Organization and communication in populations of normal and leukemic hemopoietic cells

Biochimica et Biophysica Acta, 355 (1974) 260-299 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - Printed in T h e N e t h e r l a n d s BBA 87011

ORGANIZATION NORMAL

AND

E. A. M c C U L L O C H ,

AND

COMMUNICATION

LEUKEMIC

HEMOPOIETIC

IN POPULATIONS

OF

CELLS

T. W. M A K , G. B. P R I C E a n d J. E. T I L L

Departments of Medicine and Medical Biophysics, University of Toronto and The Ontario Cancer Institute, Toronto, Ontario M4X IK9 (Canada) (Received A u g u s t 9th, 1974)

CONTENTS I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

26 I

II.

Experimental A p p r o a c h e s : A d v a n t a g e s a n d Pitfalls . . . . . . . . . . . . . . . .

262

Colony methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

262

Physical techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

267

Genetic techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

268

Cell culture techniques

. . . . . . . . . . . . . . . . . . . . . . . . . . .

269

Biochemical m e t h o d o l o g y . . . . . . . . . . . . . . . . . . . . . . . . . .

271

Multidisciplinary m e t h o d s . . . . . . . . . . . . . . . . . . . . . . . . . .

272

III.

Cellular Lineages in Myelopoiesis . . . . . . . . . . . . . . . . . . . . . . . .

273

IV.

Cellular Interactions affecting G r a n u l o p o i e s i s in Culture . . . . . . . . . . . . . .

276

V.

R e g u l a t i o n o f Erythropoiesis . . . . . . . . . . . . . . . . . . . . . . . . . .

281

VI.

Leukemic Hemopoiesis

283

General considerations

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Classification o f leukemia on the basis o f p r e d o m i n a n t cell type T h e cellular basis o f multiple f o r m s o f leukemia

VII,

. . . . . . . . .

. . . . . . . . . . . . . . . .

283 284 284

C o l o n y f o r m a t i o n in cultures o f cells f r o m patients with leukemia . . . . . . . . .

286

L e u k e m i c cells in s u s p e n s i o n culture . . . . . . . . . . . . . . . . . . . . . .

288

Putative L e u k o v i r u s e s in H u m a n L e u k e m i a

. . . . . . . . . . . . . . . . . . .

291

VIII. C o n c l u s i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

293

Acknowledgements

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

295

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

296

References

261 I. INTRODUCTION The majority of hemopoietic cells are incapable of sustained proliferation; rather, they contain specialized structures required for their function or are in the process of developing such structures while undergoing a small number of terminal mitotic divisions. Because they are numerous and morphologically characteristic, these short-lived populations predominate in classical descriptions of hemopoiesis; observations of them provide the basis for most diagnoses in clinical hematology. However, the existence of these terminal populations and the distributions of cell types within them are dependent on the proliferation and differentiation of small numbers of hemopoietic progenitors. Such cells are engaged in two processes: on the one hand, cellular proliferation provides for both the renewal of the progenitor populations and the provision of large numbers of progeny. On the other hand, steps in differentiation determine the acquisition of restrictive specialized functions while limiting potential for cell divisions. In the normal hemopoietic system, these two apparently opposing processes are balanced and flexible, leading to a regular supply of functional cells combined with appropriate responses to injury or demand. In leukemia, transformation leads to excessive proliferation accompanied by absent, decreased or inappropriate differentiation. Both normal and leukemic hemopoietic cells have properties that make them attractive candidates for studies of mammalian cell behaviour. These populations can be prepared as single cell suspensions and many techniques are available for studying their proliferation and differentiation either in vivo or in cell culture. Such studies have analogies to the methods applied to bacteria and bacteriophage that led to most presently accepted concepts concerning the molecular biology of intrinsic control. However, in contrast to free-living and independently behaving populations of micro-organisms, hemopoietic tissue exists as a community that must behave coherently; communication within this community may depend on novel biological mechanisms that underlie much of the integrated behaviour of multi-cellular organisms. If the lesions in leukemia affect, at least to some extent, communication mechanisms, study of different leukemic populations may prove as useful in the unravelling of the basis of intercellular communication as the study of mutant bacterial or phage populations played in the charting of intracellular regulation. The present review was prepared with the objective of summarizing some of the information available about cellular organization in normal and leukemic hemopoiesis. In doing so, it has proved necessary to describe briefly developmental methods for the study of hemopoietic progenitors and a model of hemopoiesis derived from such studies. This model provides a description of the parent-progeny relationships that constitute the cellular basis of hemopoietic events occuring early in hemopoiesis before the appearance of maturing and mature functional cells. The model will be used to develop concepts of cellular communication based on the properties of cell membrane components that may mediate information transfer in cultures of normal hemopoietic cells. After a brief description of current concepts and controversies

262 about the nature of leukemias, an attempt will be made to compare intercellular communication in normal and leukemic populations. In doing so, the role of leukoviruses will be discussed in relation to possible causation and maintenance of leukemic phenotypes. The major emphasis will be on human hemopoiesis because of its immediate medical relevance and intrinsic interest.

I1. EXPERIMENTAL APPROACHES: ADVANTAGES AND PITFALLS

Colony method~ Cellular populations in the hemopoietic system are heterogeneous; not only are granulopoietic, erythropoietic and lymphoid cell lines intimately admixed, but also classes within each population vary both in their differentiation state and proliferative activity. The methodological problems inherent in such heterogeneity are greater in progenitor cell populations than in the terminal states of differentiation, for the former are numerically small and devoid of characteristic morphological markers; the progenitor populations are, however, crucial for the continuing existence and function of the system. Most experimental approaches for the identification of progenitors depend on identifying a population homogeneous in respect to at least one property (marker), while recognizing that other properties within that population may be diverse. The most useful cellular methods have been developmental in nature; that is, cells have been allowed to express their potential for growth and differentiation with their numbers and properties being deduced from those of their progeny. For example, erythropoietic progenitors have been studied by the capacity of their descendants to incorporate radioactive iron into hemoglobin; the initiation of this erythropoietic differentiation may be triggered either by injections of the erythropoietic hormone erythropoietin or by transplantation of marrow into irradiated hosts (for a recent review see ref. 1). Similar less specific growth assays have been devised that depend upon a measurement of increasing DNA synthesis in transplanted or regenerating hemopoietic populations [2]. However, assays that depend on the detection of the progeny of single progenitor cells are usually considered to have greater analytical power because they provide a quantitative measure of the number of progenitors, and because they reveal most clearly the proliferative and differentiative potential of individual progenitor cells. Such clonal populations have been identified by the presence within metaphase cells of specific chromosomal markers [3}; this cytogenetic method, while very precise, is time consuming, limited to metaphase cells and not readily applicable to the quantitation of progenitors. Methods that depend on colony formation do not suffer from these limitations: in these procedures, single progenitor cells give rise to progeny that remain localized and become sufficiently numerous to be recognized as discrete clones. Such methods are available for pluripotent stem cells in the mouse and committed granulopoietic or erythropoietic progenitors in a number of species including man. These methods and references to detailed descriptions of them, are summarized in Table 1.

263 TABLE 1 PROGENITORS OF HEMOPOIETIC CELLS AND METHODS OF CULTURE DETECTION Progenitor

Medium component*

Stimulant

Significance in differentiation

References

CFU-S or S

Mouse spleen

Mouse environment

Pluripotent stem cell

4, 5

CFU-C or C

Agar

Feeder layer of cells

Methyl cellulose

BFU CFU-E or E

Plasma clot Methyl cellulose Plasma clot Methyl cellulose

Conditioned medium Serum from endotoxin treated animals Urinary glycoprotein Feeder layer of cells (same as above) Leukocyte conditioned medium Human embryo kidney conditioned medium Erythropoietin Erythropoietin

37,38,39, 40, 55, 56 39, 65 Granulopoietic progenitor

57 58

98,103 108 Early progenitor of erythrocytes Late progenitor of erythrocytes

74 137 77 76,137

* Essential supportive medium in addition to usual nutritional support and serum.

Observations of colony formation yield two different types of information about progenitor cells. First, analysis of the number and character of cells within individual colonies provides an estimate of the potential for proliferation and differentiation of the cells of origin of such colonies. However, to validate this information, proof is required that each colony is derived from a single cell. The most satisfactory demonstration of clonal origin is obtained using cytogenetic techniques. It is accepted that the cells that give rise to macroscopic colonies in the spleens of heavily-irradiated [4] or genetically anemic mice [5] are pluripotent stem cells because cells within these colonies can be identified as granulopoietic, erythropoietic or megakaryocytic [4], and cell suspensions prepared from individual colonies yield further colonies when injected into suitable test animals [6]. However, the interpretation of these findings is based on cytogenetic proof of the clonal origin of spleen colonies [7]. After appropriate doses of radiation, some stem cells retain colony-forming capacity and a minority of these have randomly radiation-induced chromosomal abnormalities. Analysis of metaphase cells in spleen colonies derived from irradiated cell suspensions showed that, where marker chromosomes were identified in a single colony, they were present in between 95 and 100 ~ of metaphases. The presence of a small minority of unmarked cells in such colonies was not unexpected, since the colonies grow in the spleens of animals, continuously exposed to circulating blood. However, detailed studies, based on quantitative analyses of differentiated cell populations in colonies

264 containing cytogenetic markers [8], were necessary to show that the contaminating unmarked populations were not sufficiently numerous to account for either the erythropoietic or granulopoietic component of the colonies. The single cell origin of colonies developing in culture can be determined directly by microscopic observation of the development of individual colonies from single cells. This method was used by Metcalf and his collaborators to demonstrate that colonies of granulocytes and macrophages derived from mouse marrow grown in semi-solid agar are derived from single cells [9]. The second use of colony assays is to obtain quantitative information about progenitor population size. This application is valid only where a linear relationship can be demonstrated between the number of cells injected or plated and the number of colonies observed. When such a relationship holds, the colony forming efficiency of the cell suspension can be expressed in terms of colonies formed by a given number of nucleated cells (for example, spleen colonies per 105 nucleated marrow cells). In animal experiments, this information can be used to calculate the colony-forming potential of a total organ since it is usually feasible to determine the nucleated cell content of that organ. Such determinations permit kinetic experiments, including the construction of growth curves. However, the interpretation of such data is limited because the efficiency of colony formation of hemopoietic cell suspensions, either in vivo or in culture, cannot be determined unequivocally, In mice, it is customary to measure the fraction (f) of injected colony-forming cells that can be recovered from spleen or marrow at short intervals after cell injection [6]. The f fraction is less than unity because of cells with colony-forming potential that cannot express that potential because they never reach a suitable environment after injection but instead lodge in the lung or other tissue not able to support the proliferation and differentiation of hemopoietic stem cells. Such measurements are important since it has been demonstrated that significant variations in the value o f f can occur [10]. However, corrections for f account for cells that have colony-forming potential but fail to exhibit it only if the proportion of such cells does not change throughout the experimental determination o f f since multiple cell transfers are required. The problem of detection efficiency for the in vivo assay has been discussed elsewhere [l 1,12]. The procedure contains another unmeasured component since no method is available to correct for cell damage, or for the proportion of undamaged progenitor cells which are potentially able to form colonies but for some reason fail to do so. In man the limitation is more serious since it is not feasible to estimate total nucleated cell number, either in a specific organ or in the whole body. For this reason, kinetic measurements, such as those demonstrating changes in granulopoietic progenitors following treatment for leukemia [13], can be considered to yield only relative qualitative patterns of change. Caution is also required in extrapolating information obtained from colony formation to biologically meaningful progenitor cell properties. For example, the distribution of cell types within colonies provides only a minimal estimate of the differentiative potential of progenitors. Spleen colony formation in vivo will not detect progeny that migrate from developing colonies. This may explain why a link

265 between the pluripotent progenitor of myeloid cells (erythrocytic, granulocytic and megakaryocytic) and the lymphoid system was not detected by analysis of spleen colonies, but rather required the identification of metaphases with characteristic chromosome markers both in spleen colonies and immunologically active lymphoid tissues [14,15]. Even this link though established in the mouse, is unsatisfactory since the methodology does not distinguish between the direct differentiation of lymphocytic and myeloid cells from a single progenitor and the alternative model based on self-renewing stem cells limited to either lymphopoiesis or myelopoiesis but with a c o m m o n ancestor in adult life. These two alternatives are shown schematically in Fig. l. No data are available that allow the choice of one model over the other,

Cellulor • nmune responses

Humorol immune responses

Erythrocytes

b)

rQnulocytes

Cellulor immune responses

Humorol immune responses

Ery rocy es io~1c

Fig. 1. (a) Model of differentiation from different pluripotent stem cells for lymphopoiesis and myelopoiesis. (b) Model of differentiation of lymphocytic and myeloid cells from individual selfrenewing stem cells but with a common ancestor. Abbreviations: T, Thymus-derived lymphocyte; B, bone marrow derived lymphocyte; SL, lymphocyte progenitor; SM, myeloid progenitor. (dashed line indicates tissue influence on SO For other abbreviations, see Table I.

although the finding in human chronic myeloblastic leukemia of a specific chromosomal abnormality (the Philadelphia chromosome) in myeloid but not lymphoid cells favours the second model (model (a) of Fig. I) for human hemopoiesis [16]. The relevance of data from assays of colony formation can be established at least in part, by correlating such data with physiologically meaningful in vivo events. Such a correlation has been achieved in studies of the proliferative state of progenitors.

266 The method depends on the exposure of cell suspension to a short pulse of highly labelled [3H]thymidine; radioactivity in the labelled precursor destroys the proliferative capacity of those progenitors in which it is incorporated [17]. Typical results are shown in Fig. 2 which depicts the effect of increasing amounts in [3H] thy-

i:°o I00 t-

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rnol adult tissue 8O I

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4O

/ Regeneroting spleen

T

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I00 2 0 0 300 4 0 0 500 600 Amountof tritioted thymidine(NCi/ml)

f032 C

Fig. 2. Radioactive thymidine suicide of CFU-S from normal adult spleen and marrow (.... ), and from regenerating spleen of marrow-transplanted irradiated recipients (----). Different symbols indicate different experiments. [17]

midine on the ability of stem cell-dependent colony-forming units (CFU-S) (Table I) to survive a 20-min pulse. Cells inactivated by the exposure are considered to be in the D N A synthetic (S) phase while those able to survive are considered to be in other phases of the cycle. The results reflect the physiological state of the marrow; for cells obtained from normal animals, almost all of the cells survived exposure while 65 of progenitors regenerating in spleen were killed. The findings, obtained using the technique of colony formation, are consistent with in vivo events; under steady state conditions, the majority of pluripotent progenitors are quiescent (the Go state of Lajtha [18] and Quastler [19]), while in response to the proliferative stimulus of

267 transplantation the majority proceed rapidly through the cell cycle. The capacity of the assay procedure to detect this change is evidence for its validity in assessing in vivo cell function. Other correlations are available, based on analysis of genetically anemic animals [5,20] or for colony-stimulating-activity-dependent colony-forming units (CFU-C), from humans suffering from neutropenia [21 ].

Physical techniques The heterogeneity of hemopoietic cell populations makes physical cell separation procedures particularly useful methods of study (for a review see ref. 22). A variety of such procedures have been developed, but two of these are most commonly used; these are velocity sedimentation at unit gravity [23] and equilibrium density centrifugation [24,25]. Both types of procedures can be used either analytically or preparatively. For analytical purposes assays are expressed per fraction; for example total nucleated cells or total colony-forming capacity per fraction. An example of this application is provided in Fig. 3: the object of the experiment was to analyze normal mouse marrow by velocity sedimentation in order to determine whether or not CFU-S and C F U - C (see Table I) have identical sedimentation velocities. A

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Fig. 3. Separation of CFU-S (3.9 ram/h) and CFU-C (4.9 mm/h) obtained by velocity sedimentation at unit gravity. Upper panel is the total nucleated cell distribution. Lower panel contains profiles for CFU (.--.) and CFU-C (O--O). [134]

268 partial separation was demonstrated between the two activities and this provided evidence that the progenitors detected by each assay were not identical. Where the objective is to prepare relatively homogeneous, or at least enriched populations of a particular cell type, the results may be expressed in terms of concentration (for example, nucleated cells or colony forming potential/cell number), and this data presentation provides an estimate of the usefulness of given fractions for further study based on their enrichment for desirable cell classes or contamination with other cells. An example of this use of cell separation procedures is supplied by a problem in marrow transplantation. This procedure between non-identical donors and recipients is complicated because immunologically active ceils in the transplant react against host tissues resulting in secondary disease (for a review see ref. 26). Cells for transplantation have been prepared by either discontinuous albumin density gradients [27] or by velocity sedimentation [28] in an attempt to elimate immunologically competant cells or their precursors. This approach has proved successful in model systems using rodents [27] and sporadic successes have been reported in man [29]. Genetic techniques

In face of the heterogeneity and complexity of the hemopoietic system, it is not surprising that the analytical power of genetic methodology has proved useful. The role of cytogenetics in identifying clonal populations has already been described. Mice with specific mutations affecting hemopoiesis have also proved to be of value. Results obtained by comparing mice with such mutations to their co-isogenic littermates are presented in Table II. The defect in spleen colony-formation by hemoTABLE Ii Genotype

Linkage group

Colony formation

Environmental function

Interpretation

W/W'"

XVII

Defective

Defect in CFU-S 5

S1/SI d

IV

Normal

Present even with irradiation Defective

f/f

XIV

Reduced erythropoietic content

Normal in irradiated recipients

Defect in organ environment Defect in rapid erythropoietic response

References

20, 30 3 I, 32

poietic cells of anemic, radiosensitive mice of genotype W / W v provided evidence that the spleen colony assay detects progenitors whose functions are necessary not only for the maintenance of normal hemopoiesis but also for recovery after radiation injury [5]. The hemopoietic tissues of phenotypically similar mice of genotype S1/ S 1 a contain normal progenitors; the defect in these animals lies in the capacity of their hemopoietic organs to support growth; these animals, therefore, provided

269 proof that hemopoiesis depends on an interaction between progenitor cells and genetically specified organ components [20,30]. The defect in mice of genotype f/f [31] is localized to an erythropoietic function required only during hemopoietic growth or regeneration [32]. The existence of this specific, genetically determined function is part of the evidence for control of erythropoiesis independent of granulopoiesis or stem cell renewal and may provide a further insight into the detailed regulation of the erythropoietic pathway.

Cell culture techniques Cell culture methods are used increasingly in the study of hemopoiesis. All but one of the colony assays for hemopoietic cells listed in Table I depend on cell culture, and a variety of suspension culture procedures have also been described [33,34]. The use of cell culture techniques has many advantages but some significant limitations. The advantages include the ability to make repeated observations on the same cells in culture as a function of time, detailed control of the cellular nutrition, sensitive tests for specific regulatory molecules that promote growth or inhibition, and finally the potential for observing the activity for both lytic and transforming viruses. Of particular importance, cell culture methods are applicable to man and provide a necessary link between model experiments in animals and clinical application. However, the data may always be criticized as reflecting in vitro artifacts having little or no relevance in vivo unless specific validation is supplied. In spite of their advantages from an analytical viewpoint, cell culture methods have been used widely only since 1966; prior to that, the literature contained numerous reports of marrow cultures in which differentiated cells disappeared to be replaced by cells resembling fibroblasts (for a review see ref 35). Only Osg0od claimed the propagation of differentiating cells [36], and his methods were not used successfully in other laboratories. The change came when Bradley and Metcalf [37] in Australia and Pluznik and Sachs [38] in Israel reported the development of granulopoietic and macrophage colonies in cultures of mouse marrow. Their success appeared to depend on two features of their cultures: first, the cells were immobilized in semi-solid medium, allowing the cells in the developing colony to remain localized. Second, growth and differentiation depended upon products derived from other cells. In some variations of the technique the cells or "feeders" were immobilized in hard agar and overlaid with marrow cells in soft agar. In other variations, the feeder cells were grown in separate vessels, the "conditioned" media were collected from them and used to stimulate colony formation in other cultures [39]. Shortly after its original description the technique for granulopoietic colony-formation was applied successfully to human marrow [40], and other culture methodologies, based on either semi-solid media or suspension techniques were introduced. Some of these are summarized in Table I. However, the technique for granulopoiesis in culture will be discussed in more detail because it provides examples of requirements that must be fulfilled before data obtained from culture experiments can be interpreted with confidence. First, the mechanism of colony-formation must be identified. Evidence is

270 needed that the colonies result from proliferation rather than aggregation. This requirement may be met by repeated direct observations either by microscopy or cell counting. When these approaches are not feasible, radiobiology provides the method of choice. The proliferative capacity of mammalian cells is highly sensitive to ionizing radiation and the dose-response relationship has been well characterized (for a review see ref. 41). Proliferative capacity is lost exponentially with increasing radiation dose and the values derived from the slopes of the negative exponential (Do) fall within a narrow range regardless of cell source. Demonstration that a culture phenomenon has a radiation survival curve characteristic of cellular proliferation provides excellent indirect evidence that the phenomenon is based on growth. Further, even where growth is not an issue, the construction of a satisfactory radiation survival curve is reassuring evidence that a culture method can be used for quantitative purposes in a consistent manner over a wide range of cell concentrations. When cell proliferation has been demonstrated, the next objective is to characterize the cells within the cultures. Recognition of the final progeny usually presents few problems since their morphological or functional properties can be compared to those of known differentiated populations. The cells originating growth in culture may be more difficult to characterize; as described above, the numbers and distribution of their progeny provide a minimum estimate of their potential for proliferation and differentiation. Their relationship to established progenitors with similar capacities may require considerable experimentation. For example, cells forming granulopoietic or macrophage colonies in culture were compared to pluripotent spleen colony-forming cells by physical means (note the experiment of Fig. 3), numerical correlations [42], cytogenetic relationship [42], physiological behaviour [43], and function in genetically anemic mice [44] before it was concluded that the two assays (colony-formation in culture and colony formation in spleen) detected distinct although possibly overlapping populations in murine myelopoiesis. The third problem in interpreting data from cultures is more difficult and relates to the significance of those cultural components that are required for growth and differentiation. Some components, such as asparagine, have only nutritional significance. Others may provide protection or support, preventing non-specific loss or destruction of cells. The more significant culture components are those suspected of playing a regulatory role in vivo analogous to that in culture. The problem is simple where the in vivo regulatory role of a compound, such as erythropoietin, is established. Similarly the importance of general cell mechanisms, such as the cyclic AMP-adenylcyclase system, are well known. However, apparently regulatory substances whose activity was first detected in culture are difficult to assign an in vivo significance. In hemopoiesis, the outstanding examples are those molecular species which are required for stimulation of granulopoiesis in culture. Although a number of attempts have been made to correlate levels of these substances with changes in vivo [45,46] or to alter granulopoiesis by exogenous administration of suspected regulators [47] none of these procedures have yielded unequivocally convincing data. Such conviction will come when it has been demonstrated that

271 deficiency of a particular compound is associated with an in vivo defect and that this defect is corrected by administration of the compound. Recently, Senn et al. [21] reported a patient with chronic deficiency of granulocytes associated with an absence of cells that produce substances active in stimulating granulopoiesis in culture. If the procedure proves to be ethically acceptable, injection of material active in culture into such a patient might provide an example of proven in vivo relevance. Finally, it should not be assumed that a culture procedure established and validated in one species will have identical significance in another; particularly, relevance of data obtained from cultures of human cells must be established by experimentation, guided, but not governed, by comparisons with physical, physiological and cultural characteristics of cells from rodents. Detailed organization of control systems may vary from species to species. For example, as will be described in more detail below, human granulopoietic progenitors exist in close proximity with significant numbers of cells actively producing granulopoietic-stimulating substances, while in the mouse the stimulatory components of granulopoiesis appear to be reduced in number relative to the granulopoietic progenitors. Perhaps it is to be anticipated that where a variety of mechanisms of regulation share similar survival values, different variants may have been selected in different species. Yet, it is often lesions affecting such regulatory mechanisms that underlie disease processes and it is necessary, therefore, to study the details in the species of interest.

Biochemical methodology The heterogeneity of hemopoietic cell populations presents a formidable barrier to biochemical analysis. For example, subtle, but significant changes in enzyme or substrate occurring in critical cellular populations may be lost in the sea of biochemical machinery required for the generalized cellular activities associated with macromolecular synthesis. In spite of these limitations, knowledge of biosynthetic pathways has proved to be of practical importance; details of DNA synthetic pathways have been used to design specific chemotherapeutic drugs and agents known to act at specific phases in the cellular replicative cycle (for example methotrexate acting at the S phase or vinblastine acting at mitosis), have been shown to be more lethal to continuously proliferating leukemic cells than to normal progenitors, capable of existing in a quiescent or Go state [48]. Because of possible differences in proliferative state between normal and leukemic cells, quantitative biochemical differences between these populations must be interpreted with caution. The differences can often be attributed to proliferative state rather than differentiation events, and comparisons between normal and leukemic populations must take differences in proliferative state into account as well as stage of differentiation, before any differences in biochemical activities can be considered specific either to normal or leukemic hematopoiesis. While quantitative biochemical changes in hemopoietic tissues are difficult to interpret, qualitative findings may be of great significance. Recently, reports have been published that leukemic but not normal cells contain RNA-dependent DNA

272 polymerase (reverse transcriptase) [49]. Since this enzyme, which permits the copying of DNA products from RNA templates, is usually associated with RNA viruses important in leukemogenesis, its finding in human cells might provide evidence for a viral etiology of human leukemia. Initially, the enzyme was also thought to be present in normal human lymphocytes, stimulated by phytohemagglutinin [50]. However, subsequent studies have shown that this ribonuclease-sensitive DNA polymerase is distinct from reverse transcriptase isolated from human leukemic cells [51]. In this instance, however, the possible hematological significance of the finding provided motivation for more detailed biochemical analyses; this activity led to the description of a variety of DNA polymerases [52]. The area remains controversial; however, it is anticipated that satisfactory resolution will come not from biochemical methods alone, but rather by associating enzymatic findings with other viral and cellular characteristics. A more traditional role for biochemistry is emerging in the analyses of the molecular species that interact with hemopoietic progenitors and affect their proliferation and differentiation. For example, erythropoietin has been the subject of detailed chemical study and extensive purification [l]. Its glycoprotein nature is now well established but the relation of structure to function is still unknown. The factors that are required for, or stimulate granulopoiesis in culture have been shown to be heterogeneous, both chemically and biologically. These materials thus provide attractive bases for relating chemical structure to biological function (granulocytic differentiation) and may provide the raw material for novel biochemical contributions to the biology of communication between cellular populations.

Multidisciplinary methodologies The preceeding description of methodological approaches to the study of hemopoietic cell populations makes no claim to completeness. Methods that combine cell culture concepts with an in vivo environment have not been included. These usually consist of placing cells in a chamber made of material permeable to macromolecules but not to intact cells and implanting the chamber within the peritoneal cavity of an animal [53]. By this manoeuver, defined cell populations, including mixtures of cells from various sources, can be confined anatomically while being exposed to the full molecular environment of an animal host. Unfortunately, many of the advantages of cell culture are lost; the environment can be manipulated only within narrow limits, repeated observations are not possible, postulated regulator substances can be tested only with difficulty and reactions of the animal host to the implanted chamber complicate free exchange between cells and environment. Accordingly, the yield of information from this approach has been limited. Throughout, the limitations inherent in methods available for studying heterogeneous populations have been emphasized. A consequence of these limitations is that no single approach can be expected to display more than a portion of the complexity of the system. Thus, successful methodological tactics for analyzing the hemopoietic system, usually use more than one approach. Equally, before a conclusion

273

can be considered to be firmly established, it is usually necessary to confirm it by examining evidence originating from multiple methodologies. This need for multiple methodologies, although widely apparent in biomedical science, is is particularly acute in studies related to hemopoiesis.

III. CELLULAR LINEAGES IN MYELOPOIESIS

The diverse functional cells of the blood trace their eventual origin to pluripotent stem cells that continue to function in adult life. There is general agreement that cellular populations with specific functions are regulated independently by mechanisms that depend on the cellular organization of the hemopoietic system. The lineages in hematopoiesis are illustrated diagrammatically in Fig. 4. The figure depicts a number of transitions; some of these are reversible where populations with similar properties may exist either in a quiescent state or in a state of active proliferation. Others are shown as irreversible; that is, when progenitor cells undergo differentiation events yielding progeny more limited in their capacity for differentiation and proliferation. It is a basic assumption of the model shown in fig. 4 that such transitions are either irreversible, or reversion occurs so rarely as to be insignificant in both normal and pathological hematopoiesis. This assumption will be accepted throughout the rest of this review; however, it is based on negative evidence; that is, reversibility in hemopoietic differentiation has not been demonstrated convincingly. The reversible transitions between populations predominately at rest to active proliferation are depicted on the basis of experiments using the [aH]thymidine

Granulocytes

Erythrocytes

10;~7C

Fig. 4. Cell lineage diagram of the structure of the myelopoietic component of hemopoiesis. 1, refs 4, 5; 2, ref. 17; 3, ref. 8; 4, refs 74, 78, 137; 5, ref. 137; 6 (see Table I); 7, ref. 136; 8, refs 76, 77, 79; 9, ref. 79.

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Fig. 5. Correlation of the number of CFU-C and the number of CFU-S in single colonies of spleen obtained 14 days after marrow transplantation. Colony counts in the spleen have been multiplied by the factor 1/0.17 to correct for cells which never reached the spleen. Results for 65 of 96 colonies are shown. The dashed line is the straight line which best fits the data for all 96 colonies. [42] suicide technique or analogous methods employing cell cycle specific drugs. The major parent-progeny relationships derive from analyses of clonal populations. Both classes of information are not, at the moment, centres of controversy. Less secure is the derivation of the lengths of the lines connecting the cell populations within lineages. These distances are intended to depict numbers of differentiation events intervening between cell populations. To date, no satisfactory method has been developed for measuring such events. Biological precedent would indicate that the differentiation events are discrete and that each might represent the activation or suppression of specific genetic information. The proper units for the lengths of the connecting lines, therefore, would be "gene activation events". Such a system of measurement does not appear to be on the threshold of feasibility, in its absence, cruder methods might suffice. One such method is based on the observed randomization that occurs during the course of differentiation. That is, as increasing numbers of differentiation events separate parental populations from progeny, the number of alternative fates of the progeny will increase; the fates of the progeny cells will be influenced by random interactions between co-existent populations and external stimuli applied to the system. Such events might be considered as the basis of randomization, and the number of such randomizing events might be expected to correlate

275 with the number of differentiation events separating parent populations from their progeny. This concept has the advantage of experimental applicability. It is possible to measure correlations between populations in a differentiation pathway; from this point of view, a close correlation would indicate a small number of randomizing events and thence few steps in differentiation. In contrast, a lack of correlation would indicate many randomizing events and numerous differentiation steps. This concept is illustrated in Fig. 5 and Table III, which show correlations between cell populations. In Fig. 5, a clear correlation is indicated between pluripotent stem cells and granuIopoietic progenitors detected in cell culture. In Table III, a lack of correlation is indicated between pluripotent stem cells and erythropoietic progenitors detected in culture. Findings such as these form the basis of the distances shown in Fig. 4 between pluripotent stem cells and their granulopoietic or erythropoietic committed progeny. The cell lineage diagram of Fig. 4 is an example of the structure of the myelopoietic component of hemopoiesis. It is evident from the structure that multiple alternative pathways exist for early cells in the system. Thus, pluripotent stem cells may be quiescent or proliferative; they may renew themselves or undergo differentiation transitions. If such transition occurs, a number of alternative pathways are available, but of these only one may be selected. The total of these multiple decisions yields both the maintenance of populations of specific cells and also appropriate responses to external demands. In contrast to the orderly behaviour of the whole system, within isolated clones, particularly spleen colonies, distributions of pluripotent stem cells and differentiated progeny are extremely heterogeneous. This heterogeneity may be based on random events at each branch point within the system; the system would then be ordered through mechanisms that determine probability for each choice [54]. However, the information derived from studies of genetically anemic mice of genotype S1/S1 d [30], described above, can be interpreted most readily as indicating that the occurrence of proliferating and differentiating events is also influenced by interactions between hemopoietic cells and genetically determined elements in hemopoietic organs. The mechanisms of information transfer between cells are beginning to be investigated and the results of such studies will be emphasized in the rest of this review. TABLE III CORRELATION OF CFU-E AND CFU-S IN INDIVIDUAL 10-12 DAY CFU-S SPLEEN COLONIES Table values represent number of spleen colonies with indicated content of CFU-S or CFU-E in each colony. A total of 144 individual colonies were examined. A Chi-square test of independence indicated no significant association between CFU-E and CFU-S (P ~ 0.25). [109] CFU-S/colony 8 8 Totals

CFU-E/colony 500 :> 500

Totals

56 12 68

114 30 144

58 18 76

276 IV. C E L L U L A R I N T E R A C T I O N S A F F E C T I N G G R A N U L O P O I E S I S IN C U L T U R E

The original method for granulopoietic colony formation by mouse hemopoietic cells in culture specified requirements for a viscid or semisolid medium and the presence of some cell-derived source of colony-stimulating activity (CSA) (for more details see above). In cultures of murine cells, CSA was absolutely required for colony formation but could be obtained from a wide variety of cells [55], cell extracts [56], serum from endotoxin treated animals [57], or human urine [58]. When the general method originally developed for mouse cells was adapted to human marrow, significant differences were observed from results obtained using mouse cells [59]. Colony formation was observed in the absence of either feeder cells or culture supernatants although the addition of either yielded a modest increase in number of colonies and an increase in their size. Moreover, activity was usually detected only in preparations derived from human cells and particularly cells of hemopoietic origin (peripheral leukocytes or adult spleen) although cultures of human embryo yielded supernatants containing CSA. The apparently marginal effects of CSA on granulopoiesis in cultures of human marrow was explained when it was demonstrated that human marrow aspirates contain not only cells capable of granulopoiesis in culture but also significant numbers of cells that produce CSA [60,61]. The two populations could be separated by a

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277 simple glass or plastic adherence procedure [61 ]; cells that failed to adhere to glass or plastic (non-adherent cells) usually formed few, if any, colonies in the absence of adherent cells or medium conditioned by human leukocytes (leukocyte-conditioned medium). Fig. 6 demonstrates the effect of separating cells from marrow into nonadherent and adherent populations and the use of non-adherent cell preparations to quantitate CSA in leukocyte-conditioned medium. Non-adherent populations may be used in a similar fashion as an assay for CSA-producing cells. It is evident that granulopoietic colony formation by human marrow cells depends upon an interaction between CFU-C and adherent CSA-producing cells (adherent cells) and that the interaction is mediated through "factors" that are added by the adherent cells to the medium in which hemopoietic cells are cultured. Further, the amount of CSA in leukocyte-conditioned medium can be increased substantially by adding phytohemagglutinin to the cultures [62,63]. It became necessary therefore to examine factors in leukocyte-conditioned media responsible for colony-stimulating activity, their chemical nature, their cellular and subcellular origins, the mechanism of their interactions with CFU-C and their biological specificity. Purification of CSA from leukocyte-conditioned medium was facilitated in its early stages by extensive information available from studies of mouse colonystimulating factors derived from human urine [64] or from mouse L-cell culture supernatants [65] and from tissue extracts [66]. Factors purified from these sources were shown to be glycoproteins with molecular weights varying from approx. 100 000 to 15000 depending upon source. Leukocyte-conditioned medium was carried through similar purification steps, including precipitation with ammonium sulphate, absorption on DEAE-cellulose, and absorption on hydroxylapatite. Material eluted from hydroxylapatite was filtered through Sephadex G150 and 3 peaks of CSA were detected. One of these appeared close to the void volume with an apparent molecular weight of between 100 000 and 60 000, the second had a molecular weight of approx. 35 000 and a third of 15 000. Polyacrylamide-gel electrophoresis following treatment with sodium dodecylsulphate has not provided evidence that the three apparent molecular species represent polymers of a single active component. However, this possibility remains to be examined by more detailed studies on the components [67]. The purification procedures based on studies of factors stimulating mouse granulopoiesis, failed to detect a molecular species of low molecular weight active on human granulopoietic progenitors. This material can be separated from leukocyteconditioned medium by dialysis or ultra-filtration and found to have a molecular weight of less than 1300 [68]. Low molecular weight CSA (low tool. wt CSA) was extractable into ether or chloroform and this property permitted the preparation of a sufficiently homogeneous fraction for it to migrate as a single band in thin-layer chromatography. Low mol. weiglat CSA can be iodinated with 125I and chromatographic evidence is compatible with the view that the radioiodine is associated with the bioactivity [69]. This iodinated low mol. wt CSA has proved an important probe in studies of the reaction between CFU-C and CSA, (see below). The subcellular origin of molecules with colony-stimulating activity has been

278 studied by fractionating human peripheral leukocytes. A procedure designed for the preparation, under mild conditions, of relatively intact and pure membrane components was particularly useful [70]. With this method it was possible to demonstrate that the highest concentration of the three molecular species of high molecular weight CSA, were found in membrane fractions rather than in fractions containing other cellular components. However, preparations of CSA from membranes were not always entirely similar to those obtained from leukocyte-conditioned media. Particularly, in certain cases of acute myeloblastic leukemia (AML), leukocyte-conditioned medium was found to contain only one of the species of high tool. wt CSA, while preparations of membranes made from the same leukemic peripheral leukocytes yielded all three species of high mol. wt CSA. It is thus apparent that membraneassociated materials can carry the information required to stimulate granulopoiesis in culture; however, these materials may vary in their availability as judged by the ease with which they leave the membrane and enter culture media. The action of phytohemagglutinin in increasing the concentration of CSA in leukocyte-conditioned medium may represent an effect of this lectin on the release of materials from the membrane. Experiments with ~2Sl-low mol. wt CSA are compatible with the view that the interaction between this particular CSA and granulopoietic progenitor cells is dependent on specific cell membrane components. The iodinated CSA can be used to demonstrate cell binding by a method analogous to the [3H]thymidine suicide technique used to detect cells in DNA synthesis. That is, marrow suspensions are incubated for a short time with 125I-low mol. wt CSA, washed carefully and then plated under optimal conditions for granulopoietic colony formation [69]. Loss of colony formation is an indication of binding of the iodinated material to target cells. A typical experiment, showing the effect of increasing concentrations of 1251-1ow tool. wt CSA in the incubation mixture, is shown in Fig. 7. Two kinds of controls

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279

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help to establish the specificity of the effect; first the addition of an excess of unlabelled low mol. wt CSA prevents loss of colony formation, indicating that inactivation is not caused by non-specific radiation affects. Second, incubation of marrow suspensions with 12Sl-low mol. wt CSA does not affect erythropoietic colony formation (Tepperman, A. D., unpublished data), indicating that the effect is specific to granulopoietic progenitors. While the short incubation period used in these experiments (2 h) would allow entry of 125I-Iow mol. wt CSA into cells, the cellular specificity of the effect would indicate that all cells are not equally permeable to low tool. wt CSA. Thus, it seems likely that some membrane component of CFU-C either binds low mol. wt CSA or facilitates its entry into cells. If this conclusion is correct, the CSAproducing cells may involve the cell membranes of both the stimulator and target cells. This implies that the interaction may be modulated by changing membrane configurations of either the stimulator or the target cells, for example by altering the availability or accessibility of membraneous components. The hypothesis presented above would be of greater interest if it could be demonstrated that specific cell types are involved in the interaction. For the target cells, the granulopoietic progenitors, this requirement is readily met since only a specific minority population in marrow is capable of granulopoiesis in culture (for example see Fig. 3). Specificity of CSA-producing cells is less easy to demonstrate, but is at least suggested by the observation that they may be selected by their capacity to adhere to glass or plastic, and that they constitute a small proportion of the total population. Additional evidence is available from experiments in which marrow was separated by velocity sedimentation and the fractions tested for their capacity to promote colony formation by autologous non-adherent cells. The experimental procedure is illustrated diagrammatically in Fig. 8. and a typical result shown in Fig. 9. It is evident that capacity to promote colony formation, either by producing CSA or by cellular interaction, is a property of specific subpopulations of adherent marrow cells rather than macrophages or monocytes as a whole. The velocity sedimentation results presented in Fig. 9 are in the form of en-

280 richment profiles; that is, fixed numbers of cells from each fraction were used either in mixtures with non-adherent cells or cultured alone to produce leukocyte conditioned media. The separation between the peaks of the active cell distributions would not be so marked if the results were plotted on a per fraction basis rather than a per cell basis. However, the former presentation was used because cells in the first peak possess a biological property not found in those of the second. When human marrow cells are placed in suspension in the presence of leukocyte conditioned media containing CSA, the number of CFU-C increases as a function of time for up to 7 days [71]. In the absence of exogenous leukocyte-conditioned medium the number of CFU-C is maintained but does not increase. Similar experiments may be conducted using factor-producing cells rather than leukocyte-conditioned medium itself in mixtures with non-adherent bone marrow cells. When such factor producing cells are obtained from fractions with sedimentation velocities similar to those of the first peak in Fig. 9, an increase in CFU-C is observed; in contrast, the larger cells characteristic of the second peak failed to yield such an increase, although leukocyteconditioned media preparations from either peak contain equivalent activities when

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281 assayed for their capacity to promote colony formation by non-adherent cells. These findings suggest a functional specificity for different populations of CSAproducing cells. However, the apparent specificity disappears when leukocyteconditioned medium prepared from each population is used in the suspension culture assay; under these conditions leukocyte-conditioned medium, containing CSA, permits an increase in CFU-C regardless of whether the leukocyte-conditioned medium was prepared from cells of the first or second peak. This capacity to promote an increase in CFU-C in suspension is retained by leukocyte-conditioned medium unless it is extensively dialyzed against distilled water (Niho, Y., unpublished data). After such dialysis, leukocyte-conditioned media failed to promote increase of CFU-C in suspension, a finding similar to that observed in mixtures of non-adherent marrow cells and CSA-producing cells of large size. It appears, therefore, that growth of CFU-C in suspension depends upon the presence of a non-dialyzable component in association with smaller molecules that can be removed by dialysis. Both components are present in all cells identified as capable of producing CSA since both are present in leukocyte-conditioned media regardless of its cellular source. However, some but not all CSA-producing cells make both components available to target cells in cell mixtures. Although the cellular mechanisms responsible for regulating the availability of these components has not yet been investigated, these findings are consistent with the view previously expressed, that CSA-producing cells contain membrane-associated information required for the in vitro regulation of granulopoiesis and exert their regulatory effect, at least in part, by varying the availability of this information. From this point of view, the presence of molecules with regulator effects at the cell surface may be a marker of the differentiation of CSA-producing cells, and the fluid, rapidly changing nature of the cell membrane [72] may provide a mechanism for altering the extent to which membrane components are available for interaction with granulopoietic progenitor cells.

v. REGULATION OF ERYTHROPO1ESIS The regulation of erythropoiesis has been studied extensively for almost two decades. The system is attractive to investigators because it lends itself readily to manipulation. Erythropoietin, a hormone-like material, glycoprotein in nature, is known to be essential for erythropoiesis both in vivo and in culture. Production of erythropoietin can be eliminated in mice by rendering them plethoric by transfusion. Administration of erythropoietin to plethoric mice is followed by an orderly wave of erythropoietic differentiation that can be observed morphologically and quantitatively in spleen and bone marrow. Further, the response of plethoric mice to erythropoietin has provided an assay for erythropoietin useful in the quantitative measurement and purification of the hormone. This in vivo method has been complimented to advantage by culture techniques in which erythropoiesis occurs in cultures of rat or mouse marrow in the presence of erythropoietin [1 ].

282 In spite of these advantages, a number of controversies exist; for example, it is not certain whether or not erythropoietin is required continuously throughout the maturation of the erythropoietic series or only for a short period. The former view is supported by observations that repeated doses of the hormone are more effective than the same quantity given as a single injection [73] and by evidence for a continuing requirement of erythropoietin for cells in culture [74]. The opposite view is supported by observations using antibodies against erythropoietin; Schooley and co-workers [75] have shown that a marked effect of a single dose of erythropoietin in vivo can be observed even if its action is terminated after 6 h by injection of potent anti-erythropoietin. However, the interpretation of this result is limited by concerns related to the specificity of the antibody. A further technical complication has been introduced by the use of urine obtained from anemic humans as a source of erythropoietin; recent studies have shown that this material also contains potent granulopoietic-stimulating substances [76] and the presence of these contaminants may obscure the interpretation of results of experiments where urinary erythropoietin was injected repeatedly. Perhaps the greatest obstacle confronting students of erythropoiesis has been the lack of colony assays. Recently, this deficiency has been corrected. Stevenson and Axelrad and their co-workers have devised a method for detecting small erythropoietin-dependent erythropoietic colonies in culture and have observed that such colonies are collected in clusters or '~bursts" when large and repeated doses of erythropoietin are used [74,77]. Large erythropoietic colonies have also been observed in cultures containing high concentrations of erythropoietin in both murine [74, 137] and human systems [76, 78]. The progenitors of small erythropoietic colonies are indicated in Fig. 3 as erythropoietin-dependent colony-forming units, (CFU-E or E), and those of the large erythropoietic colonies as erythropoietin-dependent burstforming units, (BFU). The availability of these two colony assays has made it possible to construct comparative erythropoietin dose-response curves. These curves provide evidence that the generation of large colonies has a greater requirement for erythropoietin than that of small colonies. Further, within the E population, newly derived E detected in rapidly proliferating transplanted marrow require more erythropoietin than E obtained from normal adult mice [79]. Erythropoietin-receptors on the surface of sensitive responding cells have been hypothesized [79]. It may be that as a consequence of differentiation erythropoietin-receptors develop at the cell surface, and further that the modulation of the erythropoietic response (wherein erythropoietin levels were maintained)could be achieved by changing the density or accessibility of receptors. Experimental determination of such regulatory controls would be important to studies of other regulated differentiation systems.

283 Vl. LEUKEMIC HEMOPOIESIS General considerations The major consequence of transformation from normal to leukemic hemopoiesis is an increase in proliferation relative to differentiation. This imbalance has two effects; first, deficient production of functional blood cells leads to the manifestations of marrow failure; these are anemia, because of reduced erythropoiesis, susceptibility to infection because of granulocytopenia and hemorrhages because of thrombocytopenia. Second, excessive cell proliferation leads to the accumulation of cells not only in normally hemopoietic organs but also in other sites such as liver, kidney and the meninges. These cellular accumulations are detected clinically as organ enlargement, abnormal masses or the functional consequences of infiltration (for example, the meningeal symptoms associated with leukemic infiltrates in the central nervous system). Failure of marrow function combined with cellular accumulations provide the bases for most of the clinical manifestations of leukemia and usually supply the evidence required for diagnosis. Leukemic cell populations possess the general properties usually associated with neoplasia. One of these, ability to metastasize (grow in many anatomical sites) has already been mentioned. Two others are of particular importance; first, leukemic populations exhibit the genetic instability characteristic of malignant cells; this leads to the random series of genetic changes termed progression [80]. Thus, the course of leukemia is often associated with striking qualitative changes such as the development of aneuploidy and the selection of specific aneuploid clones with growth advantages. Other cell parameters, such as doubling time, predominate cellular morphology and drug sensitivity, may also change. Progression must be considered in any attempt to relate cellular findings in leukemia to the primary lesions of the disease since any particular characteristic (for example, aneuploidy) may be the consequence of progression rather than a primary manifestation of the essential difference between normal and leukemic populations. The cellular heterogeneity characteristic of malignant populations is also present in leukemia. In previous sections of this review, the heterogeneity of normal hemopoietic populations has been emphasized. Superficially, leukemic transformation might be considered to decrease this heterogeneity since the randomizing effect of differentiation is reduced while increased self-renewal might be expected to yield more homogeneous populations. Indeed, morphological studies of acute leukemia and chronic lymphocytic leukemia usually display relatively uniform cellular populations. However, this apparent morphological simplification is misleading; changes resulting from progression have already been mentioned, and proliferating populations have an inherent heterogeneity based on the different positions ceils occupy in the cell cycle. Moreover, in addition to these sources of variation, functional heterogeneity can also be demonstrated within apparently uniform leukemic populations.

284

Classification of leukemia on the basis of predominant cell type The leukemias have been described according to the predominant cell population [81 ] and this criterion for classification has usually been found to correlate both with clinical course and response to treatment. The classification derives from the accepted morphological patterns of normal hemopoietic cells. If the major population appears primitive and is devoid of differentiated structures, the leukemia is considered to be acute; in the absence of treatment, the median survival of patients with acute leukemia is usually measured in weeks. Clinical hematologists attempt to divide acute leukemia into two varieties, acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML) [81]; the latter diagnosis is possible when occasional granules are recognized in the cytoplasm of at least some apparently undifferentiated leukemic cells, and the presence of large metachromatic granules, termed Auer rods is considered pathognomonic of AML. The diagnosis of ALL is usually made on negative grounds; that is, Auer bodies or granulation are absent [82], although histochemical tests (PAS and Sudan Black) provide supportive evidence for the diagnosis of ALL [83]. In spite of the difficulty of separating ALL for AML, the distinction is of importance; ALL is common in children (indeed, it is the commonest malignancy of childhood), while AML is the predominant form of acute leukemia in adults. Further, ALL responds much more favourably to combination chemotherapy than AML and therapeutic regimens found to be the most effective are different for each class [84]. Where the predominant cellular population is morphologically similar to mature or maturing hemopoietic cells, the leukemia is considered to be chronic and the survival of untreated patients often extends for several years. For the chronic leukemias, classification presents few problems since the morphological features of the cells are characteristic and functional abnormalities are observed that are disturbances of the normal behaviour of the mature cells. Thus, in chronic lymphocytic leukemia (CLL), large accumulations of mature lymphocytes are readily identified throughout the body and these are often associated with immunological abnormalities, including immune deficiency and occasionally the production of large amounts of homogeneous gamma globulin. In contrast, in chronic myelogenous leukemia (CML) mature granulocytes predominate although their recognizable precursors, normally restricted to bone marrow, are found circulating in the peripheral blood. In CM L, rather than having immunological disturbances, patients suffer from anemia reflecting the disturbed balance between erythropoiesis and granulopoiesis characteristic of this leukemia.

The cellular basis of multiple forms of leukemia The cellular forms of leukemia may be considered to coincide with levels of differentiation within normal hemopoietic tissues. From this point of view, undifferentiated acute leukemia would result from transformation occuring in one or more undifferentiated stem cells, perhaps the common progenitors of myelopoiesis and lymphopoiesis postulated in Fig. lb. Leukemias capable of assigment to either the lymphoid or myeloid component of the system would derive from normal pro-

285 genitors in that pathway; the degree of differentiation exhibited in a particular disease (that is its chronicity) would further reflect the differentiation state of the cells prior to transformation. At least for the myeloid leukemias, an alternative hypothesis is that leukemic transformation always occurs at the level of pluripotent stem cells or earlier; from this point of view, cellular distribution in leukemic populations would reflect disordered differentiation within transformed clones rather than the differentiation stage of the original transformants. Support for this hypothesis derives from studies of CML; in this disease the predominant cell population contains mature and maturing granulocytes. However, leukemic cells can be distinguished readily from normal cells by the presence of a characteristic cytogenetic marker, the Philadelphia chromosome [85]. This marker has been identified unequivocally in maturing erythroblasts [16] as well as granulopoietic cells, and indirect evidence is available for its presence in megakaryocytes [16]. However, lymphoid populations that respond to phytohemagglutinin do not contain the Philadelphia chromosome [16]. This cytological evidence strongly supports the view that cellular populations in C M L originate from pluripotent stem cells of the myeloid component of the hemopoietic system. The predominantly granulocytic composition of the population within the leukemic clones reflects an abnormal pattern of differentiation from pluripotent stem cells rather than transformation at the level of committed granulopoietic progenitors. In approximately half of AML patients, chromosomal abnormalities are present although a characteristic marker is found in each patient rather than a common chromosomal abnormality such as the Philadelphia chromosome of CML. Jensen and Killman have examined cells from patients with AML where such markers were present [86]. By correlating the incidence of marked cells with the incidence of early erythroblasts they provided evidence that the marker was present in at least some of the red cell precursors. This is indirect though compelling evidence that at least some red cell precursors are leukemic and therefore that capacity for erythropoiesis persists in AML. If this conclusion is valid, transformation must have occurred in a pluripotent population, and pluripotent stem cells may undergo transformation leading to either A M L or CML; if so, the balance between proliferation and the various differentiation pathways in these two diseases is determined by the nature of the transforming event rather than the class of cells in which it occurs.

If the variety of morphological types of leukemia does not reflect levels of transformation, but rather the nature of transformation, certain implications follow. First, since differentiation persists in leukemic populations, the genetic information for differentiation has not been either deleted or irreversibly damaged during leukemogenesis. Instead, the genetic change may be considered to affect mechanisms regulating differentiation rather than the process itself. Second, a finite, relatively small number of genetic events would be required to account for the observed clinical varieties of the disease. These considerations affect models of viral mechanisms in leukemogenesis, and will be discussed further in a subsequent section of this review.

286

Colony formation in cultures of cells from patients with leukemia The concept of continuing capacity for differentiation in leukemic cells was enforced when methods, developed for the study of normal granulopoiesis in culture, were applied to leukemic populations. Early animal studies were particularly revealing; Ichikawa demonstrated that cells from a murine myeloblastic leukemia, showing no evidence of differentiation in vivo, were capable of giving rise to granulocytic colonies in culture in the presence of suitable conditioned media [87]. Work on this tumor has continued to be fruitful and evidence has been presented that at least two types of clones co-exist in the tumor, one capable of differentiation and the other remaining undifferentiated [88]. Similar studies were reported for cells derived from patients with leukemia. Robinson et al [89] found that the peripheral blood of patients with A M L often contains cells capable of giving rise to large numbers of colonies in culture; further, these colonies consist of apparently normal granulocytes. These findings led to extensive studies of leukemic cells in culture in many different laboratories*. General agreement was quickly achieved in respect to the findings in patients with CML. Peripheral blood was found to contain increased numbers of cells with colony forming capacity [90]. These colonies contained maturing granulocytes and were dependent for their growth on the presence of either CSA or a cellular source of CSA. The Philadelphia chromosome was identified readily in most [91,92] but not all [91] colonies derived from patients with CML, indicating that leukemic granulopoietic progenitors were differentiating in culture. Much greater variation was found in reports from different laboratories describing culture findings in AML. In part, the laboratory-to-laboratory variation could be explained in technical terms, since both the culture procedures and the criteria for scoring colonies were different in almost every laboratory. In addition, an overriding problem faced every investigator; when cells from patients with acute leukemia were placed in culture and growth observed, the source of that growth was uncertain; that is, developing colonies might derive from leukemic progenitors or from co-existent normal marrow cells. Two general approaches to this problem have been used: first, Moore and his collaborators have compared normal and leukemic ceils in respect to their buoyant density [90]. They found that leukemic populations often contained progenitor cells of lower density than normal and that these lowdensity populations disappeared following treatment. Second, Duttera et al. [93], Moore et al. [90] and Aye et al. [94] have examined aneuploid metaphose cells in culture. Metaphase preparations with marker chromosomes characteristic of an acute leukemic clone were identified in some but not all cultures. The evidence is consistent with the conclusion that some leukemic cells form colonies in culture; * For the diversity of results and the multitude of methods employed, the reader is referred to the proceedings of two workshops on hemopoiesis in vitro. The first was held in Rijswijk in September 1971 (In vitro culture of hemopoietic cells (1972) van Bekkum, D. W. and Dicke, K. A. eds., Publ. of Radiobiological Institute, TNO, Rijswijk, Netherlands) and the second at Airlie House near Washington in May, 1973 (Hemopoiesis in culture, Second International Workshop (1973) Robinson, W. A. ed. Publ. Natl. Inst. Health, U.S.A.).

287 unfortunately, colonies from cells of leukemic patients containing diploid cells cannot be unequivocally identified as normal since aneuploidy is not a consistent finding in AML; as discussed previously, aneuploid clones in this disease may result from malignant progression; it is possible, therefore, that diploid cells detected in culture may represent either normal hemopoiesis or, alternatively, leukemic clones that have not progressed to obvious aneuploidy. The discrepancies between different laboratories reporting culture data from patients with leukemia fall into two main categories, some related to colonies and others related to the stimulation of colony formation. Most investigators agree that marrow from some patients yields greater than normal numbers of colonies while marrow from other patients produce few if any colonies [95]. However, the percentage of patients falling into either group varied greatly in different reports. Further, Metcalf and his collaborators observed small clusters containing 2-20 cells and reported that these were numerous in cultures of leukemic marrow where colony formation was depressed. An agreed correlation has not been established between clinical manifestations of leukemia and findings in culture although the Australian investigators have suggested that a number of cultural characteristics in combination, including density distribution, numbers of colonies or clusters can be used to identify a small group of A M L patients with a poor prognosis [96]. Unfortunately, in their large series of patients a variety of treatment regimens were used and this complicates the interpretation of the results. Other investigators have usually failed to collect all of the data in each patient that would be required to confirm the study from Australia. Nonetheless, it is generally agreed that successful remission-induction usually is associated with culture findings (after remission-induction) similar to those observed in normals [97]. Unfortunately, the patterns of change with therapy have been found to vary greatly from patient to patient [97]; and changes observed in culture do not regularly precede clinical manifestations of either remission or relapse and have not usually been found to be useful in patient management. The second area of controversy concerns the role of various forms of CSA in leukemia. In part, the problems of interpreting data arise from the unusual species specificities of stimulatory factors. Stimulators of mouse granulopoiesis may derive from a wide variety of sources, including not only the supernatants of cultures of mouse cells [55,65] but also supernatants of cultures of human leukocytes [98] and a glycoprotein present in human urine [58]. In contrast, human cells are fastidious, requiring factors derived from human cells and particularly, (see above), materials associated with the membranes of cells present in human peripheral blood. The urinary glycoprotein active in cultures of mouse cells has little or no effect on human granulopoietic progenitors [99]. Unfortunately, the convenience provided by readily available mouse marrow as a test material led to the use of such cells in investigations of human granulopoiesis. This practice gave rise to reports of high, normal or low stimulator levels in the urine of patients with leukemia and the presence or absence of a correlation between urinary stimulatory levels and peripheral blood granulocytes. Recent comparisons of results obtained using urinary-like factors and

288 factors from human leukocytes have failed to show correlations [100]; particularly, measurements of leukocyte factors have correlated with clinical findings in neutropenia while no such correlation was observed in concurrent measurements of activity in urine [21 ]. Finally, preliminary evidence is available that factors in human leukocyteconditioned medium that stimulate mouse granulopoietic progenitors are separable from factors on human cells [101 ]. It seems probable, therefore, that elucidation of the role of human granulopoietic stimulatory factors in leukemia must await further detailed studies in which human cells are used to assay factors of human origin. In spite of the conflicting data, two points of general agreement emerge. First. marrow specimens from patients with acute leukemia contain some leukemic cells capable of colony formation in culture. These colonies exhibit differentiation although morphological assessment of the cells provides evidence that the pattern of differentiation may be distorted or incomplete [102]. Second, colony formation by leukemic cells is dependent on the presence in the cultures of stimulator molecules. These molecules may be supplied directly by adding normal leukocytes to the cultures as feeder layers [89] or indirectly by adding medium conditioned by either normal [103] or leukemic leukocytes [63,104]. Taken together, all the data support the view that leukemic cells retain at least some capacity for differentiation and that this capacity can be demonstrated in culture by the addition of appropriate cell-derived molecules. Leukemic cells in suspension culture The data discussed in the previous section was obtained using a culture procedure that detects committed granulopoietic progenitors in normal hemopoietic tissues, along with what may be a subpopulation of leukemic cells. However, leukemic transformation probably occurs in cells less differentiated than the normal granulopoietic progenitors, and consequently a number of randomizing events may intervene between the progenitors detected in semi-solid medium and the self-renewing, proliferating leukemic cells (leukemic stem cells) that maintain and expand malignant populations. It is desirable, therefore, to search for and employ culture methods applicable to leukemic stem cells. A number of such methods have been proposed, based on the observation that hemic cells often grow in suspension. Investigators at the Roswell Park Memorial Institute [105] have developed a number of lines of cells, originally derived from marrow or blood of patients with leukemia and now growing continuously in suspension culture. Cells such as these have been useful both in studies of the Epstein-Barr Virus and for the production of antileukemic antisera for studies in tumor immunology. However, these lines are separated from their cells of origin by many generations and may not reflect cellular or molecular events in vivo. A second approach is to study short term cultures of peripheral blood or marrow containing a high percentage of undifferentiated cells considered to be leukemic. One series of studies of this design has yielded evidence of cellular communication in leukemic populations analogous to those detected in cultures of normal granulopoietic progenitors [63,106].

289 In these experiments [63], peripheral leukocytes were collected from patients with leukemia whose blood contained a high proportion of undifferentiated cells (blasts). These cells were cultivated in fluid medium for from 2-10 days, pulselabelled with [3H ]thymidine and incorporation into acid-insoluble material measured. Tritiated thymidine incorporation was found to increase with time, and, although little or no change in cell number with time was detected, autoradiographs contained increasing numbers of labelled nuclei. The increased [3H]thymidine incorporation was found to have a sensitivity to irradiation in the range associated with the radiosensitivity of the proliferative capacity of mammalian cells; therefore, the radioautographic data, combined with the radiobiological findings are consistent with the view that a small population of cells proliferated in the cultures. Further, where cytogenetically marked clones were present in the patients, cells with the karyotypes typical of the leukemic cells increased and predominated in the cultures [106,107]; it seems probable, therefore, that proliferation of leukemic cells occurs in this culture system. The system has a further technical advantage; cells stored at --70 °C in 5% dimethylsulfoxide retain their cultural characteristics, making it possible to perform repeated experiments of various designs on populations derived at one point in time from a single patient. Although reproducible results could readily be obtained using different samples of a single pool of frozen cells, great patient-to-patient variation was observed [63]. While this variation related in part to the doubling time of the proliferating population, the major variable was the cultural requirements for growth. In some patients, growth was observed in the presence of fetal calf serum and culture medium alone (growth medium); in others, growth was either entirely dependent on or markedly influenced by the addition of stimulators. Two such stimulators of growth were medium conditioned by normal or leukemic leukocytes (leukocyteconditioned medium) and phytohemagglutinin. The latter substance was chosen for study not only for its known mitogenic properties, but also because it increases the production of CSA by normal or leukemic leukocytes. Because of this latter property, it was postulated that phytohemagglutinin was sometimes effective in stimulating growth in cultures of leukemic ceils by an indirect mechanism, involving the production of growth factors by cells co-existing with the proliferating population. The hypothesis was tested by the use of cells with unusual properties, from a patient with AML. The patient had a high peripheral blast count, and had been bled on two occasions, separated by nine days of extensive treatment with chemotherapy [63]. Cells from both bleedings proliferated in culture only in the presence of leukocyte-conditioned medium; cells from the first bleeding, but not from the second, also responded to phytohemagglutinin. It was therefore possible to compare leukocyteconditioned medium preparations made in the presence of phytohemagglutinin from cells of each bleeding. Phytohemagglutinin-stimulated cells from the first bleeding yielded growth-stimulating leukocyte-conditioned media, while similar preparations from the second bleeding were inactive. The test of [3H]thymidine incorporating activity used cells of the second bleeding since these were known to be unresponsive

290 to phytohemagglutinin and therefore positive responses could not be attributed to phytohemagglutinin remaining in the leukocyte-conditioned media preparations. These findings were consistent with the view that phytohemagglutinin was affecting a subpopulation initiating release of factors that stimulated growth in leukemic ceils. From a further patient, evidence is available that the phytohemagglutinin responsive population contained aneuploid cells, and was therefore of leukemic rather than normal origin [106]. Since the hypothesis proposed the co-existence of interacting cell populations, it could also be tested by cell separation procedures. Cells from two patients with growth requirements for leukocyte-conditioned medium or phytohemagglutinin were separated by velocity sedimentation and fractions tested with leukocyte-conditioned medium or phytohemagglutinin and with growth medium alone. In both instances, the sedimentation velocity profiles of growth obtained with each simulator were not identical, and in neither instance did the peak response for either stimulator coincide with the major nucleated cell population. However, sufficient separation was achieved to permit the preparation of pools of cells enriched for either phytohemagglutinin or leukocyte-conditioned medium responsive cells. Reconstitution experiments provided evidence that pools enriched for leukocyte-conditioned media responsive cells would respond by proliferation to the addition of phytohemagglutinin-responsive populations only when the lectin was present [106]. These results are consistent with the view that some leukemic populations contain 3 subpopulations: (I) a small subpopulation capable of proliferating in suspension culture, (2) a second population capable either spontaneously or with lectin stimulation, of interacting with the first subpopulation causing its proliferation; (3) the major population observed in velocity sedimentation profiles, and identified morphologically as leukemic blasts, which is not functional in the cell culture system in these experiments. The concept of interacting cells regulating growth in leukemia is very similar to the one advanced earlier for the regulation of normal granulopoiesis in culture. In that instance, it was proposed that committed granulopoietic progenitors proliferate and differentiate in response to specific protein components associated with the cell membranes of a second co-existing stimulator-cell population. Further, the interaction was subject to modulation by factors that influence the assessibility of membrane-associated stimulatory factors and membrane-associated receptors at the surface of responsive granulopoietic progenitor cells. Preliminary evidence is available for a similar mechanism in leukemia. As mentioned earlier, conditioned media containing bioactive substances can be prepared from leukemic cells; bioactivity is usually detected as CSA, and the CSA content of leukocyte-conditioned media preparations of leukemic cells is greatly increased by adding phytohemagglutinin to to the media. In contrast with the three molecular species of different size detected in leukocyte-conditioned medium prepared from normal leukocytes, leukocyteconditioned media prepared from leukemic blast cells usually contain only 1 high molecular weight species of CSA. However, when membrane were prepared from

291 the same leukocytes used to condition media, all 3 species of high molecular weightCSA were detected. This apparent discrepancy may be explained if it is assumed that the capacity of cells to excrete already synthesized bioactive molecules into the culture medium correlates with the availability of such molecules for interaction with other cells. From this point of view, leukemic populations contain subpopulations differentiated to contain a full complement of CSA molecular species associated with their cell membranes; however, as part of the leukemic phenotype, only some of the bioactive molecular species are available for interaction. Changes at the cell surface are known to be common in leukemia, and the effect of such changes on cellular communication may represent part of the mechanism for defective regulation.

vii. PUTATIVE LEUKOVIRUSES IN HUMAN LEUKEMIA The abnormal cellular phenotypes in leukemia, described above in terms of defects in cellular communication, may reflect genotypic changes. Increasingly, evidence is available from animal models that the genome of hemopoietic cells may be altered by the insertion of DNA complementary to RNA contained in certain leukoviruses. A survey of the available information on RNA-containing tumor viruses is beyond the scope of this review; however, recent information indicating that viral mechanisms are operative in human leukemia cannot be ignored in considering cellular mechanisms in that disease. Viruses as etiological agents in leukemia have been well established throughout many animal species including fowl [110], mice [111], cats and other animals [112]; the generality of the observation has been extended to subhuman primates with the isolation of leukoviruses from gibbons [113]. In parallel, extensive searches have been made for virses in human cells; until recently, these have failed to yield convincing results because the lack of a biochemical or a direct bio-assay for the leukoviruses. Indirect evidence, based on electron microscopic observations, while encouraging [114,115], has been unconvincing because particles were rarely found and those detected could not be identified unequivacaUy as leukoviruses. The discovery of RNA-dependent DNA polymerases (reverse transcriptase) in Rous Sarcoma virus [116], and murine leukemia virus (Rauscher) [117], provided a new direct method for detecing viral components within cells. As mentioned earlier, reverse transcriptase was identified in cells of the peripheral blood of patients with acute leukemia by groups headed by Gallo [118] and Spiegelman [119]. These investigators found not only the enzyme, but evidence of RNA of large molecular size (70 S); such RNA is usually found only in association with RNA leukoviruses. Its relationship to tumorogenic viruses was substantiated by the finding that DNA copied from such RNA by reverse transcription (cDNA) showed homology to RNAs obtained from viruses known to cause tumors in subhuman primates (simian sarcoma viruses SSV) [120] and mice [119,120]. The connection was further enforced by the finding that antisera

292 prepared against reverse transcriptase isolated from SSV would neutralize reverse transcriptase purified from human leukemic leukocytes [121]. Molecular components associated with leukoviruses have also been investigated in cultures of leukemic cells. Reverse transcriptase in leukemic marrows was found to increase in short-term suspension cultures in the presence of medium conditioned by leukocytes from a patient with hemochromatosis [122]. In association with this increase in culture, the enzyme was also identified in culture supernatants. Such supernatants were found to contain particles with reverse transcriptase activity that banded at densities of 1.17 (g/ml) and 1.22-1.24 (g/ml) [123]. Electronmicroscopic studies of material in the 1.17 (g/ml) region revealed particles similar in morphology to complete leukoviruses while the 1.22-1.24 (g/ml) region contained large numbers of particles with morphology similar to the cores of leukoviruses [123]. The sucrose density peaks containing particles also were found to contain high molecular weight (70 S) R N A [124] and D N A synthesized by an endogenous reaction from the particles also showed homology with RNAs obtained from leukoviruses of simian and murine origin. Typical hybridization results are given in Table IV. The results showed that though there was homology between the D N A synthesized by the human leukemic particles and simian or murine tumor viruses, considerably more homology was found with the viruses of simian origin [124]. The availability of culture methodology made it possible to examine marrow obtained from patients in remission. While little reverse transcriptase was identified prior to culture in marrows from such patients, the amount increased following cultivation and particles were identified with properties similar to those found in relapse. Thus, cells from patients in remission respond to culture conditions by producing leukovirus-like particles, indicating that this aspect of the leukemic phenotype persists following successful chemotherapeutic remission induction. The capacity of ceils from patients in remission to generate leukovirus-like particles in culture supports the view that viral information persists [122]. TABLE IV PERCENT HYBRIDIZATION OF [3H]DNA PRODUCTS OF ENDOGENOUS REVERSE TRANSCRIPTASE FROM HUMAN LEUKOVIRUS-LIKE PARTICLES TO RNA ISOLATED FROM RNA TUMOR VIRUSES SSV, simian sarcoma virus; MuSV, murine sarcoma virus; MuLV, murine leukemia virus, N.T., Not tested. Source of viral RNA

Source of Ha-DNA MET*, ~ LAI*, ~

DEW*, ~

SSV (NC/37)

SSV (NRK) SSV (NC/37) MuSV (Kirsten) MuLV (Moloney) MuLV (Gross)

43 33 16 11 N.T.

N.T. 40 17 N.T. 12

N.T. 52 N.T. N.T. N.T.

* AML patients

47 37 12 10 N.T.

293 The origin and continuing function of such information is highly controversial. The "oncogene theory" proposed by Huebner and Todaro [125] postulates that genetic material for the synthesis of the viral components is present in germ cells and transmitted vertically. The information for the production of tumor viruses is coded as a "virogene"; a region of this gene, the "oncogene", provides oncogenicity. Normally, the virogene, analogous to an operon, is repressed, but may become derepressed by physical or chemical means with the production of infectious and transforming virus. In contrast, Temin has used studies of avian RNA-containing virus to propose a "protovirus theory" [126]; this states that a normal process of RNA to DNA to RNA information transfer is present in cells and is operative in development. This process might include the passage of information from cell to cell as part of a mechanism for cellular communication. Such a function has been postulated in some theories for the regulation of the immune response [126,127]. If normal function required the "packaging" of information in the form of RNA for intracellular or intercellular communication, mutation or some other mechanism might lead to the development of a particle capable of acting as a transforming leukovirus. The two hypotheses resemble each other in that both postulate the existence of potentially oncogenic material in normal cells; they differ in that the oncogene theory postulates fully competent oncogenic material present in germ cells and represented in all somatic cells, while the "protovirus theory" postulates the repeated evolution of leukoviruses by mutations in genetic regions that normally serve regulatory functions. Major experimental support for the universal presence of potentially oncogenic material within animal genomes derives from the observations of Lowy et al. [128] and other workers [129,130]; these investigators showed that C-type viruses could be induced by treatment of animal cells with physical and chemical agents. In primates, the evidence is less conclusive; C-type particles have been identified by electronmicroscopy in the placenta of baboons and recovered by co-cultivation with canine thymus cells [131]. These particles contain a reverse transcriptase, but this enzyme is not immunologically identical with the reverse transcriptase of either human or baboon oncogenic leukoviruses (Gallo, R. C. personal communication). In man, only electronmicrographic evidence is available for occasional budding leukovirus-like particles in the placenta [132]. Although far from conclusive, th~ evidence presently available on endogenous C-type viruses is compatible with the view that the packaging of RNA at the membranes of mammalian cells may represent a normal and continuous process; if this hypothesis is correct, the preservation of this process through evolution would certainly favour the existence of a physiological role for such "particle packaged" information. VIII. C O N C L U S I O N

The orderly behaviour of the hemopoietic system reflects a complex series of events, involving cellular proliferation and differentiation, and controlled by intra-

294 cellular and intercellular communication. The independent regulation of the various cellular lineages derived from single pluripotent stem cells is achieved by a mechanism that depends on differentiation; that is, each cellular lineage is headed by committed progenitors, separated from their pluripotent ancestors by one or more differentiation steps [133]. As a result it seems likely that committed progenitors develop specialized surface structures that allow them to respond to the specific regulators of their lineage. On the basis of studies in cell culture, it appears that for granulopoiesis, regulation may be achieved by intercellular communication based on specific components associated with the cell membrane of co-existing stimulator cells that interact with binding sites on committed progenitors. These complex events can be traced to at least two classes of genetic mechanisms One class consists of genes and their products operating intracellularly; some of the genes of the first class are structural, coding for functional molecules such as hemoglobin. Others appear to be regulatory; the W gene may be an example of this type, acting within pluripotent stem cells and required for the expression of their potential for proliferation and differentiation [5]. The second set of genes code for intercellular messengers. The most clearly defined of such genes is SI; this gene regulates an essential interaction between pluripotent stem cells and their environment [20]. It seems likely that proteins mediating intercellular communication are specific gene products; the activation of such genes in differentiation is necessary for the development of regulatory mechanisms acting at the level of committed stem cells. The hemopoietic system operates in two distinct modes. In adult life most pluripotent stem cells are quiescent [17]; renewal of differentiated elements and responsiveness to relatively minor increases in hemopoietic demand are achieved through factors acting at the committed progenitor level, that is, a stable mode. In fetal life or after extensive hemopoietic injury, pluripotent stem cells enter a growth mode, characterized by rapid proliferation [17]. In normal hemopoiesis this growth mode and the stable mode may co-exist and factors determining the relative prominence of each, though yet unidentified, must play a significant role in regulation. A relatively well developed model has been presented for regulation of the stable mode, based on cellular communication between classes of differentiated cells. An equally detailed model for the growth mode is not available. The heterogeneity of cellular populations within individual clones has led to a stochastic hypothesis, in which individual pluripotent stem ceils either renew themselves or choose specific differentiation pathways at random, governed only by definite probabilities [54]. These probabilities must be susceptible to modification, in order to account for environmental influences on the growth mode exemplified by the function of the S1 gene. The demonstrated importance o f organ environmental factors for the growth mode makes it reasonable to postulate that cellular communication plays a role in the regulation of this mode, just as it does in the stable mode. If this hypothesis is correct, the protovirus postulated by Temin [126] provides an attractive mechanism for such communication, based on the transfer of genetic information between

295 cells in the form of RNA transcribed from the genome of one class, passed to cells of a different class in the form of particles, and intergrated into the recipients' genome by reverse transcription. However, it is not essential that genetic information be transferred; regulation of gene expression could suffice, modulated by mediators of cellular communication that do not contain nucleic acid. In leukemia, processes analogous to the normal growth mode predominate and transitions to the stable mode are less common. The existence in leukemic marrow cells of leukovirus-like particles and their increase in culture may represent the effects of leukemic transformation on a normal control mechanism that operates in the growth mode. The various models of normal and leukemic hemopoiesis outlined in this review are advanced as guides to further experimentation. In evolution, the development of mechanisms for cellular communication was a requirement for the emergence of multi-cellular organisms. The elucidation of these mechanisms presents a challenge to biologists. The hemopoietic system and diseases that affect it provide attractive material for meeting this challenge. The attack must be made on many fronts, combining genetic, molecular, virological, cellular and clinical skills. No small part of the challenge is the requirement to build collaborative enterprises combining these skills under conditions that favour individual initiative. The biological importance of the problem and the clinical implications of its solution are perhaps of sufficient magnitude to provide the motivation for such enterprises.

ACKNOWLEDGEMENTS This review was supported by grants from the Medical Research Council of Canada, the National Cancer Institute of Canada, and the Ontario Cancer Treatment and Research Foundation. Reproduction of Fig. 2 (Becker, A. J., McCulloch, E. A., Siminovitch, L. and Till, J. E. (1965). The effect of differing demands for blood cell production on DNA synthesis by hemopoietic colony-forming cells of mice. Blood 26, 296-308), Fig. 6 (Messner, H. A., Till, J. E. and McCulloch, E. A. (1973) Interacting cell populations affecting granulopoietic colony-formation by normal and leukemic human marrow cells. Blood 42, 701-710), Fig. 9 (Messner, H. A., Till, J. E. and McCulloch, E. A. (I 974) Blood, in press), is by permission of Grune and Stratton, Inc., New York, N.Y. Reproduction of Fig. 3 (Worton, R. G., McCulloch, E. A. and Till, J. E. (1969). J. Cell. Physiol. 74, 171-182) and Table III (Gregory, C. J., McCulloch, E. A. and Till, J. E. (1973). J. Cell. Physiol. 81,411420) is by permission of the Wistar Institute Press, Philadelphia, Penn. Reproduction of Fig. 5 (Wu, A. M., Siminovitch, L., Till., J. E. and McCulloch, E. A. (1968). Proc. Natl. Acad. Sci. U.S. 59, 1209-1215) is by permission of the Nat. Acad. Sci. Washington, D.C., U.S.A.

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ERRATUM B I O C H I M I C A ET B1OPHYSICA ACTA, Vol. 355 (1974) The publishers wish to apologize for a printing error in a recent Cancer Review: The Immune Response to Vitally Determined Tumor Associated Antigens, by E. W. Lamon. Lines 1-4 on p. 166 were transposed and should occur at the bottom of this page, following 'Though this phenomenon . . .'.