucose uptake by diaphragm muscle, in the ab certain of its characteristics have been defined. sence and presence of added insulin (Table 2). In Thus, the sugar-transfer mechanism does not ap contrast to galactose (which merely enters and pear to involve a hexokinase-type phosphorylation leaves the cell until the concentration of sugar in step, since the insulin effect occurs with sugars the medium and cells is equilibrated), any glucose which are not metabolized in the test preparation which enters the muscle cell is removed via meta employed; the transfer mechanism has specificity bolic sequences, so that a continuous concentration in that it differentiates between sugars closely re gradient exists. If the transfer rates of galactose lated chemically; moreover, among insulin-respon and glucose entry into cells are of the same order sive sugars (i.e., those sugars whose entry into of magnitude, the values of galactose entry calcu cells is facilitated by insulin) competition exists lated from the kinetic equations should be of the for the transfer mechanism. same order as the rate of glucose uptake by According to the view of Levine and Goldstein, diaphragms, in both the presence and absence of the entry of sugar into the cells is the rate-limiting insulin. Examination of Table 2 shows that in all step in the over-all metabolism of sugar. To deter- the cases cited where diaphragms were incubated

with 100-200 mg. per cent glucose for 1 hour or sequences), the significance of limiting sugar entry more, this is the case. While it is appreciated that into the cell for DNA and RNA synthesis may be the transfer coefficients obtained with galactose of special significance. If the action of DOC to need not be precisely equivalent to those of glu limit the entry of sugar into the Neurospora cell cose, the fact that the values of glucose uptake has consequences for growth which can be ration reported in the literature are of the same magni alized in terms of established biosynthetic se tude as the values for galactose entry obtained in quences, this is not the case with respect to the our study lends strong support for the view that potassium requirement. All we can say is that po entry of sugar into cells is the rate-limiting event tassium (or rubidium) is essential for growth (for in the metabolism of glucose by the isolated rat reasons that are as yet obscure) and that DOC can diaphragm muscle and that the effect of insulin to act to prevent the entry of the essential potassium increase glucose uptake in this tissue is the result ion into the cell. of accelerated sugar entry into cells. It is apparent that if the cell membrane limits If insulin acts upon the cell membrane in certain the free entry of any essential factor, cell growth insulin-responsive tissues, might not other hor will be limited; contrariwise, when all these essen mones likewise affect membrane permeability in tial components are freely transferred into the suitably responsive cells? The suggestion that cell, growth will then be limited by intracellular steroid hormones might also act at the cell mem factors. If we assume that trophic hormones act brane arose as a result of investigations carried out at the cell membrane (the question of "how" is in a mold, Neurospora crossa. For reasons that are another matter) to account for the specificity of not germane to this discussion, Drs. Gabriel Les action of the various trophic hormones, it would be ter, David Stone, and I had been studying the in necessary to postulate that the membranes of the fluence of various mammalian steroids upon the various cells are different in the sense that they con growth of this mold ; the results of these investiga tain receptors which interact specifically with cer tions will soon be presented for publication. In es tain hormones but not with others. Our findings sence, it was found that, of some 30 steroids tested, with the Neurospora cell, although not fully ap deoxycorticosterone (DOC) was highly specific in plicable to the action of a steroid hormone upon inhibiting growth; under certain conditions it mammalian cells, afford a striking demonstration could be shown that concentrations of DOC of the of the very high order of specificity involved in the order of 10~5 M were effective. Analysis of this in interaction of steroid with the cell membrane. hibition revealed that DOC markedly inhibited The nature of the cell membrane.â€”If this view is the entry of sugar into the cell. Subsequent studies correct, the obvious need (if we are to proceed revealed that DOC also markedly inhibited the with an analysis of hormone action in fundamental entry of rubidium into the cell. The significance of terms) is to define the membrane and its com this latter observation became apparent when we ponents in biochemical terms. Our first difficulty is found that potassium is necessary for the growth that the cell membrane of the physiologist is a of Neurospora and that rubidium is the only ion functional concept which may or may not be identi which can substitute for potassium. cal to the cytologically visible plasma membrane Role of the cell membrane in regulating growth.â€” presumed to be lipoprotein. In order to explain the For our purposes, growth may be defined (at the marked specificity of the transfer of ions, sugars, simplest level) as the synthesis of the individual or amino acids into cells, biologists have postu components of a cell; we are fully aware that much lated the existence of "carrier" molecules which more than this is involved in cell growth. The con ferry substances across the membrane. These "car sequences of limited sugar entry into a cell are riers" are highly specific; they are able to differen manifold; not only is the primary energy source tiate between sodium and potassium (for example, necessary for biosynthetic reactions limited, but the potassium "carrier" complexes with rubidium glucose serves other functions as well. The cellular but not with other cations, and the sodium "car lipoids and steroids of the Neurospora cell are rier" complexes with lithium but not with other synthesized from sugar via "active" acetate; the cations). In the case of sugar transfer the specific characteristic cellulose-type polysaccharide pres ity is no less startling; the postulated sugar "car ent in the cell wall of this mold is derived from riers" in the red cell and in muscle differentiate, sugar; and the deoxyribose and ribose present in for example, between D- and L-arabinose (17). The DNA and RNA, respectively, are likewise derived chemical nature of these "carriers" is almost com from sugar. In view of the major roles postulated for DNA and RNA in current concepts of protein pletely obscure; if progress is to be made in under synthesis (which include the synthesis of the indi standing fundamental mechanisms involved in vidual enzymes which intervene in all biosynthetic membrane transfer systems, it is apparent that the

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1957 American Association for Cancer Research. HECHTERâ€”ReflectionsaboutHormone Action and Cancer Problem 515 chemical nature of these "carriers" must be eluci ate the rate of in vitro corticosteroid hormone syn dated. thesis. During this period, information has been For the past few years, operating from the accumulated concerning the effect of ACTH upon hypothesis that hormones may act to control cell corticosteroid biosynthesis in the cow adrenal at permeability relationships, we have tried to "get varying levels of organization : in the isolated per at" the cell membrane. For this purpose, we have fused gland, in tissue slices, and in cell-free ho- worked exclusively with systems wherein estab mogenates and fractions thereof (cf. 13). One of lished physiological effects of hormones can be re the outstanding features of these data is the fact produced in their essentials, in vitro, and have that, whereas the perfused gland is maximally studied the effect of insulin upon diaphragm muscle stimulated by concentrations of ACTH which are and of ACTH upon adrenocortical tissue. In the of the same order as those operative in vivo, slices case of diaphragm muscle, Resnick and I (21) of the same tissue require several hundred-fold studied some of the biochemical characteristics of more ACTH for maximal stimulation; cell-free galactose entry into cells. We found that two dis homogenates of cow adrenals (with or without for tinct processes are involved: one, operative in the tification with cofactors) are unresponsive to absence of insulin, has the temperature character added ACTH, although they do produce cor istics of a simple diffusion process and is relatively ticosteroid hormones at high rates. To test the pos insensitive to enzyme poisons like dinitrophenol, sibility that ACTH might not effectively diffuse iodoacetate, and cyanide, which interfere with cel into the tissue slice in contrast to the perfused lular energetics; the other, which is insulin-depend gland (where the circulatory system is operative), ent, is completely abolished by the above inhibitors Mr. John Eichhorn, a graduate student in my and exhibits a peculiar temperature dependence. group, undertook the problem of testing ACTH Between 0Â°and 10Â°C.insulin action on galactose action upon single adrenocortical cells. Eichhorn transfer is abolished; between 20Â°and 38Â°C.the was not able to obtain single cell preparations; by Qioof the insulin effect is about 1.0. One feature of passing adrenocortical brei through various silk our data seems most remarkable. Whereas in our in filters of varying mesh he was, however, able to vitro studies at 38Â°C.galactose enters the cells in prepare adrenocortical clumps which consisted of the absence of insulin sufficiently rapidly so that in about 1000 cells. Examination of these clumps by 1 hour the concentration of sugar is almost com standard histological technics showed that they pletely equilibrated between medium and cells, consisted of cells. When ACTH was incubated in vivo in the eviscerated-nephrectomized animal with these isolated cellular clumps, the clumps did (17) or in vitro in the isolated perfused hind limb not produce significant amounts of corticosteroids (14) galactose entry into cells occurs at a barely in the presence or absence of added ACTH; this detectable rate in the absence of insulin. The limit was most surprising, since unfortified homogenates ing barrier to the entry of galactose into cells, de produce corticosteroid in the absence of added Co- monstrable in the diaphragm, is a "leaky" barrier factors. For these cellular clumps to produce cor relative to those in more complex systems such as ticosteroid, it was found that cofactors such as the perfused hind limb or the eviscerated-nephrec ATP, DPN, and fumarate must be added to the tomized animal. We have incubated rat dia medium. With these additions, all of which are phragms with galactose-C14 using heparinized necessary, the clumps produce corticosteroid at blood, or plasma or serum from normal and evis rates comparable to those achieved in tissue slices. cerated rats instead of Ringer's solution as me The addition of these same cofactors to adreno dium, but the barrier to galactose entry was still cortical slices increases the rate of corticosteroid found to be "leaky." We tried other modifications production somewhat, but corticosteroid produc as well as the study of the rectus abdominis (as a tion proceeds at a significant rate in their absence. more typical skeletal muscle than diaphragm), but Since it is known that the enzymes involved in the galactose entry into the cells still occurred at sig biosynthesis of corticoids are associated with par- nificant rates in the absence of added insulin. As a ticulate intracellular structures of the cell (11), it result of our efforts, all that could be said was that is hard to escape the conclusion that, during the for unknown reasons, with our in vitro conditions, preparation of the adrenocortical cellular clumps, a barrier which should be "tight" becomes molecules like ATP, DPN, or TPN are destroyed "leaky." or escape from the cell; when these cells are pre From a completely different line of investiga sented with cofactors, these cofactors appear to tion, the question of "leaky" cell membranes re enter the cell. So once again, at an in vitro level, appeared to confront us. We have been interested we encounter a "leaky" barrier. for many years in the action of ACTH to acceler The clue as to what might be happening came

unexpectedly from a different line of investigation characteristic clumping of the chromatin material wherein we attempted to correlate cell structure at the nuclear membrane; after perfusion the with cell function in the case of the adrenal cortex, "deoxyribonucleoprotein" granules are finely dis where function may be quantitatively expressed in persed. The mitochondria before perfusion are terms of rates of corticosteroid production. I have rounded and show only a few cristae or tubules but previously mentioned that, in the case of the iso many vesicles within the paired membranes lated perfused cow adrenal, we have an in vitro bounding the mitochondrion; after perfusion, most system which responds to physiological amounts of the mitochondrial vesicles disappear, and the of ACTH. In such an experiment glands are ob usual cristae and mitochondrial shapes, familiar in tained at the slaughterhouse 15-30 minutes after well fixed tissues, reappear. For this discussion, the the death of the animal; the glands are transported changes which were most striking occurred in the to the laboratory in chilled saline, and this trip set of membranes separating the adrenal cell from takes an additional 60 minutes. We have found the circulating medium. Before perfusion, the en- empirically that, in order to obtain a good ACTH dothelium is discontinuous, the plasma membranes response, chilled glands must be "warmed up" by of the individual endothelial cells having become perfusing them with blood at 38Â°C.for an hour or fused and continuous. At the margin of the breaks so before we add the ACTH. If we add ACTH, of the endothelium, the underlying ground sub omitting this "warm-up" period of perfusion, the stance at various points seems to be "leached out," ACTH effect is minimal or absent. The "warm- and the plasma membrane of the adrenal cell is up" perfusion we have tended to regard as a re thus in direct contact with the circulating me covery period during which reconstitution of bio dium. The plasma membrane of the adrenal cell is chemical function occurs. However, current con intact and "smooth." Following perfusion, there is cepts of cell stability derived from electron micros the reappearance of an intact continuous endo copy of various tissues have clearly revealed that, thelium with overlapping between endothelial within minutes after death of the animal, each cells but without perforations; the ground sub cell structure shows characteristic degenerative stance is filled in, and the plasma membrane ex changes familiar to the electron microscopist as hibits surface ruffled shapes reminiscent of pseudo- "fixation artifacts." Since such changes could oc podia which extend into the ground substance. cur in the adrenal prior to perfusion with blood, The changes described as the result of perfusion the question arose whether the functional recon are rapidly reversed when the circulation is stitution observed during "warm-up" was associ stopped. ated with reconstitution of the cell structures re With regard to the effects of ACTH upon cell vealed by electron microscopy. We were fortunate structures, striking changes of the magnitude de to enlist the collaboration of Dr. John Luft, then scribed above were not observed. However, cer at Harvard, now at the University of Washington tain changes appeared to be evident following in Seattle, in tackling this problem. To date we ACTH; these require further study before defini have done the following experiment : contralateral tive conclusions can be drawn. Thus, following glands were biopsied at the slaughter house and ACTH treatment, the cytoplasmic RNA granules again before "warm-up" perfusion at the labora of the adrenal cell appear to be more dense or in tory; both glands were then perfused for 1 hour creased in number relative to the controls; associ with blood for "warm-up," and thereafter one ated with this, there is a tendency for the plasma gland was perfused with ACTH, while the other membrane of the ACTH-treated adrenal cell to was perfused with blood alone and served as con exhibit an increased frequency of pseudopod-like trol. Biopsies were taken after 1 hour of "warm- extensions into the outlying ground substance. up" and after various periods of perfusion, with These findings on the adrenal cortex may have a and without the addition of ACTH to the per more generalized significance. Various discussions fusion medium. The basic electron microscope of living systems have considered the necessity of a findings, together with the pictures, will soon be continuous expenditure of energy to maintain met- published (19). I wish here merely to summarize astable organized systems. This concept might ex the results obtained : Electron microscopy revealed plain some of the findings described. When the striking and consistent results in that before per energy supply is restricted, the mitochondria be fusion with blood, most cell structures were atypi come spherical, and their cristae expand into cal relative to currently accepted norms for tissue vesicles; this would appear to be an expression of fine structure; following "warm-up" perfusion, re the fact that the sphere is the minimal energy constitution of normal cell organization is ob state of elongated or flattened structures. The dis served. Thus, the nuclei before perfusion show a integration of the endothelium into a net under

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1957 American Association for Cancer Research. HECHTERâ€”ReflectionsaboutHormone Action and Cancer Problem 517 similar conditions would likewise appear to be a ground substance, since the human erythrocyte, reflection of its energy state. The clumping of nu which lacks a cuticle of ground substance, pos clear chromatin would appear to be less directly sesses these "carriers." Secondarily, it must be related to energy requirement; chromatin clump recognized that there are profound differences be ing by acid is a well established finding in cytology, tween tissue parenchymal cells with respect to the and it is almost certain that, during anoxia, lactic complexity and nature of the structural units in acid accumulates in cells. It is striking that the terposed between the parenchymal cells and blood plasma, nuclear, and mitochondrial membranes plasma. Thus, whereas most parenchymal cells are show no change (at the present order of resolution separated from the blood plasma by a complete, of the electron microscope) as a result of energy imperforate lining of the endothelial cell and a deprivation or accumulation of cellular metabo layer of intercellular ground substance, in liver and lites. It would appear that these membranes are spleen we have parenchymal cells which are di relatively more stable than the other structures rectly in contact with the blood plasma. In these described. tissues, capillaries with intercellular gaps are pres From all this, a new concept of the "cell mem ent, and the usual type of intercellular ground sub brane" seems to arise. With deprivation of energy, stance is absent. This does not mean that ground the endothelial cellular layer perforates so that the substance is completely absent, since some inter underlying ground substance may "leach out," ex cellular cementing substances which hold the cells posing the plasma membrane of the parenchymal together are clearly evident in these tissues. cell directly to the circulating medium. These It must be strongly emphasized that the con "holes" in the intercellular ground substance of cept advanced concerning the nature of the cell anoxic tissue bring to mind the "leaky" cell mem membrane as well as the view presented concern branes previously discussed. Taken together, these ing the possible mode of action of trophic hor diverse experiments give rise to some new possi mones is speculation. It represents a working hy bilities. The "naked" plasma membrane of paren pothesis whose only function is to suggest profit chymal cells, dissociated from the ground sub able areas of investigation. Operationally, a theory stance, may be an exceedingly "leaky" barrier to may be considered useful when it can be readily the entry of substances into cells. When intimately proved or disproved; from this point of view the surrounded by a cuticle of ground substance, the ideas put forward appear, to me, to be "good," complex of plasma membrane and ground sub since technics are at hand to test the basic ques stance may be the effective cell membrane which tions which arise. limits the cellular entry of extracellular substances, Some implications for the cancer problem.â€”Spec whether to ions, sugar, or like substances. In other ulation, once initiated, is difficult to control; hav words, the effective "cell membrane" of the mam ing started, at what point should you stop? This is malian parenchymal cell may be a composite of the a meeting concerned with cancer, and I should not plasma membrane of the microscopist plus the in like to close without mentioning some of the pos tercellular ground substance, whose integrity is sible implications of the speculative notions put dependent upon a continuous endothelium. If this forth. is so, the "cell membrane" chemically becomes Let us assume that around the tumor cell there "something more" than lipoprotein. Of the known is either a deficiency or a complete absence of constituents of ground substance, the complex ground substance and then consider some of the polysaccharides come to mind as the likely mate possible consequences. In such a cell the rate- rials which could interact with the lipoproteins of limiting event in sugar metabolism is no longer the the plasma membrane. The notion that complex sugar entry step but is now dependent upon some carbohydrates may be associated with lipoids is intracellular metabolic event. Given relatively currently the subject of intensive investigation in unlimited sugar entry and finite capacity to me the field of lipoids where studies of large molecular tabolize pyruvate via the "tricarboxylic acid" weight lipoid complexes isolated from various tis (TCA) cycle, the formation of pyruvate from glu sues reveal that complex carbohydrates are associ cose at rates exceeding the capacity of the TCA ated with phospholipoids or are present as gly- cycle would result in aerobic glycolysis. This vari colipoids. ant of a theme by Warburg (26) in actuality is If this picture of a composite cell membrane is much closer to the views of Weinhouse (27), since valid, then certain thoughts follow as a matter of no impairment of the respiratory apparatus need course. The "carriers" postulated in the membrane be involved. This "explanation" for the associa must be associated with the plasma membrane of tion of high rates of aerobic glycolysis with tumor the parenchymal cell and not with the associated cells is strikingly consistent with the extraordinÃ¤r-

ily high rate of aerobic glycolysis observed in t h tion among the various RNA templates for the Ehrlich ascites tumor cell: Recent studies by Co ri available intracellular components then becomes a and his co-workers (4) have shown that glucose possible mechanism for determining which specific pours into this cell; at 37Â°C.,equilibrium of sugar RNA templates are active and which are inactive. between cells and medium is achieved within 1 With limited pools of amino acids, energy, etc., minute. Only by lowering the temperature to there is competition among various templates so 20Â°C. could the actual rate of penetration be that some are favored, others restricted. Let us measured; at this temperature, the rate of sugar now remove the restrictive barrier of the cell mem entry was found to be rapid enough to sustain the brane so that the entry of sugar, essential amino observed rate of lactic acid formation at 37Â°C. acids, etc., into the cell is no longer limiting. The The electron microscope shows that the Ehrlich intracellular pool of energy, building blocks, etc., ascites tumor cell has a "naked" plasma mem increases and ceases to become limiting. Previously brane. inactive RNA templates now have an opportunity This view of the tumor cell provides a basis for to synthesize enzyme. This unleashing of the full understanding why the tumor cell grows so effec potential of the templates, however, is modified by tively relative to normal cells in the same tissue. associated effects such as increased aerobic lactic All the same extracellular requirements for growth acid production. To the degree that the synthesis are rather freely available for the tumor cell and of individual enzymes is pH sensitive, one may restricted for the normal cell. In the competition expect a decrease in the production of certain en between them, the tumor cell has the advantage. zymes, whereas the synthesis of others should in Differentiation is a key aspect of the tumor crease. This could result in the emergence of a com problem as I understand it, in that highly differen mon biochemical pattern in cells of diverse origin. tiated cells tend to converge to a more or less un- The notion that differentiation of cells is achieved differentiated pattern, both morphologically and by limiting substrate availability, essentially the biochemically, in terms of enzyme activities (9). basis of the above thesis, is not a new one. It has Let us, therefore, consider the possible role of the been shown in micro-organisms that, when growth cell membrane in cellular differentiation at a bio is restricted by limiting the nitrogen source of the chemical level. In any cell there are a certain num medium, various enzymes are differentially ef ber of specific templates (which may be RNA) ac fected, some are maintained, others are decreased, tive in the synthesis of specific enzymes and pro and still others disappear (5). The same notion re teins (22). We may postulate that the specificity of appears in tissue culture experiments. Dr. Pomerat each template is controlled by genetic information has previously discussed with this Committee (2) transmitted by specific units of DNA. In the cell, findings which demonstrate that, in tissue culture, all the templates for enzyme synthesis are not nec various cell types supplied with a medium rich in essarily operative at any particular time, and this growth factors grow rapidly and converge towards may be taken as the basis of differentiation. The a common nondifferentiated type; to maintain dif postulation of inactive templates arises from the ferentiated cells in tissue culture, one must se experiments on enzyme induction in micro-organ verely restrict the nutrient supply of the medium. isms; morphologically, there is a counterpart in The possible alteration of the cell membrane as regeneration experiments in newts and in tissue a basis for the property of invasiveness of the can culture experiments in which differentiated mam cer cell has previously been considered by Coman malian cells can be grown, by suitable means, to a (3). Here I wish to point out that a cell which loses primitive nondifferentiated form. The question its intercellular ground substance (as we have pos now arises: what is the mechanism operative in tulated) takes on the qualities of a macrophage in mammalian cells which restrains the potential of that it is no longer restricted in its movement by certain RNA-templates so that, while present, imprisonment within a cage of intercellular ground they are nevertheless inactive? While the absence substance. of suitable inducers, similar to those operative in There are other implications of this "cell-mem enzyme induction in micro-organisms, may be the brane" thesis which I could discussâ€”the effect of answer, the concepts previously put forth suggest cortisone upon the transplantation of tumors and an alternative explanation. Consider a large but the fact that some tumors are "hormone-independ finite number of templates all possessing much the ent" whereas others are modified by hormonal in same requirements for synthesis of their respective tervention. However, I think I have already made enzymes, and a limited supply of intracellular the point that the concept advanced here is a way amino acids and energy, the latter limitation being of integrating diverse lines of investigation in bio imposed by the normal cell membrane. Competi chemistry, cytology, genetics, and physiology into

a unitary pattern. I do not suggest that it is the Diaphragm. Proc. Soc. Exper. Biol. & Med., 82:60-62, way, nor that there are not other ways. The pic 1953. 11. HATANO,M.;SABA,N.; DORPMAN,R.I.; and HECHTER,0. ture put forth is primitive and, in many respects, Some Aspects of the Biogenesis of Adrenal Steroid Hor naÃ¯ve;inherent in these views are inadequacies mones. Recent Prog. Hormone Research, 12:79-118, 1956. which, if not already present, will be soon revealed. 12. HECHTER,O. Concerning Possible Mechanisms of Hor This will neither surprise nor disturb me, consider mone Action. Vitamins and Hormones, 13:293-346, 1955. 13. HECHTER,O.,and PINCUS,G. Genesis of the Adrenocortical ing the complexity of the problem involved. If Secretion. Physiol. Rev., 34:459-96, 1954. these speculations should serve no other function 14. HUYCKE,E. J., and KRUHOFFER,P.Effects of Insulin and than to act as an irritant, to stimulate others to Muscular Exercise upon the Uptake of Hexoses by Muscle destroy the concept put forth, progress will never Cells. Acta Physiol. Scandinav., 34:232-49, 1955. 15. KRAHL,M. E., and CORI,C. F. The Uptake of Glucose by theless have been achieved, because I think the the Isolated Diaphragm of Normal, Diabetic, and Ad- questions asked, if not the answers, are important renalectomized Rats. J. Biol. Chem., 170:607-18, 1947. at this stage. Only by integration of the various 16. KRAHL,M. E., and PARK,C. R. Uptake of Glucose by the ideas in the diverse disciplines now involved in Isolated Diaphragm of Normal and Hypophysectomized cancer research will the concept arise to explain the Rats. J. Biol. Chem., 174:939-46, 1948. origin and development of the tumor cell. 17. LEVINE,R., and GOLDSTEIN,M.S. On the Mechanism of Action of Insulin. Recent Prog. Hormone Research, 11:343-75, 1955. REFERENCES 18. Li, C. H.; KALMAN,C.; and EVANS,H. M. The Effect of 1. BROWN,D. H.; PARK, C. R.; DAUGHADAT,W.H.; and Adrenocorticotropic and Growth Hormones on Glucose COHNBLATH,M. The Influence of Preliminary Soaking Uptake and Glycogen Synthesis by the Isolated Diaphragm on Glucose Utilization by Diaphragm. J. Biol. Chem., 197: with and without Insulin. Arch. Biochem., 23:512-14,1949. 167-74, 1952. 19. LUFT,J., and HECHTER,O. An Electron-microscopic Cor 2. COHEN,P. P. Summary of Informal Discussions of First relation of Structure with Function in the Isolated Per Day. Symposium Sponsored by Am. Cancer Soc. Cancer fused Cow Adrenal. J. Biophys. & Biochem. Cytology Research, 16:668-74, 1956. (in press). 3. COMAN,D. R. Mechanisms Responsible for the Origin 20. PERLMUTTER,M.,and GREBP,R. O. The Uptake of Glu and Distribution of Blood-borne Tumor MÃ©tastases:A cose and the Synthesis of Glycogen by the Isolated Review. Cancer Research, 13:397-404, 1953. Diaphragm of Normal and Pituitectomized Rats. J. Biol. 4. CORI, C. F. Problems of Cellular Biochemistry, /n: D. E. Chem., 174:915-23,1948. GBEEN(ed.). Currents in Biochemical Research, pp. 198- 21. RESNICK,O.,and HECHTER,O.Studies on the Permeability 214. New York: Interscience Publishers, 1956. of Galactose in Muscle Cells of the Isolated Rat Dia phragm. J. Biol. Chem., 224:941-54, 1957. 5. DcLET, J. Enzyme Activity and Nitrogen Content of E. coli. Nature, 164:618-20, 1949. 22. SPIEOELMAN,S.,and CAMPBELL,A.M. The Significance of Induced Enzyme Formation. In: D. E. GREEN(ed.), 6. DEMIS, D. J., and ROTHBTEIN,A.Absence of an Insulin Currents in Biochemical Research, pp. 115-61. New York: Effect on Sugar Uptake by Rat Diaphragm under Anaero bic Conditions. Am. J. Physiol., 178:82-84, 1954. Interscience Publications, 1956. 23. STADIE,W. C. The Relation of Insulin to Phosphate 7. <:KMMti.i..C. L., and HAMMAN,L.,JK. The Effect of In Metabolism. Yale J. Biol. & Med., 16:539-59, 1944. sulin on Glycogen Deposition and on Glucose Utilization 24. VILLEE,C. A., and HASTINGS,A. B. The Metabolism of by Isolated Muscles. Bull. Johns Hopkins Hosp., 68:50-57, CK-labeled Glucose by the Rat Diaphragm in Vitro. J. 1941. Biol. Chem., 179:673-87,1949. 8. GREEN, D. E. Enzymes and Trace Substances. Adv. Enzymol., 1:177-98, 1941. 25. WALAAS,E., and WALAAS,O. Effect of Insulin on Rat Diaphragm under Anaerobic Conditions. J. Biol. Chem., 9. GREENSTEIN,J. Some Biochemical Characteristics of 196:367-73, 1952. Morphologically Separable Cancers. Symposium Spon 26. WARBURG,O. On the Origin of Cancer Cells. Science, sored by Am. Cancer Soc. Cancer Research, 16:641-53, 123:309-14, 1956. 1956. 27. WEINHOUSE,S. Discussion of Dr. Greenstein's Paper. 10. HAFT,D.; MIRSKT,I. A.; and PEHISÃœTTI,G.Influence of Symposium Sponsored by Am. Cancer Soc. Cancer Insulin on Uptake of Monosaccharides by the Isolated Rat Research, 16:654-57, 1956.

O. Hechter

Cancer Res 1957;17:512-519.

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