Chapter 7 Biochemical Control of Erythroid Cell Development

CHAPTER 7

BIOCHEMICAL CONTROL

OF ERYTHROID CELL DEVELOPMENT Eugene Goldwasser ARGONNE CANCER RESEARCH HOSPITAL A N D DEPARTMENT O F BIOCHEMISTRY, UNIVERSITY OF CHICAGO, CHICAGO, ILLINOIS

I. Introduction . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . , . 11. Systeni under Study . . . . . . . . . . . . . . . . . . . . . . , . . A. Hornional Control of Erythropoiesis . . . . . . . B. The Nature of Erythropoietin . . . . . . . . . . . . . . . . . . 111. The Role of Other Hormones in Control of Erythropoiesis A. Pituitary Hormones . . . . . . . , . . . . . . . . . . . . . . . . . . B. Steroid Horniones . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Thyroid Hormones . .. . . . . . . . . . . . . . . . . . . . . .. .. IV. The Role of Nonhormonal Substances in Erythropoiesis A. Batyl Alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . B. Cmobalt Salts . . . . . . . . . . . . . ...... . . . . . . . . . . . . .. V. Erythropoietin as Inducer of Red Cell Differentiation . . A. Regulation of Normal Erythropoiesis . . . . . . . . . . . . B. The Nature of the Erythropoietin Target Cell . . . . C. The Mode of Action of Erythropoietin . . . . . . . . . . VI. Models of Erythroid Differentiation . . . . . . . . . . . . . . . . VII. Summary . .. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .. References . . . . . . . . . . . . . _.......... ......

173 174 175 177 182 182 182 183 184 184 184 185 185 187 188 200 206 206

1. Introduction

The task of the biochemist studying the process of differentiation is to determine the detailed molecuIar mechanisms that account for the appearance and disappearance of specific groups of synthetic capabilities 173

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and properties in differentiated cells. In general, the material of choice for this type of investigation has been the developing embryo, or cellular systems derived from it. Another approach to this problem is through the study of stem cell differentiation in mature animals, of which the development of hemopoietic cells is an outstanding example. The processes of formation of recognizably distinct circulating blood cells from more primitive precursor cells are, in a formal sense, the same as those in embryonic development. There is, in addition, one significant difference: the selfrenewal property of the stem cell. For this reason, any complete description of the mechanism of stem cell differentiation must eventually explain both differentiation and the steady-state maintenance of the undifferentiated stem cell population. It is not known whether all types of circulating blood cells derive from a single type or from separate types of precursor cells that are already differentiated, but not recognizably so. Still another difficulty is our almost complete ignorance of the morphological or biochemical properties of the precursor cells. Where the term “stem cell” is used in this article, it can be understood to mean only the ultimate primitive hemopoietic precursor cell or cells. I propose to discuss what is now known about the biochemistry of the control of erythrocyte formation. A closely related subject, the synthesis of hemoglobin in erythroid cells, is the topic of Marks’ chapter, in this volume. Although information on the biochemical properties of the red cell is immense, considerably less is known about the molecular mechanisms for the formation of erythrocytes from precursors. This is the problem with which our work is concerned. 11. System under Study

The numerous biochemical events that accompany the change from stem cells to erythrocytes are understood only in broad outline (Thorell, 1947; Grasso et al., 1963); the morphological changes during this process are well documented. Induction of erythroid differentiation in a stem cell results in its descendants’ eventually acquiring the ability to synthesize and store hemoglobin as their predominant product. During the same period of time the cells lose functions such as the ability to divide, to synthesize DNA and RNA, to maintain mitochondria and their complement of enzymes, and to synthesize proteins. The end product of the process is the erythrocyte with its long life span, designed specifically for oxygen transport.

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There are a number of reasons for choosing red blood cell formation as a model for the biochemical investigation of differentiation. Some of

them may be summarized as follows: a. There is a large body of data on the composition of erythrocytes, including various enzymes. b. Information about the physical and chemical properties of hemoglobin, the major component of the red cell, is abundant, as is knowledge of the biogenesis of both heme and globin. c. The genetic control of globin structure is fairly well understood. d. There is some understanding of the mechanisms involved in the regulation of erythropoiesis in the adult animal. This last point will be the focus of this article, within the larger context of how erythrocytes develop from nonerythrocytic precursors. CONTROL OF ERYTHROPOIESIS A. HORMONAL The basic assumption in the following account is that the hormone, erythropoietin, is the primary inducer of erythroid differentiation. Before examining the evidence for this assumption it might be helpful to outline the physiology and biochemistry of erythropoietin. The original description of a plasma-borne factor that is formed in response to anemic or anoxic stress and that causes increased red cell formation dates back some 60 years (Carnot and Deflandre, 1906). After a long period of dormancy the concept is now widely accepted and ampIy documented. Background information on erythropoietin may be found in a number of reviews and symposia (Grant and Root, 1952; Gordon, 1959; Jacobson et al., 1960; Jacobson and Doyle, 1962). Although the role of erythropoietin in stimulating red cell formation as a response to anemic or anoxic stress is clear, its possible regulatory action in the normal, unstressed animals is still not completely established. The assumption that erythropoietin does regulate normal erythropoiesis is supported by the following considerations: The regulatory mechanism proposed by Fried et al. (1957) states that, in situations where the oxygen requirement of the animal is greater than its capacity to provide oxyhemoglobin to the responsive tissue, erythropoietin production is increased. Conversely, when the ability to carry oxygen in the circulating blood exceeds the need of the responsive tissue, erythropoietin production decreases. In polycythemic, fasted, and hyperoxic animals the rate of erythropoiesis is appreciably lower than in the normal animal, in which, in turn, it is lower than in anemic or hypoxic

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animals. Because of the great difficulty in the quantitative estimation of small amounts of erythropoietin, determinations of erythropoietin titers lower than in normal plasma under conditions of suppressed erythropoiesis have not yet been reported. Qualitative information shows less erythropoietic activity in plasma from polycythemic rats than in plasma from normal rats ( Reichlin and Harrington, 1960). There is also evidence of an indirect nature which suggests that erythropoiesis in the normal animal is under the control of erythropoietin. In mice made polycythemic by injection of red cells, the rate of erythropoiesis falls to an extremely low level and there is no morphological evidence of red cell formation (Jacobson et al., 1957) or appreciable incorporation of labeled iron into circulating cells (DeGowin et al., 1962). Such plethoric mice, whose erythropoietin production is assumed to be negligible owing to absence of erythropoietin, can respond to small amounts of exogenously administered erythropoietin by increased formation of hemoglobin and reticulocytes. Three lines of evidence indicate that erythropoietin is present in normal plasma: ( 1 ) Polycythemic mice injected with normal mouse plasma show a definite increase in circulating reticulocytes compared to controls injected with saline solution (Jacobson et al., 1957). In addition, polycythemic rats show increased iron uptake into circulating red cells when given normal plasma in place of saline solution ( Reichlin and Harrington, 1960). ( 2 ) Rabbits immunized to human erythropoietin develop an anemia, which suggests that their own erythropoietin is being neutralized by the antibody (Garcia and Schooley, 1963). Similarly, mice given rabbit antierythropoietin develop an anemia (Schooley and Garcia, 1962). The conclusion drawn from these experiments with the antierythropoietin is that anemia develops probably because the antibody neutralizes the endogenous erythropoietin of the recipient animals. (3) Normal plasma samples from nonanemic rabbits were concentrated by a heat denaturation method based on that used by Borsook et d.( 1954). The data from three experiments (Goldwasser, Kung, and Shin, unpublished) given in Table I show that normal rabbit serum contains approximately 0.02 units of erythropoietin per milliliter.* This figure is in fair agreement with the value (0.04-0.14 units/ml ) calculated from indirect immunological measurements by

* The definition of a unit of erythropoietin used here is that of Goldwasser and White (1959) and is equivalent, in biological effect, to 5 bmoles of CoCI2. Assay methods, in uiuo, are usually based on measurement of incorporation of labeled iron into circulating red cells of fasted or polycythemic animals.

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Schooley and Garcia ( 1965). These conclusions must be considered tentative since we do not have sufficient data to rule out all influences on erythropoiesis, other than erythropoietin. TABLE I ERYTHROPOIETIN CONTENT OF NORMAL RABBITSERU& Experiment A B C

D

Preparation Normal Anemic Anemic Normal Anemic

rabbit serum concentrate rat serum rat serum concentrate rabbit serum rat serum Concentrate

Volume of original, ml

Total units found

175 10 10 175 10

4.5 12.0 10.2

12.6

,a The data from experiments B and C show 85% recovery in the concentration process. Correction of the amount found in A by 0.85 yields a total of 5.3 units/ 175 ml or 0.03 units/ml. Correction of experiment D by 0.85 yields a total of 14.8 units, of which 12 were added as anemic serum so that the remainder, 2.8 units, was due to the 175 ml of normal serum or 0.016 units/mI. The mean of these values is 0.02 units/ml of normal rabbit serum.

Recently Finne (1965) has reported that normal human urine contained a measurable amount of erythropoietin. Calculations from his data indicate a daily excretion of about 3 units. Although the role of erythropoietin in the regulation of normal erythropoiesis cannot be yet considered firmly established, the suggestive evidence is good enough to be accepted as a working hypothesis.

B. THE NATUREOF ERYTHHOPOIETIN Studies of the chemical nature of erythropoietin have been hampered by two major difficulties: inadequate methods of assay and inadequate sources of material for purification of the hormone. A discussion of problems of assay would be beyond the scope of this review. One should indicate, however, that the methods of assay in vivo, including that used in this laboratory (Fried et al., 1957), require too much material or too much time or are inaccurate. 1 . Sources of Erythropoietin Although erythropoietin appears to be formed in the kidney, there seems to be too little present in normal kidney tissue or in the kidneys of stimulated animals to make it a practical source of the hormone. The data of Contrera et al. (1965a) show that there is about 0.4 units/gm of kidney from anemic rats and none in kidneys from norma1 animals; the

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data of Kurnick, Dukes, and Goldwasser (unpublished) show that normal rat kidney contains approximately 0.25 units/gm. Kuratowska ( 1965) fractionated kidneys from normal, anemic, and cobalt-treated rabbits and found erythropoietic activity predominantly in the large particle (nuclear) fraction that was sedimented at 600 g. The activity was slight, unless the preparation was incubated with an aglobulin fraction from normal plasma. Somewhat similar experiments were reported by Contrera et al. (1965b) in which the activity was found in the “light mitochondria” fraction of kidney. This activity increased upon incubation with normal plasma. In contrast to the minor amounts of the hormone detectable in its apparent tissue of origin, the plasma of sufficiently anemic laboratory animals contains appreciable quantities. Under the proper conditions plasma erythropoietin titers may reach 10 or more units/ml. Some years ago, we developed methods for the large-scale preparation of partially purified erythropoietin from the plasma of sheep made anemic by treatment with phenylhydrazine (White et al., 1960). We obtained preparations with potencies in the range 5-20 units/mg of protein. That became the widely used standard A, which was the first standardized erythropoietin in general use since the discovery of the hormone. These preparations have been further purified ( Goldwasser et al., 1962a,b), and more recently we obtained fractions with potencies of about 3000 units/mg of protein, albeit in quite low yield (Goldwasser and Kung, unpublished). Since the starting plasma had a potency of 0.007 units/mg of protein, this represents a purification factor of approximately 400,000. Not enough of this high-potency erythropoietin has yet been accumulated for even the simplest tests for homogeneity. However, such a test might well be premature since on occasion we have achieved a still higher potency. The minute quantities of very highly purified erythropoietin that are at present obtainable leave the investigator almost no recourse but to depend on the potency, or specific activity, as an indication of heterogeneity. That is to say, only a preparation that cannot be further purified to a higher potency should demand any effort to determine whether it is homogeneous and, if so, what the chemical and physical properties are. Assuming that the pure hormone has a potency of 3000 units/mg of protein, and if we assume a 1% over-all yield from the original plasma, in order to get 10 mg (which would be enough for the partial physical and chemical characterization of the hormone), we would require plasma

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containing 3 million units. With present methods, this would correspond to approximately the yield from 300-400 liters of high-activity plasma. Erythropoietin can also be found in substantial quantities in urine of some severely anemic patients. However, it should be noted that methods of fractionation that have worked for plasma have not yielded equally good results when applied to urine concentrates. Experience in this laboratory has confirmed that of Graham et al. (1963), who found that urinary erythropoietin fractions appear to be less stable than plasma fractions with similar potencies. 2. Properties of Erythropoietin

The chemical properties of the hormone from the two sources appear to be different, but these differences may be more artifactual than intrinsic. The activity found in urine is almost certainly derived from the plasma, and since there is evidence that the activity in plasma does exist in more than one form (Goldwasser et al., 1962b), possibly as complexes with normally occurring proteins, the differences observed may reflect differences in the nature of the materials that can form a complex with erythropoietin. In the absence of pure erythropoietin for the determination of its physical and chemical properties, some information can be gleaned from the study of specific reagents upon the biological activity of the hormone. Erythropoietin activity in plasma is resistant for short times to temperatures as high as 100°C (Borsook et al., 1954). The activity is also stable at 0°C to 0.5 N perchloric acid for short periods of time (Goldwasser et al., 1957). Both of these observations provide a method for the preparation of small quantities of plasma erythropoietin concentrates, since they result in the removal of large amounts of the inactive proteins. Lowy and Borsook (1962) have shown that partially purified erythropoietin from rabbit plasma is inactivated by reaction with fluorescein isothiocyanate, which substitutes on phenolic hydroxyl groups ( tyrosine residues) and on free amino groups (lysine and/or the N-terminal residue of a polypeptide chain). They also showed that esterification with anhydrous formic acid (reaction with serine or threonine hydroxyl groups) caused no loss of activity. Substitutions with iodine, acetic anhydride, formaldehyde, and methanol, however, inactivated rabbit plasma erythropoietin (Lowy et al., 1960). W e have found that p-chloromercuribenzoate did not inactivate erythropoietin ( Goldwasser and Kung, unpublished observations ) . Although the presence of serine, threo-

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nine, or cysteine in erythropoietin is not ruled out, if they are present they either are not required for biological activity or are buried in the tertiary structure, Trypsin treatment causes loss of activity, indicating that erythropoietin is composed of a protein or polypeptide-containing lysine and/or arginine (Slaunwhite et al., 1957; Lowy et al., 1958). Erythropoietin also appears to contain a carbohydrate portion containing sialic acid. Rambach et al. (1958) showed that mild acid hydrolysis, under conditions known to remove sialic acid from glycoproteins, inactivated erythropoietin. More specific data from the experiments of Lowy et al. (1960) showed that sialidase inactivated both urinary and plasma erythropoietin preparations. Recent studies of inactivation by sialidase treatment ( Dukes and Goldwasser, unpublished) have raised new questions concerning the role of sialic acid in erythropoietin activity. The enzyme inactivates erythropoietin only when the hormone is assayed in vivo. If the assay is done in vitro by the marrow cell culture method, there is no loss of activity when either hemoglobin synthesis (Krantz et al., 1963) or stroma synthesis (Dukes et al., 1964) is measured. This is true whether the sialic acid is removed enzymatically or by acid hydrolysis. When tryptic hydrolysis is used, both in vitro methods show the loss of activity. These results might be interpreted as indicating that the in vitro assay methods respond not to erythropoietin but to some adventitious impurity. This seems very unlikely, since the in vitro responses agree quantitatively with the in vivo assay for preparations that differ by a factor of several hundred thousand in potency. It would seem highly improbable that the fractionation would concentrate two entirely different activities in parallel. There are two possible explanations for the divergence of results with sialidase-treated erythropoietin. Erythropoietin may be held in a complex form in which the sialic acid is a part of a hypothetical binding component. Removal of sialic acid dissociates the complex (or shifts the equilibrium toward dissociation). The resulting “free” erythropoietin is excreted or inactivated in the assay animal, while in vitro it remains in the culture medium and can act on its target cells. To test this we attempted to “revive” the activity of sialidase-treated erythropoietin by addition of possible binding components. None of the large number of plasma fractions tested had a “reviving effect. Alternatively, sialic acid may be an intrinsic part of the erythropoietin molecule and is required in the animal only for transport or for protection

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against excretion or inactivation; it is not needed for interaction with the cells or for its effect upon them. In this case, there would be no possibility of “reviving” the in vivo activity short of replacing the sialic acid on the hormone molecule. There is some indirect support for this view ( Schooley and Garcia, 1965). In the absence of the pure hormone, the only feasible methods for determining molecular weight are those that utilize measurements of biological activity. Inactivation by ionizing radiation has been used (Rosse et al., 1963) with somewhat conflicting results. With X-ray inactivation of crude human erythropoietin they found an apparent molecular weight of 66,000, but with high energy electron inactivation it was 27,000. They assumed that the difference was due to an asymmetric target with an axial ratio of 10, and that the molecular weight was 27,000. In work that is as yet unpublished, Hodgson (personal communication) used 6oCo y-irradiation of rabbit plasma erythropoietin and found a target molecular weight of about 68,000. In this laboratory (Goldwasser and Kung, unpublished) we determined the sedimentation coefficient of highly purified sheep plasma erythropoietin by the ultracentrifugal separation-cell method ( Yphantis and Waugh, 1956) and by sucrose-gradient centrifugation (Martin and Ames, 1961). Values obtained by the two methods agree fairly well at approximately 5 S. If we use the molecular weight of 27,000 we can calculate, from it and from the sedimentation coefficient, a frictional ratio of 0.6, which is a physical impossibility. The frictional ratio of an ellipsoid with an axial ratio of 10 would be 1.54 (Scheraga, 1961). A similar calculation with the molecular weight of 66,000 yields a frictional ratio of 1.01, indicating a nearly spherical molecule. All of these data are much too inaccurate to yield more than a rough estimate of the actual molecular size of the hormone, but at present it would appear to be of the order of 60,000-70,000. The fact that there is little or no species specificity in erythropoietin action suggests that the hormone is a poor antigen. When we attempted to prepare an antiserum to sheep plasma erythropoietin (Goldwasser et al., 1962a), the antibody activity we found was directed against some of the impurities in the antigen mixture, not against the hormone. On the other hand, Schooley and Garcia (1962), using crude human urinary erythropoietin as the antigen, did succeed in producing a rabbit antiserum capable of neutralizing the biological activity. Antiserum to human erythropoietin, they found, can neutralize rat, rabbit, mouse, sheep, and human erythropoietin (Garcia and Schooley, 1963). Although it

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might seem desirable to use the specific antibody as an aid in purifying the hormone, in developing an immunoassay for erythropoietin, and in localizing the site of erythropoietin production, the fact that the original immunizing antigen was highly impure would severely limit its use for these purposes. I doubt whether the use of absorption techniques to purify the antibody would overcome this disadvantage. The implicit assumption that the only new antigenic component arising because 01 the anemia is erythropoietin has never been put to a test. The antibody however, has been used to great advantage in the study of erythropoietin action (Schooley and Garcia, 1962, 1965; Schooley, 1965) and also to show that angiotensin (Fisher and Crook, 1962) and ceruloplasmin (Hatta et ul., 1962, 1963) are not identical with erythropoietin. Ill. The Role of Other Hormones in Control of Erythropoiesis

A. PITUITARY HORMONES There is now fairly wide agreement that there is no specific pituitary hormone that regulates erythropoiesis. The actions of such hormones as corticotropin in increasing the red cell mass (Garcia et al., 1951) are understood to be indirect, probably via the erythropoietin mechanism. The apparent anemia resulting from hypophysectomy is considered to reflect the lowered metabolic activities of an animal without a pituitary, not the absence of a pituitary erythropoietic factor. The recently reported effect of prolactin in increasing the rate of erythropoiesis in nonlactating mice (Jepson and Lowenstein, 1965) may also be an indirect effect, although experimental tests of this interpretation have not yet been published.

B. STEROIDHORMONES It has long been known (Crafts, 1941; Volmer and Gordon, 1941) that testosterone causes an increase in red cells. Recent evidence indicates that this effect is probably mediated through the erythropoietin mechanism. Fairly large amounts of testosterone cause increased erythropoiesis in plethoric mice (Fried et ul., 1964; Naets and Wittek, 1964), and a similar effect was found with the synthetic androgen, nandrolone phenylpropionate (Gurney and Fried, 1965a). Although it first appeared that the androgen acted to potentiate the effect of erythropoietin on its target cells (Naets and Wittek, 1964; Gurney and Fried, 1965a), more recent experiments indicate that androgen treatment increases a plasma eryth-

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ropoietic activity that may be erythropoietin (Fried and Gurney, 1965a,b). Janda et al. (1965) have shown that cobaltous ions (see below) and testosterone act synergistically when the androgen is given to mice 2-3 days before the cobalt and suggest that, while both may cause an increase in circulating erythropoietin, this increase may arise by way of different mechanisms. Since testosterone is known to increase kidney growth (Selye, 1939), possibly by increasing the mitotic rate of some kidney cells, it may well be that the synergism is the result of more potential erythropoietin-producing cells that have been stimulated to synthesize erythropoietin by cobalt. If this were the mechanism, one would expect to see the synergism abolished by a mitotic inhibitor such as colchicine. In contrast to androgens, estrogens cause a decrease in erythropoiesis. As little as 2 pg of estradiol given to a male rat can significantly lower the rate of hemoglobin synthesis (Dukes and Goldwasser, 1961). The data suggested that estradiol can act on the target tissue of erythropoietin to interfere with erythroid digerentiation; however, the possibility still exists that estrogens inhibit the formation of erythropoietin in a manner reciprocal to the effect of androgens. Studies of the effect of adrenal cortical steroids (Fruhman and Gordon, 1956) have shown that hydrocortisone increases erythropoiesis. Fisher and Crook (1962) found that small amounts of corticosterone, ll-dehydrocorticosterone, and hydrocortisone given to hypophysectomized rats stimulated the incorporation of radioiron into circulating red cells, whereas aldosterone and large amounts of hydrocortisone had no effect. It is not yet certain whether those cortical steroids that stimulate erythropoiesis do so by directly increasing the erythropoietin level, since they also increase the metabolic rate of hypophysectomized animals. C. THYROID HORMONES Information on how thyroxin and triiodothyronine increase erythropoiesis (Fried et al., 1957; Meineke and Crafts, 1959; Fisher and Crook, 1962) is also incomplete. There are indications that thyroid hormones may act indirectly by increasing the metabolic rate, as does dinitrophenol (Fried et al., 1957): There has been, as yet, no direct demonstration of increased circulating erythropoietin in the treated animals. It would be of interest to determine whether antibody against erythropoietin would abolish the effects of testosterone, cortisone, and thy-

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roxin, in which case the direct participation of erythropoietin in the action of these hormones would be on a firmer basis. IV. The Role of Nonhormonal Substances in Erythropoiesis

A. BATYLALCOHOL There have been reports of the effect of nonhormone lipids, particularly batyl alcohoI, on erythropoiesis (reviewed by Linman and Bethell, 1960; Linman and Pierre, 1962) that suggested the following conclusions: There are two erythropoietin factors, a thermolabile protein that influences hemoglobin synthesis, and a thermostable lipid that influences the rate of erythroid cell division, The lipid factor administered to animals by itself caused an increased number of circulating erythrocytes, smaller than normal and with a shorter life span, but no increase in the amount of circulating hemoglobin. These contentions, however, have not been confirmed, and there is a brief published account of a failure to reproduce the results (Evenstein et al., 1958). Furthermore, lipid-free erythropoietin concentrates can cause an increase in the total amount of circulating hemoglobin as well as increases in the number of reticulocytes and red cells (Gurney et al., 1961). In addition, Gurney (personal communication) failed to find a reticulocytosis in polycythemic mice treated with batyl alcohol.

B. COBALTSALTS It has been known (Waltner and Waltner, 1929) that cobalt salts can cause increased red cell formation, but the detailed mechanism of this action is not completely established. Cobaltous ions are thought to induce the formation of erythropoietin (Goldwasser et d.,1957, 1958); however, this point will not be definitely settled until the hormone is purified and shown to be the same whether from plasma of anemic or of cobalt-treated animals. Evidence concerning the reactivity of active plasma from cobalt-treated animals with erythropoietin antibody would be helpful in this context. Assuming that erythropoietin production is stimulated by cobalt, another interesting problem arises. What is the mechanism by which cobaltous ions act on the site of erythropoietin production? Cobalt has been shown to inhibit glycine incorporation into heme (Laforet and Thomas, 1956) and to dissociate heme and globin synthesis in the marrow by inhibiting the former (Morel1 et al., 1958). It is possible that the effect of cobalt on erythropoiesis is due to a specifically localized anoxia that

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causes increased erythropoietin formation. The possible role of cobalt as a constituent of erythropoietin has not been entirely excluded, but we failed to find any label in active fractions of plasma from animals injected with 60C0C12.Other data show that vitamin B12 does not increase erythropoiesis in fasted rats, making it unlikely that cobalt salts act via this compound. V. Erythropoietin as Inducer of Red Cell Differentiation

A. REGULATIONOF NORMAL ERYTHROPOIESIS The suggestion that erythropoietin is directly concerned with initiation of normal red cell differentiation was first made by Alpen and Cranmore (1959) and by Erslev (1959). Data in support of this suggestion have come from several other laboratories. Among the more compelling arguments for assigning to erythropoietin the role of primary inducer of erythroid differentiation are those derived from the experiments of Jacobson et al. ( 1957, 1961). As noted earlier, artificially polycythemic mice can be kept devoid of any recognizable erythroid cells in their hemopoietic tissues, and of circulating reticulocytes, for as long as they are kept plethoric. Regardless of the duration of the plethoric state, the maximum number of circulating reticulocytes, after erythropoietin administration, appears at the same time ( 3 days). These observations suggest that in the absence of red cell formation, after all previously induced cells have progressed to the erythrocyte stage, the stem cells can be kept in a quiescent state. In this state they remain capable of responding in a normal fashion to the presence of erythropoietin by eventually giving rise to erythrocytes. It is true that the hemopoietic tissues in the plethorasuppressed mouse contain a number of different types of cells that are not identifiably erythroid and that there are no morphological criteria for stem cells, so that the “quiescent” cells may represent an already differentiated population, capable of becoming erythroid cells only when erythropoietin is present. Bruce and McCulloch (1964), in fact, have suggested that this is the caye after studying the time course of endogenous erythropoietin action on the number of colony-forming cells in the spleen and marrow of mice. This problem will be discussed in a later section; for the present it will suffice to stress our ignorance regarding the nature of the target cell for erythropoietin. In an operational sense there is little difference between an unrecognizable “erythroid cell becoming a recognizable erythroid cell under the influence of erythropoietin, and an

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unrecognizable “stem” cell becoming a recognizable erythroid cell under the same influence. The question of the nature of the stem cell is, of course, an important one, and will have to await the application of new techniques before it can be answered. The presence of erythropoietin in animals making red cells under normal conditions also provides supporting evidence for its roIe as a primary inducer. Other information provides negative evidence. Erythropoietin does not appear to act upon nucleated erythroid cells or upon reticulocytes ( Erslev, 1964). Data from this laboratory (Goldwasser et al., unpublished), seen in Table 11, show clearly that, while rat reticulocytes can take up labeled TABLE I1 LACKOF EFFECTOF ERYTHROPOIETIN ON RETICIJLCCYTES~ Control Incorporation of glycine-2-14C into heme Incorporation of glucosamine-lJ4C into cells

Erythropoietinb

14,300 (+- 3400) 15,400 (+- 1200) 1890 ( & 150) 2200 (+- 215)

a Cell suspension consisted of 2.2 x 109 total cells per ml, from phenylhydrazinetreated rats, containing 7.7 x 108 reticulocytes per ml. Medium was 50% newborn calf serum, 50% NCTC 109. Cells were preincubated for 2.5 hours without erythropoietin or label, washed, resuspended, then incubated 21 hours with the hormone and labeled precursor. Numbers in parentheses are standard deviations of the mean. b Measured in 0.2 units.

glucosamine and glycine readily, erythropoietin has no effect on these processes. In marrow and spleen cells, however, the same processes are markedly affected by erythropoietin, as will be discussed in a later section. Other studies in vitro also support the concept of erythropoietin as primary inducer. Marrow cells in culture lose their capacity for heme synthesis in the absence of added erythropoietin (Krantz et al., 1963); in its presence this synthetic capacity is maintained for some time. This, too, will be discussed subsequently. Whether red cells newly formed under the influence of erythropoietin are normal erythrocytes is still not entirely settled. There have been reports (Stohlman, 1961a,b; Brecher and Stohlman, 1961; Borsook et al., 1962; Borsook, 1964) that red cells and reticulocytes formed in response to severe anemia (large amounts of erythropoietin) have a greater diameter and are shorter-lived than normal. This suggested that erythrocyte development is more rapid under conditions of high erythropoietin concentration and, possibly because one or more mitoses are by-passed, the

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resulting cells have not had time to attain their final (smaller) size. Changes in the red cell membrane also accompany anemic stress (Hillman and Giblett, 1965). Whether this is also true with physiological concentrations of erythropoietin has not been determined. If so, then obviously, erythropoietin cannot be the agent for physiological regulation: Conversely, if it does act in the normal process, the resulting red cells must be of normal size and life span. Some recent data (Ito and Reissmann, 1965) show that protein-depleted rats can be maintained in normal steady state erythropoiesis with 1.8 units of erythropoietin per day and that the erythrocytes have a normal life span. For the purposes of this essay, I will continue to assume that the hormone does regulate normal red cell formation and will avoid the complications introduced by nonphysiological amounts of the hormone. B. THE NATUREOF

THE

ERYTHROPOLETIN TARGET CELL

As was indicated earlier, an important problem in the study of erythropoietin-induced differentiation centers about the target cell for the hormone. It is outside the scope of this article to discuss the enormous (and often contradictory) body of work on the nature and behavior of stem cells, but since our postulate has been that erythropoietin acts upon stem cells, whatever they may be, some mention must be made of the problem. There have been only a few experimental approaches to determine what type of cell is acted on by erythropoietin. Takaku et al. (1964) found that some degree of cell specificity was involved in the erythropoietin stimulation of stroma synthesis (see below), Mixed populations of rat marrow cells and polycythemic mouse spleen cells respond to the hormone, but peritoneal granulocytes do not. Marrow cells separated into five crude fractions in a polyvinyl pyrollidone gradient showed the greatest erythropoietin effect in the fraction that contained the highest proportion of immature cell types. This method of cell fractionation has too little resolving power to accomplish the task of separating out the target cells, but the small degree of success obtained with it, and the development of newer techniques such as those involving continuous gradient centrifugation (Anderson, 1962), stable flow electrophoresis and sedimentation (Mel, 1964), and separation methods based upon volume difference ( Fulwyler, 1965), make it clear that the problem is potentially soluble. The indirect experimental approach, such as that used by Bruce and

188

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McCulloch (1964), based on the study of competing pressures for differentiation and self-renewal, has suggested that the cells acted upon by endogenous erythropoietin are already differentiated and not kinetically identical with what are generally assumed to be stem cells (i.e., cells capable of both differentiation and self-renewal). On the other hand, Liron and Feldman (1965) found that when polycythemic mice were used for the assay of colony-forming cells (stem cells) by the method of Till and McCulloch (1961) there was a reduction in the number of erythroid colonies and a concomitant increase in the number of granuloid colonies. When erythropoietin (as anemic plasma) was given, the suppression of erythroid colony formation was reversed (Bleiberg et al., 1965). These findings suggested that the same cells gave rise to one or the other type of colony depending on the stimuli that were present in the animal. Unfortunately, the change in the number of colonies of the granuloid type was too small to allow an unequivocal interpretation. C. THE MODEOF ACTION

OF

ERYTHROPOIETLN

1. Biochemical Actions in vivo Several studies of biochemical effects of erythropoietin in vivo have been reported. In general, these have all demonstrated an increase, in the hematopoietic tissues, of one or more indicators of cellular activity such as DNA synthesis (Rambach et al., 1957; Linkenheimer et al., 1959; Kurtides et al., 1963), RNA synthesis (Pieber-Perretta et al., 1965), RNA polymerase, DNA polymerase, thymidylate kinase, and 8-aminolevulinic acid ( ALA) dehydrase (Fischer, 1962). The increase in ALA dehydrase was shown not to be due to an activation of a previously formed proenzyme (Cooper and Gordon, 1964).

2. Biochemical Actions in vitro Because of the complexity of the erythropoietic system in vivo, interpretations of data concerning, for example, the effects of erythropoietin in elevating the levels of enzyme activity are not without hazard. There are no simple methods of dissociating possibly indirect and nonspecific effects of the hormone from what might be direct and specific ones. This difficulty may be exacerbated by the necessary use of impure preparations of the hormone. For these reasons (and other obvious ones) much effort has been spent in the development of a system that can respond to erythropoietin in vitro. (The phrase in vitro is used here in the sense of culture outside the whole animal, not “cell-free.”)

7.

ERYTHROD CELL DEVELOPMENT

189

Some marrow cell culture methods have used morphological and/or autoradiographic techniques to evaluate the intactness of erythroid function and effect of erythropoietin (Lajtha and Suit, 1955; Suit et al., 1957; Astaldi and Cardinali, 1959; Rosse and Gurney, 1959; Berman and Powsner, 1959; Matoth and Kaufmann, 1962). Others have measured the uptake of labeled iron into marrow cells (Erslev and Hughes, 1960; Erslev, 1962, 1964) or incorporation of glycine (Powsner and Berman, 1959) or iron (Korst et al., 1962) into the heme fraction of marrow cells. Most of these studies showed that erythropoietin could stimulate increased iron uptake and heme synthesis, although one such study (Thomas et al., 1960) showed no effect of the hormone. We could demonstrate an erythropoietin effect on marrow cells in vitro (Krantz et al., 1963) by measuring the rates of heme synthesis at intervals after erythropoietin introduction, and with different hormone concentrations. Under these conditions there was a dose-response curve similar to that found with the in vivo assay performed with erythropoietin (in the form of anemic rat plasma or of a highly purified fraction). With partially purified fractions of relatively low specific activities the situation was different, as indicated in Fig. 1. In this experiment, a fraction with a potency of about 2 unitslmg caused a marked inhibition of heme synthesis at levels of hormone above 0.5 units/ml. This depression must have been due to some nonspecific inhibitory substance in the crude erythropoietin fractions which was lost on further purification. A similar inhibition was found when incorporation of glucosamine (see below) was studied (Dukes et al., 1964). Two observations (Krantz et al., 1963) with this type of marrow cell culture suggest that the cells can act in a nearly physiological manner under the conditions we use: (1) There was increased heme synthesis when the amount of added erythropoietin was approximately equal to that in normal plasma. ( 2 ) The amount of heme synthesized per day by rat marrow in vitro, as calculated from the initial rate of iron incorporation into heme, is of the same order of magnitude as that calculated , about 1.4 for the system in vivo. The rate of heme synthesis, in u i t r ~is mg/day/lO1° nucleated rat marrow cells, while the corresponding estimate in vivo (assuming that there are 8 x lo9 total nucleated marrow cells in the adult male rat) (Cohrs et nl., 1958) is 10 mg/day/lO1° cells (Bozzini, 1965). While the discrepancy between these amounts appears large, the approximations are so uncertain that only an order of magnitude is really pertinent.

190

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We evaluated marrow cell heme synthesis by extracting the newly formed heme, which had been labeled with a suitable precursor, into a nonaqueous solvent after acidification. The need for acidification suggested that the heme was protein-bound and not free (Krantz et al., 1963). Subsequent studies ( Gallien-Lartigue and Goldwasser, 1964) showed that at least 93% of the labeled heme was derived from hemoglobin. These data provided a basis for using this technique with the

0.01

0.1

1.0

5

UNITS PER ml FIG. 1. Effect of impure erythropoietin on heme synthesis. Cultures started with 2 x 106 nucleated marrow cells/ml from 3-day-fasted rats. Medium: NCTC 109-rat plasma (1:l) preincubated 47 hours with step I11 erythropoietin (lot 192A), 2 units/mg protein. Heme synthesis assessed for the next 8 hours with "Fe-labeled rat plasma.

marrow cell system in uitro in studying differentiation as indicated by erythropoietin-induced hemoglobin synthesis. Another functional aspect of erythrocyte development studied in this laboratory is the erythropoietin-stimulated incorporation of labeled glucosamine into cell stroma. By using fluorescein-labeled antibody against human erythrocyte stroma, Yunis and Yunis (1963) showed that even the earliest erythroid cells already had some of the specific antigen on their surfaces, whereas nonerythroid cells did not. Dukes et al. (1963, 1964) found that erythropoietin increased glucosamine incorporation

7.

ERYTHROID CELL DEVELOPMENT

191

into a stroma-like fraction; this stimulation was dependent upon the amount of hormone in the medium, and the stimulated increment was linear with time of incubation. The biochemistry of this process is still obscure, since the details of biosynthesis and the structure of stroma have not been worked out. In the marrow cell system, about one-third of the incorporated glucosamine was found, after acid hydrolysis of the stroma, in N-acetyl and N-glycolyl neuraminic acids. None of the label appeared in free glucosamine and the remaining two-thirds has not yet been identified. The increase in hemoglobin or stroma synthesis in marrow cells in response to erythropoietin has been further studied in order to learn more about the action of the hormone (Krantz and Goldwasser, 1965a). The cells were removed at intervals up to 15 hours after incubation in erythropoietin-containing medium, and the amount of erythropoietin remaining in the medium was determined by addition of marrow cells (preincubated in erythropoietin-free medium ) . There was no detectable depletion of the hormone from the medium by the cells, although by this method we should have detected a fall of 20% or more. Nevertheless, cells exposed to erythropoietin for 15 hours, then transferred to erythropoietin-free medium, continued to synthesize hemoglobin for a number of hours. [In these experiments, preincubated media were used to control against the possible effects of medium conditioning (Parker, 1961).] The results suggest that a small fraction of the hormone to which the marrow cells are exposed is sufficient to initiate and maintain, for some time, the process of hemoglobin synthesis in culture. In a somewhat similar experiment ( Dukes and Goldwasser, 1965), the marrow cells were exposed to erythropoietin for 1hour in the presence of labeled glucosamine. The medium was then replaced with preincubated medium containing labeled glucosamine but no erythropoietin. During the next 6 hours, the rate of glucosamine incorporation into these cells was the same as that of cells exposed continuously to erythropoietin. The rate then declined to the control level seen in cells that had not had contact with erythropoietin at all. The l-hour exposure to erythropoietin was sufficient to induce a period of stimulated glucosamine incorporation which lasted another 6 hours, suggesting that induction of differentiation (as measured by this particular function) may occur in waves or bursts. The effect of cell number on erythropoietin stimulation of hemoglobin synthesis, at several levels of hormone, showed a sigmoid rather than the linear relationship expected from a simple model involving the in-

192

EUGENE GOLDWASSER

teraction of hormone molecules with susceptible cells. The curves in

Fig. 2, derived from the original data (Krantz and Goldwasser, 1965a),

show the effect of cell number on hemoglobin synthesis per cell. Since there is no detectable depletion of erythropoietin from the medium, the fact that hemoglobin formation is greater per cell as cell number increases cannot be explained by a “multihit” kind of mechanism. The number of cells used in these experiments, even at the low levels, makes 45 I B

0 X

-I

40-

J

35-

5a

30-

W V

D

’ y

25-

LL

W

5I

20-

150.085 UNITS

2 - I 50

I

2

3

4

5

0 6

7

8

9

0.0210 UNITS 0 . 0 UNITS

l

NUCLEATED CELL NUMBER X

FIG.2. Relationship between rate of heme synthesis per cell and number of cells. (Data from Krantz and Goldwasser, 1965b.)

unlikely an explanation based on simple nutritional support among the cells (Eagle and Piez, 1962). There would seem, then, to be a cooperative interaction between the marrow cells in these cultures, and the following speculation is offered to explain this cooperation. Rat marrow cells have a tendency to aggregate even when care is taken to start the cultures with singly dispersed cells. The hypothesis postulates that there is an aggregate size optimal for erythropoietin-stimulated hemoglobin synthesis. As the total cell number increases, the probability that such optimal clusters will occur would increase according to Poisson statistics. If the rate of hemoglobin synthesis were a direct function of the number of such clusters, an increase in cell number would result in a sigmoid

7.

ERYTHROID CELL DEVELOPMENT

193

increase in the rate of hemoglobin formation. The curve would tend to flatten at the higher cell numbers, possibly because of limited entry of erythropoietin and/or nutrients needed for hemoglobin synthesis when the cluster became too large. A rationale for the optimal-cluster idea can be derived from the phenomenon of contact inhibition ( Abercrombie, 1962; Levine et al., 1965). It is likely that cells in the smaller-thanoptimal clusters will be dividing more actively than those in larger clusters. Assuming that a cell (potentially erythroid) cannot be induced by erythropoietin when it is dividing, the probability would be greater for the larger, optimally sized aggregates to contain inducible cells. When erythropoietin-stimulated hemoglobin synthesis was measured, the relationship to cell number discussed above was found, but when glucosamine incorporation was measured, we found a linear relationship between stimulated incorporation and cell number. Another instance of a difference between these two methods of study of erythropoietin action comes from evaluating the media. We found maximum hemoglobin formation in marrow cell cultures when the medium contained serum of either fetal or newborn calves. However, glucosamine incorporation was substantially greater when normal calf serum (heated to 56°C for 30 minutes) was used. We have no explanation for these differences. The mode of action of erythropoietin was further analyzed by studying the effect of specific inhibitors on the hormone-induced changes. Actinomycin D, which inhibits DNA-dependent RNA synthesis, interfered with the action of erythropoietin in mice (Gurney and Hofstra, 1963), and we have found that it also inhibits erythropoietin-induced changes in vitro. These observations suggest that erythropoietin action may be mediated through DNA-dependent RNA synthesis, and raise the possibility that the hormone acts on genetic transcription. In culture the effect of erythropoietin on hemoglobin synthesis became actinomycin resistant if an interval of 24 hours elapsed between addition of hormone and inhibitor ( Gallien-Lartigue and Goldwasser, 1965). These experiments demonstrated that whatever RNA syntheses were involved in mediating erythropoietin action had ceased by 24 hours, and that the RNA species formed had a rather long life span. A stable messenger RNA for globin synthesis by recticulocytes has been known to exist for some time (Nathans et al., 1962) and has also been suggested to occur in fetal liver cells (Grasso et al., 1963). Erythropoietin-dependent glucosamine incorporation is also sensitive to actinomycin and to puromycin (Dukes and Goldwasser, 1965), im-

194

EUGENE GOLDWASSER

plicating both RNA and protein synthesis in this process too. We have used actinomycin inhibition to evaluate the mean life span of those mRNA’s involved in the complex process of stroma synthesis. Marrow cells preincubated for 15 hours with erythropoietin, before addition of adinomycin, continued to incorporate glucosamine at an undiminished rate for 3.5 hours after addition of the inhibitor. After this time, increased incorporation ceased. This short life span (3.5 hours) is appreciably shorter than that of the hemoglobin mRNA. When the culture contained 0.25 pg of actinomycin per million cells, there was complete abolition of any erythropoietin effect; when the ratio was 0.156 pg per million cells, the rate of glucosamine incorporation was reduced to about 60% of the control level (Dukes and Goldwasser, 1965). This reduced rate persisted for about 8 hours, after which increased incorporation due to erythropoietin could not be detected. These observations suggest that two or more distinct processes may be inhibited by actinomycin, one of which is only slightly affected when the ratio of inhibitor to cells is low. The relationship between degree of inhibition of glucosamine incorporation and concentration of actinomycin is expressed by a curve with at least two distinct slopes (Fig. 3 ) . While this might suggest two or more sensitive processes that are dependent upon DNAdirected RNA synthesis, a comparable study of the inhibition of purified RNA polymerase in the presence of a DNA primer shows a similar curve (Reich, 1964). It might also be interpreted as indicating that there are guanines in the DNA that can react differentially with the inhibitor. Further interpretations of these data on inhibition of glucosamine incorporation will be discussed in a later section. These data on inhibition of erythropoietin-induced changes by actinomycin prompted us to examine the possibility that the hormone acted directly on RNA synthesis. Some years ago Perretta and Thomson (1961) showed that crude urinary erythropoietin stimulated the incorporation of labeled formate into the nucleic acid (RNA and DNA) purines of spleen and liver slices but not of marrow cell suspensions. These observations were not supported by our subsequent findings, which suggested that most of the effects described in their earlier paper may have been nonspecific and unrelated to erythropoietin action (Dukes and Goldwasser, 1962). More recently, we have shown that highly purified erythropoietin does cause an increase, in vitro, of labeled uridine incorporation into marrow cell RNA (Krantz and Goldwasser, 1965b). After incubation of the cells with erythropoietin for 6 hours we detected an increased rate

7.

195

ERYTHROID CELL DEVELOPMENT

of total cellular RNA synthesis, as measured by a 20-minute uridine pulse. When the RNA was fractionated by sucrose gradient centrifugation, a small but significant increase in incorporation could be detected 15 minutes after addition of the hormone, using a 10-minute pulse. The RNA whose synthesis was stimulated was found in the 12-20 S region of the gradient, while both larger and smaller RNA species did not show

140 130

X

0.010 UNITS ERYTHROPOIETIN

0

CONTROL

0 U

I

0 0

LL 0 W 0

U

W

a

50-

30

20

-

-

------c_ _

10 -

I

I

0.1

X

I

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1

pg ACTINOMYCIN/ml

FIG. 3. Effect of increasing amounts of Actinomycin D on incorporation of glucosamine by marrow cells. Cultures started with 5.2 x 10" nucleated rat marrow cells per ml in culture medium that consisted of NCTC 109-calf serum ( 1:l); incubated for 24 hours. Erythropoietin used as 0.1 unit per rnl.

any signilkant increase in label due to erythropoietin under these conditions. The stimulated RNA synthesis was abolished in the presence of actinomycin and was not seen when the erythropoietin had been pretreated with trypsin. In addition, the stimulation was probably not due to some general stimulation of RNA synthesis unrelated to erythropoiesis, since we found that the hormone had no effect on RNA formation by suspensions of Murphy-Sturm tumor cells under the same conditions. Incorporation of formate into rat marrow nucleic acids is also increased

196

EUGENE GOLDWASSEH

by erythropoietin ( Pieber-Perretta et al., 1965). When RNA synthesis was increased by erythropoietin, we found that incorporation of thymidine into DNA for as long as 9 hours was not affected by the hormone (Table 111) (Krantz and Goldwasser, unpublished). TABLE I11 INCORPORATION OF THYMIDWE INTO DNA

BY

MARROW CELLS~

cpm/mg DNA

Time (minutes)

Control

Erythropoietin

0 30 60 180 360 570

0 6.6 12.4 58.6 124 194

0 5.3 13.0 58.4 129 196

a Cultures had 2 x 107 nucleated cells/ml from rats starved for 3 days; erythropoietin at 0.67 units/ml; total volnme of' each dish 0.9 ml; 0.04 pmoles of thymidine ( 1 . 3 kc) added per dish.

The data so far accumulated with this system are all consistent with a model, discussed below, of erythropoietin-induced differentiation derived from the Jacob-Monod model for the regulation of gene activity (Jacob and Monod, 1961, 1963), but a large number of gaps remain to be filled. The exact nature of the RNA that is formed rapidly in response to erythropoietin needs to be determined. Although the sucrose density experiments show clearly that it is neither 28 S ribosomal RNA nor 4-5 S RNA (tRNA), it remains possible that the population of newly formed RNA molecules includes 18 S ribosomal RNA as well as other species of RNA, among which might be specific messengers. We do not know yet whether the rapidly synthesized RNA can act as a template for protein synthesis, or, if it is a messenger, for which particular proteins it contains the code. 3. Control of Hemoglobin Synthesis

Investigation of the primary steps in erythropoietin induction of erythroid differentiation will be hampered for some time, no doubt, because of our ignorance concerning the controlling reactions in the various processes involved. * There are data indicating that the rate-limiting reaction An excellent summary, including many provocative speculations, of this aspect o€ erythroid differentiation may be found in the review article by Granick and

Levere ( 1964 ) .

7.

ERYTHROID (3ELL DEVELOPMENT

197

in heme synthesis is the one catalyzed by 6-aminolevulinic acid ( ALA) synthetase, i.e., the condensation of glycine and succinyl coenzyme A to form ALA. The experiments, which show that the overall rate of porphyrin synthesis is determined by the activity of ALA synthetase, were done with mammalian liver preparations (Granick and Urata, 1962; Urata and Granick, 1963), photosynthetic bacteria ( Burnham and Lascelles, 1963; Higuchi et al., 1965), and chick embryo cells (Levere and Granick, 1965), but the same considerations may hold for marrow cells. Another important observation is that heme added to reticulocytes can increase the formation of soluble protein (presumably hemoglobin) as measured by incorporation of labeled amino acid (Bruns and London, 1965). These experiments, and those on ALA synthetase, lead to a plausible model suggesting that a part of the developmental sequence initiated by erythropoietin is as follows: The hormone can react with the specific repressor which keeps the structural gene (or, if there are such in the mammalian systems, the operator gene) for ALA synthetase from being expressed. Such a derepression results in the formation of mRNA specific for ALA synthetase, followed by enzyme synthesis. Since the other enzymes in the biosynthetic chain leading to heme are present in nonratelimiting amounts, the rate of heme synthesis would increase with an increase in synthetase. As heme accumulates in the cell it stimulates globin formation so that hemoglobin formation is controlled, at least in part, by the level of a single enzyme. If the rate of globin synthesis cannot keep pace with heme synthesis owing, for example, to a deficiency of a- and P-chain messengers or of free ribosomes, there is feedback inhibition of ALA synthetase by heme (Burnham and Lascelles, 1963; Karibian and London, 1965) so that no appreciable accumulation of porphyrins, which may be deleterious to the cell, can occur. We have attempted to test these concepts by determining the level of ALA synthetase shortly after introduction of erythropoietin into the culture medium. There is a low but detectable level of the enzyme in marrow cells when they are taken immediately from the animal, but after a short period in zjitro we could find no enzyme at all. Quite evidently the cells are capable of heme synthesis, and the fault must lie in the method of estimation of the enzyme. There is fairly widespread agreement that this particular enzyme is one of the least stable and most difficult to work with. If the regulatory reaction actually were the synthesis of ALA, addition of it to cells making heme should result in a bypass of the enzyme and

198

EUGENE GOLDWASSER

an accelerated heme synthesis. Levere and Granick (1965) have shown this to be the case for chick embryo cells in culture. There are, however, some other observations which are not consistent in detail with the mechanisms proposed. The paper of Bruns and London (1965) that showed the stimulation of globin synthesis by heme also showed that this occurred only in reticulocytes obtained from iron-deficient animals; if they were not iron deficient, no effect was seen. Winterhalter and Huehns (1963) have detected a small amount of free globin in mature human erythrocytes. If confirmed, this must mean that the formation of hemoglobin cannot be regulated solely by the availability of heme. If it were, no free globin would remain at the end of the developmental process. In addition, Gribble and Schwartz (1965) found that protoporphyrin increased the amount of soluble protein formed by a cellfree system derived from reticulocytes without any change in the total amount of amino acid incorporated. These data suggest that the stimulatory role of heme seen by Bruns and London may have been confined to the release of polypeptide chains from the particulate protein-synthesizing system. The rate of formation of soluble globin would then be dependent upon heme only in an apparent fashion; the actual incorporation of amino acid into polypeptide would be independent of the presence of heme. In chick blastoderm cultures, a protein that reacts with antiglobin appears to be formed before heme is detected (Wilt, 1962), suggesting that globin synthesis is dependent upon prior heme formation. The data of Schwartz and his collaborators (Schwartz et al., 1959, 1961) showing that heme synthetase, the enzyme that adds iron to protoporphyrin, is stimulated by native globin also suggest that protein participates in the regulatory processes. We have recently found that heme synthesis in marrow cells is inhibited shortly after an inhibitor of protein synthesis ( puromycin or Actidione ) is added to the culture medium (Hrinda and Goldwasser, 1966). The rate at which heme synthesis is reduced by these inhibitors suggests either that the enzymes involved in heme synthesis by marrow cells have a very short life span or that heme synthesis is regulated by some sort of protein synthesis. Some recent observations (Tschudy et al., 1965) indicate that ALA synthetase in the livers of animals treated with allylisopropylacetamide (to induce ALA synthetase formation) has a half-life of about 70 minutes, as does its mRNA. The situation in erythroid cells, however, must be different. The half-

7.

ERYTHROID CELL DEVELOPMENT

199

life of the enzyme in those cells forming hemoglobin is probably considerably longer. From the data of Karibian and London (1965) the estimated half-life of the system synthesizing heme from glycine is 3 4 hours in reticulocytes. Our data indicating a long life span for the hemoglobin-forming system ( GaIlen-Lartigue and Goldwasser, 1965) are in qualitative agreement with those of Grasso et al. (1963) and of Wilt (1965). This latter author showed that, in the chick embryo culture system, hemoglobin synthesis, in the head-fold stage, occurs 8 hours after inhibition with actinomycin. If the inhibition was complete, this observation would be consonant with a long life span for the entire system that catalyzes the synthesis of heme and globin. If ALA synthetase and the other enzymes in the sequence have a relatively long life span in erythroid cells, it may be that globin synthesis is required for heme formation (Granick and Levere, 1964) and that the RNA formed rapidly in response to erythropoietin is composed of globin chain messengers. It may also suggest a dual control of hemoglobin synthesis. 4. Other Possible Primary Efects

Whether the primary steps of erythropoietin action are, in fact, concerned with hemoglobin formation is still an unanswered question. Some recent experiments (Hrinda and Goldwasser, 1966) now suggest that one of the early effects of erythropoietin is facilitation of the entry of iron into some cells of the marrow. This effect is discernible before the stimulation of hemoglobin synthesis can be detected, indicating that it is not due to the displacement of the equilibrium distribution of iron caused by sequestration of the iron as heme. Puromycin inhibits this erythropoietin-effected iron uptake, suggesting that the hormone has induced the synthesis of some new protein, or proteins, required for the facilitated entry of iron into the cells. This may be a membrane component that has a direct influence on iron transport, or it may be an intracellular component involved in transfer of iron from the membrane to the heme-synthesizing system. In other studies, we examined the proposed role of erythropoietin as a mitotic stimulant ( Matoth and Kaufmann, 1962). As mentioned above, erythropoietin did not increase DNA synthesis when there was already increased RNA, hemoglobin, and stroma synthesis; this indicates that cell duplication was not required prior to initiation of differentiation. The

200

EUGENE GOLDWASSER

mitotic inhibitor colchicine at 5 x lops M did not completely inhibit erythropoietin-stimulated hemoglobin synthesis; about 14% of the hormone effect remained ( Gallien-Lartigue and Goldwasser, 1965). Erslev and Hughes (1960) found earlier that colchicine (2.5 X M ) caused a small inhibition (3040%) of iron uptake by marrow cells in vitro. We interpret our data as suggesting that the induced cells could maintain hemoglobin synthesis in the absence of mitoses; by blocking cell divisions, colchicine decreased the amplification of hemoglobin synthesis due to the few cell doublings that would normally have occurred during erythropoiesis. When the effect of colchicine on erythropoietin-stimulated glucosamine incorporation was studied, quite different results were found (Dukes and Goldwasser, 1965). The inhibitor had no effect on the process. We can reconcile these findings with those on hemoglobin synthesis by referring back to the suggestion that stimulated glucosamine incorporation occurs in short bursts during some part of the cell cycle, while hemoglobin synthesis occurs continuously. Even if cell division were completely inhibited, the entire erythropoietin-induced stimulation of glucosamine incorporation would be seen, because of the continuous generation of cells in the proper phase of the cell cycle. We might expect inhibition by colchicine if the experiment were greatly prolonged, but this experiment has not yet been performed. Alternatively, it may be that the stimulated glucosamine incorporation occurs only in those digerentiated cells incapable of further division. This is doubtful in view of our finding that increased glucosamine incorporation can be seen as earIy as 4 hours after introduction of the hormone. It would seem improbable that the whole process of stimulation of primitive cells through the stage of nondividing erythroid cells could occur so rapidly. VI. Models of Erythroid Differentiation

Our observations on the mode of action of erythropoietin on marrow cells in culture can be fitted into the highly speculative models for induction of erythroid differentiation described in Figs. 4 and 5. In Fig. 4, the process of erythroid differentiation is divided into three distinct stages: sensitization, induction, and specialization. Sensitization is the process by which stem cells, potentially capable of being converted to erythroid cells, become inducible or competent. * We

* In Fig. 4 the position of sensitization in the cell cycle is not specified; it could be at any stage.

7.

201

ERYTHROID CELL DEVELOPMENT

postulate that sensitization requires the new synthesis of a species of RNA and would, therefore, be inhibited by actinomycin. This inhibition requires less of the inhibitor than do other stages, possibly because of a relative paucity of guanine in the region of the DNA being transcribed for the purpose of sensitization. When sensitization has been inhibited by an amount of actinomycin too small to affect other cellular processes materially, those sequential events initiated by erythropoietin can continue until the supply of sensitized cells is exhausted, after which the SPECIALIZATION

I

I

'

DESENSITIZATION

E'

E"

E"'

RI

RBC

FIG.4.

Proposed model of stem cell differentiation. S represents stem cells; S', sensitized stem cell; E, first stage of erythroid differentiation; E', E", E"', later stages of erythroid differentiation; Rt, reticulocyte; RBC, erythrocyte; e, erythropoietin. The stippled area on S' represents the postulated attachment site for erythropoietin.

erythropoietin effect will cease. This mechanism can explain our observations of the inhibition of the rate of glucosamine incorporation by actinomycin (p. 194). The nature of the sensitization step, if it exists, is even more speculative. It could involve the formation of a messenger RNA with a very short life span concerned with the formation of a specific attachment site (also with a very short life span) on the cell surface for interaction with erythropoietin. While the attachment site for erythropoietin existed, the cell could be "hit" by the hormone. After it was lost, erythropoietin would have no effect. * * The concept of a limited period of sensitivity is formally equivalent to the

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If the concept of specific attachment site has validity, it could account for the specificity of erythropoietin action. We would assume that completely undifferentiated cells would be the only cells capable of becoming sensitized to erythropoietin; all other cells, in various states of differentiation, would have lost (or had repressed) the capability of forming an erythropoietin attachment site, so that, for example, muscle cells could not be induced by erythropoietin to form hemoglobin eventually. Once erythropoietin is within the cell, the second stage, induction, can occur. Induction, in this context, refers to the primary effect of the relatively few hormone molecules inside the ce1l.t If there were a large number of hormone molecules in the cell, the hormone might act simply as a metabolite. Emphasis is put on the primary effect in order to dissociate the immediate result of erythropoietin action from the subsequent secondary events. It is the process of induction that involves the actual mechanism of the hormone action, and it is about this process that we know virtually nothing. After the primary event has occurred, the series of secondary, tertiary, etc. events that may derive from it constitute the third stage, specialization. In this stage the easily recognizable special biochemical and morphological characteristics of the erythroid cell arise. This view of how differentiation of stem cells leads finally to erythrocytes contains no provision for maintenance of the stem cell pool, a condition that obtains in vivo and has been the subject of several kinetic models of stem cell differentiation (Osgood, 1959; Lijtha and Oliver, 1961; Lajtha et al., 1962; Stohlman et nl., 1962; Cronkite, 1964; Till et al., 1964). I would like to propose still another speculative mechanism to explain this phenomenon. Stem cell division may be regulated by a process similar to contact inhibition of cells in uitm. When cells are induced toward erythroid differentiation by erythropoietin, one of two changes may occur. Either the surface properties of the induced cells ( E in Fig. 4) suggestion of Lajtha and Oliver (1901) that there is a part of the cell cycle in which the cells are refractory to the action of a differentiation stimulus. f A calculation based on the concentration of erythropoietin in normal blood, and assuming the following: target cell diameter, 12 p; 5000 units per mg for pure erythropoietin, erythropoietin molecular weight 66.000, and equilibrium distribution of the hormone inside and outside the cell, shows that there would be 10-20 molecules of erythropoietin inside the cell. If there were a concentration mechanism for erythropoietin in the cell, the number of molecules inside would, of course, be considerably greater.

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change SO that they are no longer recognized as neighboring cells by the uninduced stem cells, or the induced cells migrate some distance and are removed from physical contact with their former neighbors. In both of these cases the loss of recognizable like neighbors would be the signal for cell division to fill the gaps. Gurney and Fried (196513) suggest that the signal for stem cell replication may involve a decrease in the amount of a mitosis-inhibiting factor. If the period of sensitization is short with respect to the cell cycle, and if the biological life span of erythropoietin is not long, protection of the stem cell pool from depletion by high levels of the hormone will be provided. A more detailed description of a basically similar type of regulatory mechanism for stem cells is contained in the model proposed by Kretchmar ( 1966) from his analog computer simulation of erythropoietin action. In this model the integrity of stem cell number is maintained by restricting the effective action of erythropoietin to a part of the GI phase of the stem cell cycle and by making GI variable and dependent upon the negative feedback of cell division. As G1 is lengthened owing to reduced stem cell proliferation, the time the cell spends in GI is longer than the effective life span of intracellular erythropoietin, so that the hormone does not persist until S phase where it is supposed to act as a derepressor. Under conditions of maximal stem cell division, on the other hand, the GI period is so short that the probability of effective interactions between stem cells and the inducing hormone is reduced to a value SO IOW that depletion of the stem cell pool does not ensue. Further speculation on how induction can lead to specialization derives from the Jacob-Monod model. If we adopt the view that normal cellular constituents may have roles in gene regulation, as well as their more accustomed functions as enzymes, co-factors, and structural components, a model can be made in which there is no need for specific regulator genes (Waddington, 1962), which have not been described in mammalian cells, and without introducing any new genetic entities ( Barr, 1962). In this model, as schematized in Fig. 5, the primary product in induction, in addition to its other role in the economy of the cell, may also act as a repressor, a derepressor, or both. In model I, erythropoietin ( e ) by combining with the repressor x acts to initiate the synthesis of a gene product i which is a derepressor (combining with t o ) for some other repressed function H and/or a repressor for another function J which will eventually be lost by the differentiated cell. The new derepressor i has the same possibilities of action as did the original one, erythropoietin.

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It can cause the formation of a new functional component of the cell which has the same options (with differing specificities) as i did. The second product h can repress, derepress, or both. This type of process can then lead to a cascade of newly formed properties of the cell and a loss of some preexisting properties. This third stage, specialization, is the one that eventually provides the “constellation of synthesis and properties”

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FIG. 5. Proposed model for speciaEzation. Capital letters inside the vertical lines represent structural genes; Iower case letters to left of lines indicate repressors; to right of arrows indicate gene products. Erythropoietin is indicated as “e”. In model I. e is a derepressor; in model 11, e is a repressor.

mentioned earlier. It also provides for the other aspect of cytodifferentiation, which is often overlooked, the loss of specific functions. A possible alternative, but basically similar, role for erythropoietin is indicated in model I1 of Fig. 5. In this proposal the hormone ( e ) acts as a repressor of a gene I, the product of which, i, is the repressor of one of the functions ( H ) of the erythroid cell, and also has another function in the cell. The inhibition of formation of i can in turn lead to similar cas-

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cading processes, which result in gain of new functions and loss of existing ones. While neither of these models specifically requires the existence of genes that are coordinately repressed or derepressed, such operons can, of course, easily be accommodated by them. The only new concept introduced is the overlapping identities of regulator and structural genes. A similar suggestion, in a somewhat different context, has been made by Stent ( 1964). From this type of model, a prediction about the mechanism of loss of the nucleus during erythroid differentiation can be made. Nuclear loss and cessation of DNA and RNA synthesis may be the result of the repression of the citric acid cycle enzymes by some new constituent of the differentiating cell. This new constituent may be hemoglobin. Granick and Levere (1964) have already suggested that hemoglobin, because of its positive charge, may interact with DNA and act as a repressor substance. The function repressed by hemoglobin may well be the synthesis of one or more key enzymes involved in formation of stored energy from oxidized substrates. Among the enzymes conspicuously absent in the erythrocyte are those of the citric acid cycle, and if they are lost by decay after their synthesis has been repressed, the supply of deoxyribosidetriphosphates will decline due to an inadequate store of ATP for their formation. In the absence of precursors, DNA and RNA synthesis will stop, If the integrity of the DNA requires some repair of adventitious breaks in the polynucleotide, and if such repair requires the input of energy or of deoxyribosidetriphosphates, then the existing DNA will be gradually broken down within the nucleus. At some time, the presence of badly damaged and unrepairable DNA may be detrimental enough to the cell to lead to the extrusion of the nucleus by some completely unknown mechanisms. The study of biochemical mechanisms in erythroid differentiation is still in its infancy; yet the experimental approaches, data, and hypotheses now available can provide a useful point of departure for more sophisticated studies. At present, investigation of the mode of action of erythropoietin is greatly hampered by the lack of both a pure hormone and a homogeneous population of target cells, but these difficulties cannot be assumed to be everlasting. When they have been overcome, it may be possible to test some of the speculative conjectures made here, and to develop others that may come closer to accurate descriptions of the fas-

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cinating developmental process by which specialized cells are formed in metazoans. VII. Summary

Evidence has been presented indicating that the hormone erythropoietin is the direct regulator of red blood cell formation in mammals and is also the primary inducing factor for erythroid differentiation. Erythropoietin can be obtained from the plasma or urine of anemic animals and, although not yet purified, appears to be a glycoprotein of fairly large molecular size. Marrow cells can respond, in vitro, to erythropoietin by increase in hemoglobin, stroma, and RNA synthesis. These increases occur during the time when D N A synthesis is unaffected by the hormone. Synthesis of RNA, not identical with transfer or ribosomal RNA, occurs a very short time after exposure of the cells to the hormone. The data suggest that erythropoietin acts on transcription to induce specific syntheses characteristic of erythroid cells. Nothing is known of the nature of the erythropoietin target cell, but some evidence indicates a cooperative relationship among cells induced to synthesize hemoglobin. Erythroid differentiation has been arbitrarily divided into distinct phases and some speculative models have been proposed for the molecular and cytological mechanisms underlying the processes. ACKNOWLEDGMENT I am indebted to A. Kretchmar, J. Schooley, J. Lewis, and G. Hodgson for permitting me to see and use unpublished experiments even though I did not use all the material available. Thanks are also due to D. Steiner, C. W. Gurney, and M. Doyle for helpful criticisms of this manuscript and to P. P. Dukes, S. B. Krantz, M. Hrinda, M. Gross, A. Dahlberg, and C. Kung for stiniulating discussions of the problem of erythroid differentiation. REFERENCES Abercrombie, M. (1962). Cold Spring Harbor Syntp. Quant. Biol. 27, 427. Alpen, E. L., and Cranmore, D. (1959). In “The Kinetics of Cellular Proliferation” (F. Stohlnian, Jr., ed.), p. 290. Grune & Stratton, New York. Anderson, N. G. (1962). J . Phys. Chem. 66. 1984. Astaldi, G., and Cardinali, G. ( 1959). Arch. Intern. Eniatol. Sper. Clin. 2 , 17. Barr, H. J. (1962). J . Theoret. B i d . 3, 514. Berman, L., and Powsner, E. R. (1959). Blood 14, 1194. Bleiberg, I., Liron, M., and Feldman, M. (1965). Transplantation 3, 706. Borsook, H. (1964). Ann. N . Y. A d . Sci. 119, 523.

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