Avian leukemia viruses interaction with their target cells in vivo and in vitro

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Biochimica etBiophysicaActa, 516 (1978) 2 6 9 - 2 9 9 © Elsevier/North-Holland Biomedical Press

BBA 87051

A V I A N L E U K E M I A V I R U S E S

I N T E R A C T I O N W I T H T H E I R T A R G E T C E L L S IN V I V O A N D IN V I T R O

THOMAS GRAF * and HAR T M UT BEUG *

Max-Planck-Institut ffir Virusforschung, Biologisch-Medizinische A bteilung, D- 7400 Tfibingen (G.F.R.)

(Received March 7th, 1978)

C o ~ e ~ s

I. In t roduct ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270 II. The nondefect ive leukemia viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270

A. Frequency in nature, origin, and oncogenic spec t rum . . . . . . . . . . . . . . . . . . . . 271 1. The lymphat ic leukemia viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 2. The osteopetrosis viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271

B. In vitro effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272 C. Target cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272 D. Genetic properties o f nondefect ive leukemia viruses . . . . . . . . . . . . . . . . . . . . . 273

III. The defective leukemia viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 A. Frequency in nature , origin, and oncogenic spec t rum . . . . . . . . . . . . . . . . . . . . 273

1. AEV-type strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 2. AMV-type strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 3. MC29-type strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275

B. Transformat ion in vitro and target cell specificity . . . . . . . . . . . . . . . . . . . . . . 276 1. Multiple t ransforming potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276

(a) Transformat ion o f hematopoie t ic cells . . . . . . . . . . . . . . . . . . . . . . . . . 276 (b) Transformat ion o f fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278 (c) Transformat ion o f epitheloid cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 278

2. Lineage specific t ransformat ion of hematopoie t ic cells . . . . . . . . . . . . . . . . . . 279 (a) Differentiation parameters o f the t ransformed hematopoe t ic cells . . . . . . . . . 280 (b) Characterizat ion o f the hematopoie t ic target cells . . . . . . . . . . . . . . . . . . 282

3. Influence o f helper virus on lineage specific t ransformat ion by defective leukemia viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283

4. Are t ransformed hematopoie t ic cells blocked in differentiat ion? . . . . . . . . . . . . 284 C. Genetic properties o f defective leukemia viruses . . . . . . . . . . . . . . . . . . . . . . . 286

IV. Role o f hos t mechanisms in the expression o f leukemia virus-induced neoplasms . . . . . . 286 A. Genetic susceptibili ty o f the hos t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 B. Other factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287

V. Discussion, speculat ions and models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 A. What distinguishes defective avian leukemia viruses f rom nondefect ive leukemia and

sarcoma viruses? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 B. How do leukemia viruses originate? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288

* Present address: German Cancer Research Center , Neuenheimerfe ld 280, D-6900 Heidelberg (G.F.R.) .

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C. How do leukemia viruses induce a neoplastic transformation? . . . . . . . . . . . . . . . 290 1. Cellular aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290 2. Viral aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291

D. What is the mechanism of target cell specificity? . . . . . . . . . . . . . . . . . . . . . . . 294 E. Tools or toys? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2~5

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295

I. Introduction

Avian leukemia viruses comprise a large number of strains isolated from field cases of the domestic chicken. They are commonly transmitted by congenital infection and by horizontal transmission [ 1,2] and cause a variety of hematopoietic and nonhematopoietic neoplasms in chickens and other avian species (for older reviews on the pathology see refs. 3-6) . The most comprehensive review of the literature prior to 1969 is that of Vogel et al. [6], in German. Structurally, these viruses are closely related to the avian sarcoma viruses in that they have the morphology of C-type viruses, possess a single stranded RNA genome and contain a reverse transcriptase which directs the synthesis of a DNA inter- mediate during viral replication. They can therefore be assigned to the oncovirus division of the retrovirus family [9]. Their mode of replication and their genetics have recently been discussed in authoritative reviews [7,8].

The aim of this article is to provide the reader with an up to date view of the field of avian leukemia viruses, emphasizing the origin of these viruses, their oncogenic spectrum, their in vitro target cell specificity and some aspects of their genetic properties. This will be done mostly on the basis of recent work but relevant literature not appropriately covered in previous reviews will also discussed.

Operationally, avian leukemia viruses can be subdivided into two groups: '(1) The nondefective leukemia viruses which predominantly induce lymphatic leuke-

mias after long periods of latency and (2) The defective leukemia viruses which predominantly cause acute erythroid and

myeloid leukemias after short periods of latency. In order to be able to present a comprehensible picture of the variety of strains from

each of these classes, smaller groups of strains with similar properties will be discussed separately.

II. The nondefeetive leukemia viruses

Viruses of this category are competent for replication and have earlier been referred to as "slowly" or "weakly" transforming leukemia viruses [10], "lymphatic leukemia viruses" [ 11 ], "lymphoid leukosis viruses" [12], visceral lymphomatosis viruses [3 ] or simply as "leukosis viruses" [7]. They can be classified into seven subgroups, A-G, as defined by their viral envelope antigen. Subgroups A - E consist of viruses isolated from the chicken, subgroups F and G from pheasants [8]. They frequently occur as independent oncogenic agents but are also associated with replication defective strains of leukemia or sarcoma viruses for which they function as helper viruses. During the replication of nondefective strains of avian sarcoma viruses, deletion mutants which have lost their ability to transform fibroblasts and to induce sarcomas are spontaneously generated at high frequencies. These so called "nont~ansforming mutants" or "transformation defective mutants" are, however, still oncogenic and elicit an oncogenic potential similar to naturally occurring nondefective leukemia viruses [13,14].

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IIA. Frequency in nature, origin, and oncogenic spectrum In the following, strains predominantly causing lymphatic leukemia will be discussed

separately from those mainly inducing osteopetrosis and nephroblastomas. 1. The lymphatic leukemia viruses (LL V]. Viruses of this category are the most ubiqui-

tous members of avian leukemia viruses. The total incidence of spontaneous leukemia, which occurs most frequently in the form of a lymphoproliferative disease of visceral organs, varies between about 2% and 20% in chicken flocks not selected for resistance to and lack of avian leukemia viruses [5,6]. Most of these naturally occurring neoplasms contain nondefective leukemia viruses and are transmissible by cell free extracts, causing predominantly lymphatic leukemia but also osteopetrosis, nephroblastomas, erythroblastosis and occasionally sarcomas and endotheliomas [15]. The broad oncogenic spectrum observed could be explained by the fact that field isolates often consist of virus mixtures. Studies with clone-purified LLV-strains, however, indicate that cloned strains can still cause a variety of neoplasms in addition to lymphatic leukemia, such as osteopetrosis, nephroblastoma and erythroblastosis [16,13,17,14]. It is not clear from the literature whether or not there are strains which induce lymphatic leukemia only.

The earliest neoplastic changes induced by LLVs are observed in the bursa, 4 to 8 weeks after infection of newly hatched chicks. The infected birds generally start to die after 10 to 15 weeks with a peak of mortality at about 20 weeks [18]. The incidence of lymphatic leukemia can be as high as 80% in susceptible flocks inoculated with certain virus strains [ 15,17].

2. The osteopetrosis viruses. The discussion of osteopetrosis viruses as a separate entity is justified by the isolation of strains which predominantly induce osteopetrosis. It serves the main purpose of stressing their importance as an experimental model system. Most, if not all, of the osteopetrosis strains described were isolated from stocks of avian sarcoma viruses or defective leukemia viruses. The MAV-2 strain, isolated from stocks of avian myeloblastosis virus (strain BAI-A), induces an overt expression of osteopetrosis as early as 3 weeks after inoculation of chicken embryos, with an overall incidence of up to 100% [16,19]. The high incidence of disease, combined with its early Onset, probably prevents the observation of other neoplasms since chicks with osteopetrosis die before sufficient time has elapsed for the development of lymphatic leukemia, erythroblastosis or nephroblastoma. Osteopetrosis viruses cloned by plaque-purification in chicken fibroblast cultures (see next section) still induce osteopetrosis [20,21 ].

It is not clear whether avian osteopetrosis represents a truly malignant growth; many investigators consider that the lesions observed are hyperplastic rather than neoplastic. Nevertheless, avian osteopetrosis is a rapidly progressive disorder and, particularly because of the nondefectiveness of the causative agent, represents an excellent model system for the study of virus-induced diseases of the bone.

Fourcade and Ogura and their colleagues described the isolation of nondefective leukemia viruses causing a high incidence of nephroblastomas in the inoculated chicks [22,23]. Both strains isolated by these authors were derived from stocks of the BAI-A strain of avian myeloblastosis virus. Ogura's strain, when tested in another chicken flock, induced mainly osteopetrosis (Smith, R., personal communication), suggesting that it is similar, if not identical, to the osteopetrosis-inducing MAV-2 strain mentioned above. This observation suggests that host factors may play an important role in determining the oncogenic spectrum of a given nondefective leukemia virus strain. Other data, however, indicate that the tumor specificity is primarily determined by the virus; osteopetrosis viruses induce mainly osteopetrosis even in chicken flocks which are highly susceptible to lymphatic leukemia [20,16,18].

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liB. In vitro effects

There are no reports of a successful in vitro transformation with nondefective leukemia viruses. Several strains were, however, described to induce morphological changes in chicken embryo fibroblast cultures and to increase their life span. These cells were not tumorigenic and did not develop colonies in soft agar [24,25].

Viruses of subgroups B, D and F as well as some subgroup A strains cause cytopathic alterations and plaques in chicken embryo fibroblast cultures, effects which are not observed with subgroups C, E and most subgroup A viruses [26-29]. Pictures of plaques induced by nondefective leukemia viruses are shown in Fig. 1.

IIC. Target cells Despite the lack of in vitro transformation assays, the major hematopoietic target cell

of LLVs has been identified as an immature B-type lymphocyte residing in the bursa of Fabricius. Bursectomized chicks were no longer susceptible to virus-induced lymphatic leukemia [30-32,17]. These chicks could be made susceptible again by reimplantation of bursa cells [17,33]. It is interesting that in the bursectomized animals an increase in the incidence of osteopetrosis was observed whereas the incidence of erythroblastosis remained unchanged [30,31,17]. This suggests that there is an immunological control of osteopetrosis which is mediated by B-lymphocytes and further supports the notion that LLVs interact with target cells other than lymphoid cells. The finding of IgM but not of IgG or IgA on the surface of LLV-induced lymphatic leukemia cells suggests that the virus infection causes an arrest of the lymphoid target cells at a stage in development prior to the switch from IgM- to IgG- or IgA-producing cells [34].

Nothing is known about the target cells for osteopetrosis viruses. Attempts to establish an in vitro transformation system have so far been unsuccessful (Smith, R.E., personal communication). Neither has it been possible to transform hematopoietic cell cultures with several nondefective leukemia virus strains (Graf, T., unpublished observations).

Fig. 1. Plaques induced in CEC cultures by MCAV-B, a nondefective avian leukemia virus. A, culture 17 days after infection, stained with neutral red. B, mierograph of a single plaque.

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liD. Genetic properties o f nondefective leukemia viruses Since this area has been extensively covered in previous reviews [8,7], only some basic

facts about the genome organization of LLVs will be summarized here. All nondefective leukemia viruses seem to be closely related and resemble avian sarcoma viruses in their overall structure and in the organization of their genome. They contain a genomic RNA which in its monomeric form has a molecular weight of about 2.5 • 106 and which encodes 3 structural genes. These read, from the 5' to the 3' end, gag-pol-env. The gag gene codes for the viral core proteins p19, p12, p27 and p15. The pol gene codes for the reverse transcriptase and the env gene for the viral envelope glycoproteins. In addition, there is a sequence termed "c" (for constant) at the 3' end of the RNA which is present in all strains of nondefective oncoviruses and whose function is unknown. The main difference between nondefective leukemia viruses and avian sarcoma viruses is that only the latter contain the src gene, required for the initiation and maintenance of fibroblast transformation [11,8,7].

Nondefective leukemia viruses are also closely related to RAV-0, the only known type of endogenous chicken virus. However, as determined by competition nucleic acid hybrid- ization, about 20% of their genome is lacking in RAV-0. On the other hand, about 13% of the RAV-0 genome is unique [35,36]. At least part of these unshared sequences reside in the env region of the RNA [37]. Recent studies by Coffin and coworkers suggest that there is another major difference between the genome of RAV-0 and other nondefective viruses: the "c" region of the RAV-0 RNA is significantly shorter or perhaps even absent [37]. Finally, it is interesting to note that differences exist at the 3' end of the RNA between viruses which have different oncogenic spectra, such as the MAV-2 osteopetrosis virus and a field isolate of LLV [36].

III. The defective leukemia viruses

Viruses of this category are defective for replication and have also been called "acute" [11], "strongly or rapidly transforming" leukosis or leukemia viruses [10] or simply "leukemia" viruses [7]. Of the many isolates described, eight strains of defective leukemia viruses which are under intense investigation will be reviewed in more detail. These viruses are listed in Table I. For reasons which will become apparent during the course of further discussion they have been subdivided into three classes named after three typical representatives: avian erythroblastosis virus (AEV) strain ES4, avian myeloblastosis virus (AMV) strain BAI-A and avian myelocytomatosis virus strain MC29. As we now know, all of these strains consist of mixtures of the defective leukemia virus and one or more nondefective leukemia viruses acting as "helpers". In describing their oncogenic spectrum we have omitted all neoplasms which are likely to be caused by the helper viruses.

IliA. Frequency in nature, origin and oncogenic spectrum 1. AEV-type strains. There are at least 10 reports of independent isolations of viruses

which share some of the properties characteristic for the erythroblastosis-type strains [38-47]. All of these viruses were described to induce an acute erythroleukemia which kills the animals within a few weeks after inoculation. Interestingly, most of the strains described also induced sarcomas at the site of injection. In one case, a transition from a so called "pure" strain causing erythroblastosis only (strain 1) to a "complex" strain also causing sarcomas (strain 13) has been described [43]. Both the original strain and the variant maintained their specific properties over s u b s e ~ t passages during a period of

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TABLE I

DEFECTIVE LEUKEMIA VIRUS STRAINS

Virus strain a Type of neoplasm in chicken of origin

Predominant type of Country neoplasms induced, of origin attributed to the defective virus component

Year of first description

AEV-ES4 Sarcoma and myelo- Erythroblastosis, sarcomas Denmark 1933 cytomatosis

A E V - R Erythroblastosis Erythroblastosis (sarcomas) Denmark 1931

AMV-BAI/A Neurolymphomatosis Myeloblastosis U.S.A. 1941 E26 Not known to author "Erythroblastosis" Bulgaria 1962

of this review

M C 2 9 Myelocytomatosis Myelocytomatosis, liver Bulgaria 1964 and kidney carcinomas

C M I I Myelocytomatosis Myelocytomatosis G.F.R. 1964 MH2 Endothelioma Liver and kidney England 1927

carcinomas, sarcomas, monocytic leukemia (?)

OK10 Endothelioma (?) Endotheliomas (?) Finland 1975

a The prototype strain of each group is italicized.

several years. Attempts to separate strain 13 into sarcomagenic and leukemogenic com- ponents failed [43]. Similarly, it was not possible to isolate "pure" substrains of the ES4

,strain causing erythroblastosis or sarcomas only [48]. Unfortunately, most of the AEV- type strains described seem to have been lost with the exception of strains ES4 and R from Engelbreth-Holm's laboratory.

A E V strain ES4. This strain was derived from an Italian hen with a spontaneous sarcoma and several small myelocytomas [42]. In other animals the blood of this bird caused erythroblastosis and anemia when injected intravenously, and predominantly sarcomas when. injected intramuscularly [42]. Myelocytomas were never observed in later passages. Interestingly, a decrease in the period of latency with increasing number of passages was observed (from 2 months down to about 2 weeks). In recent studies the anemia-inducing potential of strain ES4 was found to be due to the associated helper virus [49]. This ruled out the possibility, suggested by Ellermann [50] and by Furth [51], that the anemia represents an early stage of AEV-ES4.induced erythroleukemia, leading to a regenerative proliferation of erythroblasts only at later stages.

A E V strain R. This virus was isolated from a Plymouth rock hen with erythroleuke- ,mia, anemia, and increased numbers of circulating myelocytes [40]. It could be effec- tively transmitted in chicks, regularly causing erythroblastosis and anemia. No increase in the number of circulating myelocytes was seen after the first passage. Thus it seems possible that the myelocytes observed in the original animal were due to an inflammatory response (either to the neoplastic cells or to another infection). As in the case of the ES4 strain, the virus from the first passage had a latency period exceeding several months which decreased to as little as 10 days after two further passages. Concomitantly, there was an increase in the proportion of takes from 15% to 100% (in the 7th passage). In the early reports by Engelbreth-Holm [40] this strain never caused sarcomas. It did, however,

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in later studies [44,52,49], suggesting that a transition had occurred similar to the one described by Furth between strain 1 and strain 13 [43]. It would be interesting to know whether or not stocks derived from early passages still exist which are unable to induce sarcomas. An alternative possbility is that AEV-R was exchanged by mistake with the ES4 strain at some time in its early history, since both strains are now virtually indistinguishable in their biological properties. As in the case of the ES4 strain, the anemia inducing potential was recently identified as a property of the associated nondefective helper virus [49].

2. AMF-type strains. At least 4 independent isolates of chicken viruses have been described which cause an acute myeloblastosis leading to death of the infected birds within a few weeks after infection [38,53-55].

AMF-strain BAI A. This strain, commonly designated as standard AMV or simply AMV, is the best described. Its origin, pathology and biological properties have been reviewed by Beard [3] and recently by Moscovici [56]. It was first isolated from a chicken with neurolymphomatosis and other symptoms of Marek's disease [55]. In subsequent passages it induced "erythroblastosis" at a high incidence and with decreasing periods of latency [55]. In the late 1940s, Beard's group introduced the name "erythro- myeloblastosis virus" for the virus and finally adopted the term "myeloblastosis virus" following a proposal made by B. Burmester in 1953 (discussed in ref. 57). These uncer- tainties in the diagnosis of the leukemia induced by AMV during its early history are understandable in view of the difficulties in classifying immature hematopoietic cells on the basis of their morphology in stained smear preparations alone. The study of other parameters of differentiation has only recently allowed the definite assignment of AMV- transformed cells to the myeloid lineage of differentiation (see Section IIIB. 2b).

The E26 strain. This strain was isolated in Bulgaria [58] and causes an acute "erythroleukemia" within 2 to 4 weeks after injection into newborn chick, turkeys or quail [59]. Below, recent evidence will be presented which indicates that the leukemia caused is a myeloblastosis rather than an erythroblastosis as had been previously assumed. Inter- estingly, it also causes myelocytomatosis when inoculated into guinea fowl-poults (quoted in ref. 59).

3. MC29-type strains. At least 4 cases of spontaneous, transmissible myelocytomatosis of the chicken have been reported. From them, several strains were derived: strain 2 [60], strains MC29 and MC31 [61], and the CMII strain [62]. Since little is known about the MC31 strain and the strain 2 is no longer available, these viruses will not be discussed further, except to say that the latter has also been reported to induce endotheliomas [60]. Instead, the MH2 and OK10 strains, although not primarily associated with myelocytomatosis, will be described in more detail in addition to MC29 and CMII viruses. The reasons for including MH2 and OK10 in the MC29 group will become apparent after their transforming specificities in vivo and in vitro have been outlined during the course of this review. Chickens inoculated with virus strains of the MC29 group die only after latency periods of 5 weeks to several months.

The MC29 strain. This virus was isolated in Bulgaria by the discoverers of the E26 virus from a hen with spontaneous myelocytomatosis [61]. The spectrum of neoplasms it induces is very complex. It not only induces myelocytomatosis but also liver and kidney carcinomas, and tumors described as mesotheliomas and endotheliomas [63-67]. The pathology of this strain has recently been reassessed and it was found that it induces hepatocarcinomas, adenocarcinomas and "soft tissue" sarcomas in addition to myelocytomas [68].

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The CMII strain. This virus was isolated in Germany from a hen with a spontaneous myelocytomatosis [62]. After injection into newly hatched chicks, it causes myelocytomatosis in about 10% of the animals within 4 to 12 weeks. The tumors are frequently located in the periosteum of the body skeleton; infiltrates are also observed in the liver, kidney and intestinal tract [69].

The MH2 strain. This strain was originally isolated by Begg from a hen with a large tumor in the ovarian region, described as an endothelioma [70]. In subsequent studies it was found to induce endotheliomas [71] and more recently also kidney carcinomas [72,68], liver carcinomas and "soft tissue sarcomas" [68]. Moscovici et al. [68] suggested that the tumors diagnosed as endotheliomas by the early investigators correspond to the carcinomas detected in recent studies. With one particular substrain of MH2 a high incidence of leukemia of probably monocytic type was also observed [68].

The OKIO strain. This strain was isolated from a liver tumor of a chicken which had been inoculated with a field isolate of "leukosis virus", and which probably represented a nondefective leukemia virus [72]. It now regularly causes tumors in the mesenterium of the gut, liver, and testes, which have been tentatively classified as endotheliomas but which may also turn out to be carcinomas [72]. There are no reports describing its possible potential to induce myelocytomatosis.

Table I summarizes the data on the origin and tumor spectrum of the eight defective leukemia virus strains described in this section.

IIIB. Transformation in vitro and target cell specificity The successful establishment of in vitro transformation assays for defective leukemia

viruses made it possible to study a number of questions, the investigation of which had not been possible using in vivo systems. The following questions will be discussed here: (1) Can the spectrum of neoplasms induced by some strains of defective leukemia viruses be explained by the assumption that they are pure strains, capable of transforming more than one target cell, or do these virus isolates represent mixtures which differ in their target cell specificity? (2) Is the specificity of tumorigenesis by defective leukemia viruses a reflection of their target cell specificity? (3) What is the nature of the target cells and how are they affected by the process of transformation? (4) Do nondefective helper leukemia viruses exert an influence on the transformation target cell specificity of defective leukemia viruses? And (5), do defective leukemia viruses induce a block of differentiation in their hematopoietic target cells?

1. Multiple transforming potential. The basis for the multiple oncogenic potential of certain strains of defective leukemia viruses has been a matter of controversy until recent years and could only be settled with the development of in vitro transformation assays, thus making it possible to work with clone-purified virus. It is now clear that clone- purified strains of AEV-ES4 and AEV-R are capable of inducing both erythroblastosis and sarcomas, regardless of the helper virus used [49,73]. Likewise, it has been possible to demonstrate that these virsuses [49] as well as clone-purified stocks of MC29 [74] and CMII [75] are capable of inducing a transformation of cloned chicken embryo fibroblasts in vitro in addition to hematopoietic cell cultures. This showed that certain defective leukemia virus strains are capable of transforming different types of target cells rather than being virus mixtures with different target cell specificities. In the following, the in vitro transformation of various types of target tissues with defective leukemia viruses will be discussed separately.

(a) Transformation of hematopoietic cells. The first successful transformation of

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hematopoietic cultures with a leukemia virus was reported for AMV in bone marrow cells [76] and was extended by Baluda a n d Goetz [77] and Moscovici [78,79] to other hematopoietic tissues. Transformation assays in hematopoietic cells were subsequently developed also for the following strains: MC29 [80,74], AEV strains ES4 and R [81,49], CMII [75], MH2 [82], and for OK10 and E26 viruses (Graf et al., in preparation). Two types of transformation assay systems have been developed: a tissue culture or focus assay [79,81,75] and a colony assay with cells seeded in semisolid media [74,75,91]. The finding that the number of transformed foci or colonies is directly proportional to the virus dilution indicates that a single particle is sufficient to induce transformation. These assay techniques are therefore not only useful for the isolation of clones of transformed hematopoietic cells but also for the quantitation of the transforming activity in a given virus stock. Photographs of foci and colonies of transformed cells are shown in Fig. 2.

Fig. 2. Transformation of bone marrow cell cultures by defective leukemia viruses. A, culture of uninfected bone marrow cells 5 days after seeding, predominantly showing normal macrophages; B, focus induced by MC29 virus, 6 days after infection; C and D, colonies of transformed cells induced by AEV-ES4 in a methylcellulose-containing culture (35 mm dish), 14 days after infection. Bars represent 100 ~m.

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At this point it is important to mention that in these in vitro studies the only criterion used to define leukemic "transformation" is the induction of proliferation of certain types of hematopoietic cells. Individual clones of hematopoietic cells transformed by AEV- or MC29-type viruses have a life span of 20 to 40 generations. By comparison, hematopoietic cultures infected by avian sarcoma viruses or nondefective leukemia viruses do not divide to a significant extent (ref. 113, and Graf, T., unpublished results).

(b} Transformation offibroblasts. The most widely used tissue culture system of the chicken are cells derived from 10 to 11 day old decapitated embryos. These chicken embryo cell (CEC) cultures mainly consist of cells with fibroblastoid morphology and have, therefore, frequently been designated also as "chicken embryo fibroblast" (CEF) cultures. In vitro transformation of such "CEF" cultures by leukemia viruses was first reported for AMV [83] but later studies with clonal strains of fibroblasts showed that this virus probably only transforms hematopoietic target cells which are present in CEC cultures even after several passages [74]. Two further strains were since described to transform CEC cultures: the MC29 strain [84] and the R-strain of AEV [52]. Studies with clonal strains of chicken fibroblasts [85] demonstrated that these viruses as well as MC29 [74,86], AEV-R and ES4 [49,86] and CMII [75] are indeed able to transform fibroblasts. In addition to the strains mentioned, MH2 [87,82] and OK10 [25] have been reported to transform chicken embryo fibroblast cultures. Attempts to transform fibroblasts with the E26 strain have so far been unsuccessful (Graf, T., unpublished observations). It is remarkable that the morphological transformation of fibroblasts induced by the AEV-type strains is strikingly different from that of flbroblasts transformed by MC29.type viruses; and that fibroblasts transformed by different members within the AEV- and MC29-groups, respectively, are virtually indistinguishable. Micrographs of fibroblasts transformed by defective leukemia viruses are shown in Fig. 3.

(c) Transformation of epitheloid cells. A morphological transformation of chick ovary cells by AMV has been described in an early report [88] but these cells were not further characterized. In another study, MC29 and AMV were found to induce a morphological alteration and growth stimulation in chicken embryo kidney cells [89]. However, while the cells transformed with MC29 had an epitheloid morphology, AMV-transformed cells had a more "fibroblastic" appearance. We have also observed a focal outgrowth of cells with epitheloid morphology after infection of embryonic kidney and liver cell cultures with MC29 and with the CMII and OKIO strains. (Graf, T., unpublished results). These cells are larger than the uninfected control cells, display large nucleoli and survive for longer times under normal culture conditions. Photographs of such epitheloid cells transformed by MC29 in vitro and in vivo are shown in Fig. 4.

The ability of MC29 to transform epitheloid cells in culture probably reflects its capacity to induce carcinomas. In addition, the in vitro results with OK10 hint at the possbility that the "endotheliomas" induced by this virus represent carcinomas. In the light of these findings it may also be worthwhile to reexamine the pathology of the CMII strain, in order to see whether or not there are conditions under which this virus might also be capable of inducing carcinomas.

It has to be stressed that the studies reported so far, including our own observations, are very preliminary and that questions such as the role of helper virus in determining the capacity of defective leukemia viruses to transform epitheloid cells have not yet been studied. Furthermore, criteria other than morphology are required to unambiguously distinguish epithelial cells from other types of cells.

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Fig. 3. Transformation of cloned chicken embryo fibroblast cultures by defective leukemia viruses; A, uninfected cells; B, cells transformed by AEV-ES4 virus; C, by MC29; D, by CMII. Bar represents 50 p,m.

The ability of the various defective leukemia virus strains to transform chicken hematopoietic ceils, fibroblasts and epitheloid cells in culture is summarized in Table II.

2. Lineage specific transformation of hematopoietic cells. On the basis of the current concept of normal mammalian hematopoiesis [90] it is assumed that also in the chicken all types of hematopoietic cells arise from a pluripotent stem cell in a series of matura- tion steps. This is illustrated in a highly simplified diagram showing the lymphoid, erythroid and myeloid lineages of differentiation (Fig. 7). The myeloid lineage is defined here as the lineage leading to the formation of both granulocytes and macrophages (monocytes).

That different strains of defective leukemia viruses regularly induce different types of leukemias could be explained by assuming that they selectively transform certain types of target cells within different lineages of hematopoietic differentiation. In favour of this hypothesis is the observation that chick bone marrow contains 10 to 20 times more target cells for MC29 and AMV-viruses (approximately one in 103 cells) than for AEV- type viruses [74,75,91], and that the presence of 1% dimethylsulfoxide is advantageous for the AEV-assay [75] while it is inhibitory for the assay of MC29 and AMV [75,79]. As will be discussed below, the above hypothesis is further supported by studies on the expression of differentiation markers in the transformed cells and by the separation and

280

Fig. 4. Transformation of chicken liver cultures by MC29 virus. Phase mierographs of A, uninfected culture from an 11 day old embryo, 2 days after seeding; B, focus of transformed cells 8 days after infection of embryonic liver ceils. At this time, all normal epitheloid islands in the urtinfeeted cultures were overgrown by fibroblasts; C, focus of transformed epitheloid ceils grown from a liver tumor in an adult hen induced by MC29. Bar represents 50/~m.

characterization of the target cells for different virus strains prior to infection. (a) Differentiation parameters o f the transformed hematopoietic cells. Earlier studies

on virus-induced leukemia cells were hampered by the lack of appropriate criteria for their assignment into a particular lineage of differentiation. In our laboratory, we have developed assays for the erythroid markers hemoglobin [91 ] chicken erythrocyte specific histone H5 [92,91] and chicken erythrocyte specific cell surface antigen(s) (Fig. 5, ref. 91 and Beug et al., in preparation) using indirect immunofluorescence techniques. The proport ion of hemoglobin-positive cells was also tested by benzidine staining (ref. 91 and Beug et al., in preparation). Myeloid markers tested were adherence and phagocytic capacity [92,91] Fc receptors as determined by rosette formation (Meyer-Glauner and

TABLE II

IN VITRO TRANSFORMATION OF VARIOUS TISSUES BY DEFECTIVE LEUKEMIA VIRUSES

Virus strain Hematopoietic ceils Fibroblasts Epitheloid cells (hypothetical target ceils)

AEV-ES4 Erythroblast-like cells Yes ? AEV-R Erythroblast-like cells Yes ?

AMV Immature myeloid ceils No E26 Immature myeloid ceils No

MC29 Macrophage-like cells Yes CMII Macrophage-like cells Yes MH2 Macrophage-like cells Yes OK10 Macrophage-like ceils Yes

(Yes) 9

Yes

Yes ? Yes

281

O

Fig. 5. Staining of in vitro-transformed bone marrow cells with differentiation specific antibodies in indirect immunofluorescence. A, C, cells transformed by AEV-ES4; B, D, cells transformed by MC29. Cells in the upper row were reacted with rabbit anti-chicken erythrocyte serum. Cells in the lower row with serum prepared similarly against macrophages. Both sera were properly absorbed with various types of chicken cells. Bar represents 20 t~m.

Graf, unpublished; and ref. 93) macrophage/granulocyte specific cell surface antigen as detected by immunofluorescence (Fig. 5, Beug et al., in preparation) and dependence of colony formation on colony stimulating factor (CSF) [114].

The results obtained were as follows: (1) Bone marrow cells transformed by AEV-ES4 (and AEV-R) have the morphology of erythroblasts [81] and are positive for all the erythroid markers mentioned above. The expression of some of these markers, however, varies with different clones and is, as a rule, far below the levels expressed in mature erythrocytes (refs. 92, 91 ; Beug et al., in preparation). (2) Bone marrow cells transformed by MC29, CMII and OK10 (ref. 75; and Graf et al., in preparation) are much larger and resemble proliferating macrophages. They express all the myeloid differentiation markers tested but are only slightly adherent under conditions of active proliferation (refs. 92, 75, and Beug et al., in preparation). (3) Bone marrow cells transformed by AMV resemble myeloblasts and express myeloid markers, although in most cases to a lower degree than cells transformed by MC29-type viruses (refs. 91, 93; and Beug et al., in preparation). In contrast to the latter, they are also positive for ATPase activity (ref. 150, and Graf, unpublished results; Langlois, AJ. , personal communication). In view of the description of E26 as an erythroblastosis virus, it was surprising to find that E26-transformed bone

282

marrow ceils behaved like myeloblasts rather than erythroblasts in all parameters tested (Beug et al., in preparation). The fact that this holds true also for leukemia ceils induced by E26 warrants its reclassification as a myeloblastosis virus.

(4) Cell transformed in rive by AEV-ES4 and AMV, when brought into culture, were indistinguishable from the corresponding in vitro transformed cells in all parameters tested, including morphology and staining properties with Wright-Giemsa (refs. 81, 49 and Beug et al., in preparation). In addition to the above similarities, the comparable rapidity with which the in vitro and in vivo transformation processes occur (the earliest AEV-transformed cells in culture or in the peripheral blood can be seen 3 and 5 days after infection, respectively [81,73]) strongly suggests that the former are the in vitro corre- lates o f the leukemia cells induced in the animal. A direct demonstration of the leukemo- genicity of the in vitro transformed cells has not been possible, mainly due to the problem of histoincompatibility with animals from standard noninbred chicken flocks. As judged by their appearance after Wright-Giemsa staining, bone marrow ceils transformed by MC29 in vitro resemble nonadherent macrophages (monocytes) and are clearly different from transformed myelocytes found in rive [80,74]. This finding remains unex- plained so far.

A summary of the results obtained with bone marrow cells transformed by AEV-ES4, MC29 and AMV is shown in Table III.

(b) Characterization. o f the hematopoietic target cells. The erythroid and myeloid nature of hematopoietic cells transformed by erythroid and myeloid leukemia viruses, respectively, suggests that these viruses transform specific target cells within the erythroid and myeloid lineages o f differentiation. The possibility existed, however, that they infect a common stem cell which is then induced to differentiate into erythroid or myeloid cells. This was ruled out by demonstrating that the target cells can be separated and that they are committed to the respective lineage of differentiation.

A large proportion of the bone marrow target cells for AEV-ES4 could be selectively

TABLE III

DIFFERENTIATION PARAMETERS IN LEUKEMIA VIRUS-TRANSFORMED HEMATOPOIETIC CELLS

Parameters of differentation

Virus used for transformation

AEV-ES4 MC29 AMV

Erythroidmarkers

Myeloid markers

Hemoglobin + a _ _ Histone H5 + - - Erythrocyte cell surface antigen + b _ _

Adherence capacity c _ + (_) CSF dependence - + (+) Phagocytic capacity - + (+) Fc receptors - + + Macrophage/granulocyte cell - + + surface antigen

a Percentage of positive cells varies with cell clone tested. Hemoglobin synthesis can be induced in all clones by treatment with butyric acid [91 ].

b Varies with cell clone tested. c MC29- and to a lesser extent, AMV-transformed cells, tend to convert into strongly adherent, macro-

phage-like cells.

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eliminated with erythrocyte-surface-specific antiserum in a complement-dependent reac- tion (ref. 91 ; and Beug et al., in preparation). Conversely, target cells for MC29 can be selectively removed from bone marrow by incubation of a cell suspension with iron filings and by pulling out the iron-containing cells with a magnet [92]. In addition, macrophage cultures established from bone marrow, yolk sac, spleen or peripheral blood can be efficiently transformed by MC29 [74,92,91] and AMV [79,91] but not at all by AEV- ES4 or R [92,91]. They are also transformable by CMII [75], MH2 [82,68] and by OK10 and E26 viruses (Graf et al., in preparation). Preliminary observations showed that macrophage cultures passaged several times were still susceptible to transformation by MC29 but that they were no longer transformable by AMV (Royer-Pokora and Graf, unpublished), suggesting that these viruses transform different target cells within the population of cells in the macrophage cultures. In addition, the target cells for AMV, MC29 and CMII could be separated on the basis of size from the bulk of the normal granulocyte/macrophage colony forming cells (Royer-Pokora and Graf, in preparation; and ref. 75).

In co ,unct ion with the finding that AMV-transformed cells seem to represent myeloid cells at an early differentiation stage, whereas cells transformed by MC29 resemble mature macrophages, these observations suggest that the target cells for AMV and MC29 represent myeloid cells at early and late stages of differentiation, respectively. This interpretation is at variance with that of Gazzolo et al. [93] who suggested that AMV is capable of transforming immature (myeloblasts) and mature myetoid cells (macrophages) thereby inducing a limited dedifferentiation in the latter. According to our view, however, infection of macrophages with AMV leads to the transformation of a small proportion of immature target cells present in the cultures. This apparent conflict might be resolved by the assumption that uninfected macrophages are capable of undergoing a limited dedifferentiation, thus generating target cells for AMV. Clearly, further work is needed to distinguish between these alternatives.

3. Influence o f helper virus on lineage specific transformation by defective leukemia viruses. Recent studies by Gazzolo et al. indicate that macrophages can be transformed by AMV only if pseudotypes with helper viruses from subgroups B and C, but not from subgroups A, D, E and F are used [94,95]. This finding was explained by the observation that only helper viruses from subgroups B and C replicate in macrophages, while helper viruses from subgroups A, D, E and F do not, although they replicate in fibroblasts of the same host. However, a few inconsistencies remain: (1) In a study with yolk sac cells infected and transformed with the defective component of AMV only, Moscovici and Zanetti [96] could rescue transforming AMV by superinfection with helper viruses of subgroup D in addition to helper viruses of subgroups B and C. (2) Ishizaki et al. [97] described a substrain of AMV with pure subgroup A specificities. This strain was still fully leukemogenic. (3) Pseudotypes of MC29, CMII and MH2 viruses with subgroup A and D helper viruses can transform macrophage cultures [82,75,68]. While an explanation cannot yet be offered for the apparent discrepancies observed with AMV, the latter findings could be explained by assuming that MC29, CMII and MH2 viruses code for some as yet unidentified envelope determinant which confers to them a host range different from that imposed by the helper virus.

With AEV-ES4 and AEV-R the situation seems to be less complicated: nonproducer fibroblasts or erythroblasts infected with the defective virus only can be rescued by helper viruses of all subgroups tested (A-D). These pseudotypes have envelope properties of viruses from the respective subgroups as tested by their host range in fibroblasts (ref. 49 and Graf, unpublished results).

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4. Are transformed hernatopoietic cells blocked in differentiation? The data discussed so far showed that defective leukemia viruses specifically transform committed cells of the erythroid and myeloid differentiation lineages. The observation that cells transformed by AEV, AMV and possibly MC29 do not express the respective differentiation parameters to an extent comparable to that of terminally differentiated cells, suggests that they are blocked at some stage within the respective lineage of differentiation. Alterna- tively, one can imagine that virus infection causes a limited dedifferentiation of the target cells or that it induces an aberrant pattern of differentiation. In the following, preliminary data relevant to this problem will be discussed.

That defective leukemia viruses may induce a dedifferentiation is suggested by the finding that embryonic antigens are expressed in the corresponding transformed hematopoietic cells [98]. However, as long as pure populations of target cells have not been tested, these data can also be interpreted by the assumption that the target cells already express these antigens before infection.

The concept of a differentiation block induced by transformation is supported by the observation that erythroblasts transformed by AEV-ES4 could be induced to synthesize increased amounts of hemoglobin after treatment with butyric acid [91]. That hematopoietic cells transformed by MC29- and to a lesser extent by AMV-type viruses revert spontaneously into adherent, macrophage-like cells (ref. 75, and Graf, unpublished results) may reflect a similar release from a differentiation block. The precise conditions favouring the latter phenomenon are not known,nor is its relationship to the regression of AMV-induced myeloblastic leukemia [99].

The observation that various AEV-ES4-transformed erythroblast clones express different levels of hemoglobin in a stable fashion [91 ] is compatible with the idea that the virus is capable of infect!ng target cells at slightly different stages of differentiation and arrest- ing them at their respective stage. However, other explanations may hold true, such as the possibility that after transformation the target cells undergo differentiation or dedifferentiation to a limited degree, or that the differences seen may simply reflect clonal variations.

Another approach in determining whether or not defective leukemia viruses exert a block in differentiation is the isolation of mutants temperature sensitive in this function. With such mutants it should be possible to study whether the infected cells, shifted from permissive to nonpermissive temperature, would undergo further differentiation as would be expected from a "differentiation block" model, or whether they would cease growing and (or) die as would be compatible with an "aberrant differentiation" or "dedifferentiation" model. Recently, we have indeed been able to isolate such mutants from AEV-ES4. These mutants are temperature sensitive for the maintenance of an early stage of erythroid differentiation in that transformed erythroblasts synthesize increased amounts of hemoglobin (as determined by benzidine staining) when shifted from the permissive to the nonpermissive temperature (ref. 100, Fig. 6).

The evidence discussed tends to favour the idea that defective leukemia viruses induce a block in differentiation in their hematopoietic target cells, thus giving rise to proliferating leukemic daughter cells very similar in their pattern of differentiation parameters to the parental target cells. This does not preclude the possibility that in addition a limited dedifferentiation occurs.

A compendium of our interpretation of the results about the interaction of nondefective and defective leukemia viruses with their hematopoietic target cells resulting in leukemic transformation is presented in a speculative model in Fig. 7. It needs to be mentioned

285

Fig. 6. Bone marrow cells (erythroblasts) transformed by a temperature sensitive mutant of AEV-ES4. Cells were stained with benzidine according to the technique of Orkin et al. [149]. A, cells kept at 35°C; B, cells 3 days after shift to 41°C.

that the branching point within the myeloid lineage which leads to the formation of granulocytes and macrophages is not known. It is, therefore, nbt possible to distinguish between the possibility that the target cells for AMV and MC29 represent cells at different stages of maturat ion and the possibility that they belong to the granulocyte and macrophage branches, respectively.

÷ I - L y . , , , . . , .

~ ~ ? - Erytlarecyte

Stem C e l i ~ Ery eilast /

) : Nyeleblest ~ArP.se -

MacrepkalJe Fig. 7. Hypothetical scheme of chicken hematopoiesis depicting the target cells for defective leukemia viruses. Arrows indicate differentiation. Capacity of self replication of stem cell and of immature committed cells not shown. Bar across arrows: block of differentiation. For explanations, see text.

286

IIIC. Genetic properties of defective leukemia viruses This is one of the areas in which much progress has been achieved recently and where

our present knowledge and concepts are likely to become rapidly outdated. It is now clear that all of the in vitro transforming leukemia virus strains tested so far

are defective for replication and require a helper virus of the nondefective leukemia virus type. These strains are: AEV-R and AEV-ES4 [49], AMV [96,79], MC29 [101]. CMII [75], MH2 [82] and E26 and OK10 (Graf et al., in preparation). The most extensive biological studies on the nature of the defectiveness have been performed with MC29 and to a lesser extent with AEV-ES4. Both viruses are deficient in their polymerase activity and do not seem to code for a functional envelope glycoprotein (ref. 102; and Royer-Pokora and Graf, in preparation). The RNA of these viruses in its monomeric form has a molecular weight of approx. 2 • 106 daltons (MC29 [102]; AEV-ES4: Royer-Pokora and Graf, in preparation; Saule and St6helin, unpublished) and is therefore even smaller than the RNA of transformation defective helper viruses. The RNAs of MC29 [103,104], AEV-ES4 [104] and of AMV [105] lack sequences homologous to the src gene of avian sarcoma viruses. In addition, these viruses do not seem to transform their host cells by inducing a transcription of the src-related sequences [104] present in the genome of normal chicken cells [106]. The RNA of MC29 [103] and ofAEV-ES4 (St6helin, D., personal communication) contain specific sequences in addition to sequences related to nondefective viruses. It remains to be seen whether part or all of these sequences define new types of transformation genes.

An analysis of proteins synthesized in nonproducer cells transformed by MC29 by amino acid labeling and immunoprecipitation with sera specific for the various structural l~roteins of nondefective leukemia viruses revealed the presence of an aberrant polyprotein of 110 000 daltons. This protein is related to the gag gene, and contains antigenic determinants of p19 and part of p27 but not of p15 proteins [102]. In contrast to the normal gag precursor protein it is not cleaved into any of the viral structural proteins. It now appears that the synthesis of aberrant polyproteins related to virus structural antigens is a common property of defective leukemia viruses. The AEV-ES4 and R strains both induce the synthesis of a 75 000 dalton protein (ref. 107, and Hayman and Graf, in preparation); MH2 of a 100 000 dalton protein [82]; CMII and OK10 of 95 000 and 75 000 dalton proteins, respectively 0-layman and Graf, in preparation). The finding that AEV-R codes for a polyprotein indistinguishable from that of AEV-ES4 further supports the suggestion made earlier that these two strains may have a common origin.

None of the strains tested (AEV-ES4, MC29, AMV, CMII and E26) seem to induce the expression of sarcoma virus-directed specific cell surface antigen (TSSA) in transformed hematopoietic cells as determined in a cell-mediated cytotoxicity assay [98]. In an earlier report, however, a TSSA-like activity was described for MC29-transformed cells by the use of sera from tumor bearing chickens inoculated with Rous sarcoma virus [108]. It is unclear whether this activity is mediated by group specific determinants of viral envelope glycoprotein, by embryonic antigens [98], or by a virus-directed TSSA.

IV. Role of host mechanisms in the expression of leukemia virus-induced neoplasms

IVA. Genetic susceptibility o f the host Peyton Rous in his early studies already noted that Rous sarcoma virus could be trans-

mitted only to certain strains of chickens [109]. As we know now, susceptibility and

287

resistance to avian sarcoma viruses and nondefective leukemia viruses is determined by dominant cellular genes. These loci probably code for receptors specific for different virus envelope antigens and therefore exert a restriction different from the Fv-1 genes of the mouse which control virus infection at the postpenetration level [7,8]. Recently, another type of genetic resistance has been described which controls the expression of the LLV- induced lymphatic leukemia and which does not affect the replication of the virus in fibroblasts. As shown by the finding that transplantation of bursa cells from a susceptible chicken to a bursectomized resistant animal confers susceptibility to the latter, genetic resistance in this system seems to be determined by specific properties of the lymphoid target cells [33]. The molecular basis of this restriction remains to be elucidated. As discussed before, another example of a differentiation-specific restriction system is the susceptibility of avian macrophages to viruses with certain envelope antigenicities only [94].

IVB. Other factors That young animals are more susceptible to virus induced leukemogenesis than older

ones [12] is probably due to their less developed immunological system. A role of the immune system in controlling the expression of certain virus-induced neoplasms such as lymphatic leukemia is also suggested by the observation that the incidence of osteopetrosis is sharply increased in bursectomized birds infected with LLV [31,17], but this phenomenon may also have other explanations.

A second critical factor for the type of neoplasms induced by a given leukemia virus strain is the route of inoculation. Thus, AEV-ES4 induces mainly erythroblastosis when injected intravenously, and induces sarcomas in addition to erythroblastosis when injected intramuscularly [48,73]. It is interesting that similar observations were also made with field strains of nondefective leukemia viruses [110]. This phenomenon has been explained on the basis of a "first come first serve" mechanism for the interaction of the virus with its hematopoietic and mesenchymal target ceils, respectively [73].

A third factor influencing the incidence and type of neoplasms induced is the way in which the virus used for inoculation had been passaged or selected during its history. Viruses from early in vivo passages after their primary isolation are, as a rule, much less oncogenic than virus selected by several in vivo passages. This is true for most nondefective [111] as well as for defective leukemia viruses such as AEV-R [40] and also occurred during the development of Rous's original virus strain [112].

Lastly, the tumor specificity of AEV-type strains can be modulated according to the type of tumor cells from which the virus is obtained: erythroblast-grown virus is more prone to induce erythroblastosis than the same virus isolated from a sarcoma [43,48]. Despite repeated attempts, "pure" sublines of strains causing erythroblastosis or sarcomas only have so far never been isolated [43,48]. It is remarkable that a modulation of the tumor spectrum by passaging the virus in different types of host cells was again also observed with nondefective strains [ 111 ].

V. Discussion, speculations and models

VA. What distinguishes defective avian leukemia viruses from nondefective leukemia and sarcoma viruses?

Previous classifications of avian oncoviruses were mainly based on their oncogenic spectrum and their period of latency. As has been outlined in this review, these criteria do not allow an accurate distinction between sarcoma viruses and the different types of

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leukemia viruses. The AEV strains, for example, cause sarcomas in addition to leukemia and in this property resemble the Kirsten and Harvey murine sarcoma strains, which are known to be able to induce erythroleukemia in mice [115]. Of course, the term "leukemia" virus is not always correct either, in view of the strains which predominantly induce neoplasms of non-hematopoietic origin such as osteopetrosis or carcinomas.

Since it now appears as if none of the avian leukemia viruses possess the sr.c gene of avian sarcoma viruses, we propose to use this property as the main criterion for the distinction between these two major virus classes.

For the following reasons, we prefer to subdivide avian leukemia viruses into "nondefective" and "defective" viruses instead of using other nomenclatures:

(1) The possibility that defective avian strains may exist which cause an acute lymphatic leukemia, such as the Abelson virus of the mouse which is also capable of transforming in vitro [ 116,117], strongly argues against the widely used classification of leukemia viruses into "lymphatic" and "acute" leukemia strains. An avian virus causing a "lymphatic" leukemia within 2 weeks after infection was indeed described some 50 years ago [118]. This strain is, unfortunately, no longer available. (2) The existence of nondefective leukemia virus strains which have a latency period comparable to or even shorter than that of some defective leukemia viruses, together with the possibility that in vitro transformation assays might still be developed for nondefective strains, is incompatible with a nomenclature which refers to the two groups as "slowly or weakly transforming" and "rapidly or strongly transforming" leukemia viruses, respectively.

A biochemical property characteristic for the defective leukemia viruses is their ability to direct the synthesis of gag-related polyproteins (ref. 107; Hayman and Graf, in preparation). The characterization of these proteins from cells transformed by different strains might serve as a basis for future classifications of viruses of this group.

The criteria which distinguish nondefective leukemia viruses, defective leukemia viruses and sarcoma viruses are summarized in Table IV.

VB. How do leukemia viruses originate? The nondefective leukemia viruses are very similar to the chicken endogenous virus

RAV-0. However, they differ in several properties: (1) In contrast to nondefective leukemia viruses; RAV.0 is virtually nononcogenic [119,14]. (2) RAV.O and a closely related defective endogenous virus (detected in "gs ÷'' or " c h f " cells) code for an envelope antigen of subgroup E antigenicity. In contrast, all exogenous nondefective leukemia viruses belong to other subgroups [8,11 ]. (3) There are differences in the nucleotide sequences of the genome of RAV-0 and nondefective leukemia viruses which are located mainly at the 3' end of the RNA, in the region of the env gene and in the "c" sequence [35,36,37]. (4) A considerable number of nondefective leukemia virus stains induce a cytopathic effect and plaques in fibroblast cultures whereas RAV-O does not [27,28,29].

These differences are reminiscent of those found between an ecotropic endogenous virus of AKR mice and recombinants of this virus in the env gene with a xenotropic endogenous virus [120-122]. Since these recombinants appear shortly before the onset of leukemia and are themselves more leukemogenic than the parental strains, they have been implicated in the formation of the spontaneous leukemia of AKR mice [120]. It is remarkable that the recombinartts not only acquired a new envelope antigenicity but that they also induce a cytopathic effect in certain types of cultured cells [120].

By assuming an analogy of the avian to the murine system, one may speculate that nondefective leukemia viruses arose by a recombination between the env gene of RAV-0

TABLE IV

DISTINCTIVE FEATURES OF AVIAN LEUKEMIA AND SARCOMA VIRUSES

289

Avian leukemia viruses

Defective leukemia Nondefective Avian sarcoma viruses leukemia viruses viruses

Presence of src-like sequences

Replication capacity

In vitro transforming capacity

Synthesis of aberrant, gag-related proteins

Period of latency

Predominant type of neoplasm induced

No No Yes

No Yes Yes a

Yes No Yes

Yes No No

Weeks to months Weeks to years Weeks

Myeloid leukemia Lymphatic leukemia Sarcoma Erythroleukemia Osteopetrosis Sarcoma Nephroblastoma Carcinoma Erythroleukemia

Sarcoma

a Except for some strains defective in the env or pol gene.

and the env gene of another, as yet unidentified, endogenous virus. Alternatively, they may have arisen directly from RAV-0 by mutation and selectio!a as suggested by Coffin [10l.

In several instances, defective leukemia viruses were described to originate from field isolates with a long period of latency, an observation also made with Rous sarcoma virus [112]. In most of these cases, virus from early animal passages already had a tumor specificity similar to the more rapidly transforming virus of later passages. A good example is the ES4 strain of AEV which already induced sarcomas in addition to erythroblas- toffs during the first passages [42]. In this regard it is interesting that field isolates of nondefective leukemia viruses selected for their capacity to induce erythroblastosis were found to induce sarcomas with a higher frequency than unselected strains; and that viruses recovered from sarcomas induced by nondefective leukemia viruses were likely to induce erythroblastosis in addition to sarcomas and other neoplasms [111], thus resem- bling AEV-type virus from early passages. In aU these cases, however, it has apparently not been possible to isolate defective type leukemia viruses from neoplasms induced by the nondefective strains.

Taken together, the above observations suggest that highly leukemogenic, defective leukemia viruses have evolved from low leukemogenic, nondefective strains in a multistep process involving the acquisition of several new properties such as a defined tumor specificity, defectiveness, and the capacity for rapid leukemogenesis and for in vitro transformation. These changes are possibly introduced through the selection of variants which may arise by mutation or recombination of parental virus(es) or by recombination of viral with cellular genes. In the light of the evidence, it is tempting to speculate that nondefective leukemia viruses are not transforming per se but that they are capable of rarely converting into unstable or incomplete forms of defective leukemia viruses which would then be the ultimate cause of transformation. The formation of stable defective

290

viruses would require yet another, even more rarely occurrying step. This idea would be strongly supported if it were possible to detect gag-related aberrant polyproteins in neoplasms induced by nondefective leukemia viruses.

VC How do leukemia viruses induce a neoplastic transformation? In the murine system, the findings mentioned earlier in this discussion and other evi-

dence not reviewed here suggest that the formation of recombinants in the env gene is a crucial event in virus-induced leukemogenesis. On this basis several groups now strongly favor the envelope antigen itself as a candidate for the transformation protein of murine oncoviruses [120,121,123,124]. Similar speculations can also be made for nondefective avian leukemia viruses. The avian osteopetrosis viruses and the anemia inducing strains, because of their short period of latency, represent promising systems to study the role of the envelope antigen and of other structural antigens as mediators of virus-induced disor- ders. It should be possible to construct recombinants of these viruses in the gag, pol and env genes with LLV strains having a long latency period, which can then be tested for their pathogenic properties. However, the development of in vitro systems allowing detec- tion of disease-specific alterations seems to be crucial for further progress in the understanding of the interaction of nondefective leukemia viruses with their host cells at a molecular level.

Because of the availability of in vitro transformation systems for defective leukemia viruses, more can be said about possible mechanisms involved here. In the following we will discuss our present ideas about two aspects of the interaction of defective leukemia viruses with their target cells, resulting in transformation: (1) changes induced in the target cell after transformation and (2) possible mechanisms of virus-target cell interaction.

1. Cellular aspects. Because of the capacity of defective leukemia viruses to induce morphological alterations in different types of tissues, the term "transformation" has to be carefully def'med according to both the virus and the target cell involved. In the best understood oncovirus system, the interaction of avian sarcoma viruses with mesenchymal cells, cell transformation in vitro is defined by the expression of a series of characteristic alterations most extensively studies in fibroblasts and known as "transformation parameters" [11,125]; and by the capacity of avian sarcoma viruses to interfere with terminal differentiation of certain types of target cells, such as myoblasts, chondroblasts and pig- mented retina cells [126-129] or to induce a proliferation in other cell types such as neuroretina cells [130]. Studies with temperature sensitive mutants indicate that these effects can all be attributed to the pleiotropic action of the viral src gene [ 126-131 ].

Recent investigations showed that after infection with the nonsarcomagenic strain MC29, fibroblasts express only part of the avian sarcoma virus-specific transformation parameters tested; whereas fibroblasts transformed by the sarcomagenic AEV-strain express virtually all of these parameters [86,114]. Whether or not defective leukemia viruses are capable of interfering with the differentiation ofmesenchymal cells is not yet known. Recent studies indicate that AEV is capable of inducing a proliferation ofneuro- rgtirla cells and that MC29 is not [132].

The situation becomes much less clear when we consider the "transformation" of epitheloid and hematopoietic ceils by defective leukemia viruses. The only criterion for transformation used so far in these systems is the induction of proliferation and morphological alterations in certain types of primary embryo cells. Transformation of liver and kidney cells by MC29 virus might be a suitable model system to det'me further trans-

291

formation parameters characteristic for epitheloid cells. Its importance stems from the fact that it seems to represent the only known in vitro model system for virus.induced carcinogenesis. It would be interesting to know whether or not epithelial cells transformed by MC29 express the full spectrum of transformation parameters characteristic for fibroblasts. If this were the case, it would suggest that the expression of transformation may be subject to a control by the host cell which varies with the type of cell infected. This idea is supported by the finding that macrophages infected by avian sarcoma virus express only some of the transformation parameters. Macrophages are, incidentially, also not induced to proliferate by sarcoma virus infection [94].

As discussed earlier, in the hematopoietic system the stimulation of cell proliferation observed after infection with leukemia viruses appears to be accompanied by a block of differentiation of the infected cells. Are these two events separable or do they represent two facets of the same phenomenon? We think that the available data, in particular those obtained with AEV-transformed cells, do not rule out the possibility that the observed induction of proliferation might simply be a consequence of blocked differentiation or vice versa. Further studies on the temperature sensitive mutants of AEV should be helpful in discriminating between these possibilities.

At this point of the discussion it seems useful to compare the process of AEV-induced leukemogenesis to that induced by Friend leukemia virus. The most typical effect of the defective component of the Friend virus complex is that in spleens of infected mice it induces a massive outgrowth of erythroid cells some of which differentiate into erythrocytes and some of which die [133]. In plasma clot or methylcellulose cultures these cells grow in the absence of erythropoietin into small colonies of nonproliferating, differentiated cells which are indistinguishable from colonies of hormone dependent cells of normal spleens [134]. Colonies of erythropoietin independent cells can also be obtained under certain conditions after infection of erythroid target cells with Friend virus in vitro [135]. A more rare effect of Friend virus is the induction in leukemic spleens of cells which can be grown into cultures of established lines [136]. These rapidly proliferating cells are arrested at an early stage of differentiation and can be induced to synthesize hemoglobin by treatment with a variety of compounds such as dimethylsulfoxide [136].

Leukemogenesis by AEV resembles the latter effect of Friend virus in that immature erythroid ceils are induced to proliferate, and do not differentiate further unless they are treated with inducing agents. AEV-transformed cells have an in vitro life span of up to about 30 generations [113], which is comparable to that of uninfected or avian sarcoma virus-transformed fibroblasts. Due to the lack at the present time of hormone preparations in the avian system, the influence of erythropoietin on AEV-induced transformation has not yet been determined. However, the observation that bone marrow cells can be transformed in vitro in the absence of normal or anemic chicken serum (presumably con-

• ~ raining avian erythropoietin) [81 ] suggests that this process is hormone independent. 2. Viral aspects. After ruling out the involvement of viral or cellular src-like sequences

[104], basically three possibilities remain to explain the effects of avian leukemia viruses which lead to transformation: (1) The virus integrates at a specific site in the host genome, thereby altering regulatory functions or activating a cellular "oncogene", with the consequence of cell transformation. In this case, the continuous expression of viral gene products is not necessary for the maintenance of transformation. (2) A viral gene product, for instance a protein, has to be continuously expressed in order to maintain the transformed phenotype. This protein could either be coded by a nonstructural gene analogous to src, or it could be related to a structural protein of the virus. (3) Transfor-

292

mation depends on both the integration of the virus genome at a specific site in the host's genome and the continuous expression of a viral gene product.

As mentioned before, studies with temperature sensitive mutants of AEV indicate that the continuous expression of a viral gene is required for the virus-induced block of differentiation in erythroblasts. Assuming that the block in differentiation is causally linked to the process of leukemic transformation, this finding rules out the possibility that a specific integration of viral sequences alone can account for transformation. However, it would still be compatible with the third possibility, that the integration of the virus at a specific site is a prerequisite for the expression of an AEV-specific transformation protein. It needs to be added that it is quite possible that different mechanisms are operative in the interaction of this virus strain with fibroblasts, or in the interaction of MC29- or AMV-type viruses with their corresponding target cells.

To date, the best line of evidence suggesting that defective leukemia viruses code for new type(s) of transforming gene(s) is the finding that MC29-specific sequences also occur in the DNA of normal avian cells and that they are better conserved during evolution than the genes from chicken endogenous virus (Bishop, J.M. and Sheiness, D., personal communication). This parallels the findings obtained earlier with the s r c gene of avian sarcoma viruses [137].

In order to stimulate further discussion and experimentation we have constructed a tentative and highly speculative model of leukemogenesis by defective leukemia viruses. This model, shown in Fig. 8, is based on three assumptions. The first assumption is that the transformation gene of defective leukemia viruses (thought to be acquired during evolution) represents a modified sequence of a cellular gene (arbitrarily termed b) required for normal differentiation. Depending on the type of virus, different types of b-genes (bE, bM, etc.) and their products (BE, BM, etc.) would be involved. For example, AEV- type viruses would synthesize a protein which is similar to a protein (BE) required during normal erythroid differentiation; and AMV- and MC29-type viruses would synthesize modified myeloid differentiation proteins (BM). The second assumption is that a necessary step in hematopoietic differentiation is the interaction of an early differentiation factor (A)

1. Modification of differentiation protein after acquisition by virus of cellular b gene by integration into virus

B,---- B,* B . - - B. B, --.- B,*

2. Normal hematopoietic cell

A-T , - [AB ]-"--C--terminal d ifferen tiation

B 3, Transformed hematopoietic cell

A lAB. ]_ b,oc. o, I d ifferentiation

Fig. 8. A model for defective leukemia virus-induced leukemogenesis. BE, BM, BL: CeLlular proteins required for erythroid, myeloid and lymphoid differentiation, respectively. For explanations, see text.

293

with cellular protein B thereby triggering the synthesis or activation of differentiation factors characteristic of later stages. That such a mechanism may indeed exist is suggested by recent studies in the Friend erythroleukemia system [138]• The third assumption of the model is that the virus-coded transformation protein B is still capable of binding to the cellular component A. However, the resulting complex AB* is nonfunctional and the late differentiation factor (C) is not produced. The transforming protein B* would thus effect a block of differentiation by competing with the normal cellular protein B. Candidates for such virus-coded modified differentiation proteins are the unique poly- peptide sequences of unknown nature in the aberrant polyproteins synthesized by defective leukemia viruses. Since these sequences seem to be always linked to various portions of the gag-precursor protein it would not be surprising if they were rendered nonfunctional.

The model presented has several attractive features: First, some aspects of it are tes- table. If antibodies could be obtained against the putative transforming proteins (e.g. the unique portion of aberrant polyproteins) these antibodies should react specifically with proteins from the corresponding normal target cells. Second, it explains the lineage specificity of defective leukemia viruses since the transformation protein can only be "recog- nized" in cells in which the corresponding differentiation protein is active• And third, it is not restricted to leukemogenesis by defective avian leukemia viruses• Spontaneous leukemias of clonal origin such as observed in man could be explained by a mutation or recombination in a b-type gene, thus leading to a block of differentiation as a result of the synthesis of a modified b gene product• Likewise, leukemogenesis by nondefective avian leukemia viruses could be explained by assuming that these agents act as specific muta- gens, capable of integrating within cellular genes of the b-type, Here again it would be interesting to know whether or not antibodies against the putative transforming proteins of defective leukemia viruses would react with a specific protein from leukemic cells of the corresponding differentiation lineage. In the light of our model, the recent data on AKR leukemogenesis discussed above could be interpreted in a novel way. Rather than concluding that the envelope antigen of the recombinant viruses confers to them their leukemogenic potential it could be speculated that a specific sequence of their RNA allows them to integrate within a cellular b-type gene required for lymphoid differentiation. Obviously, however, even if this alternative explanation for the leukemogenesis by nondefective viruses would turn out to be wrong, the model proposed may still be basically correct for defective leukemia viruses.

That particular strains of defective leukemia viruses cause a transformation in nonhematopoietic as well as hematopoietic target cells raises the possibility that they carry more than one transforming gene. However, the finding that in all strains there appears to be a close correlation between their specificity in the hematopoietic system and their ability to transform certain types of nonhematopoietic cells (see Table II) is more compatible with the idea that there is a single transforming gene which differs for each type of virus. It should be possible to test this assumption with the use of DNA probes specific for different types of defective leukemia viruses.

It seems premature to introduce designations for the hypothetical transforming genes of defective leukemia viruses before they have been defined by genetic and biochemical criteria. If designations were to be proposed, the target cell specificity of the respective virus type should be taken into account as is the case with "src". Thus, it is of little use to designate the hypothetical transforming gene of defective leukemia viruses with the general term "'luk" (for leukemia), as has been proposed recently [10], nor would it be

294

correct to capitalize on certain aspects of their transforming potential only, such as has been done by proposing a 'crc" gene (for carcinoma) for MH2 virus [139]. For the time being, it appears to be more appropriate to reintroduce the general term "onc" (for oncogene) [140] and to indicate the name of the virus carrying it in a subscript, e.g. oncMc29 for the transforming gene of MC29.

VD. What is the mechanism of target cell specificity? At first glance the ability of different strains of defective leukemia viruses to transform

a variety of hematopoietic and nonhematopoietic cell types is devoid of a predictable pattern. However, as discussed in this review, it appears that AEV-type, erythroblast- transforming viruses are always capable of transforming fibroblasts as well, whereas AMV-type, myeloblast-transforming viruses are not. Similarly, MC29-type viruses are capable of transforming fibroblasts and epitheloid cells in addition to macrophage-like cells (see also Table II). The basis for this intriguing association of target cell specificity remains a mystery.

The most obvious explanation for the transformation target cell specificity of avian leukemia viruses in hematopoietic cells is the possibility that they are able to infect and to replicate only in certain types of cells. This seems to be true for the interaction of murine radiation leukemia virus with its target cells in vivo and in vitro [141,142]. That nondefective avian leukemia viruses efficiently replicate in "nontarget" cells such as in fibroblast cultures [143] seems to argue against this possibility. In the animal, however, these viruses apparently replicate preferentially in lymphoid cells of the bursa [144]. A similar organ tropism was also observed with the Moloney strain of mouse leukemia viruses [145]. Preliminary experiments indicate that defective leukemia viruses replicate in nontarget cells in vitro (Meyer-Glauner and Graf, unpublished observations), but nothing is known about their replication specificity in vivo.

Depending on the mechanism of hematopoietic cell transformation which is assumed to exist, the transformation target cell specificity of avian leukemia viruses can be explained in several basic ways, as illustrated in Fig. 9.

If a specific integration is sufficient for transformation, then, according to hypothesis 1, different strains of leukemia viruses might be able to integrate only into the genome of their respective target cells where they would occupy different, specific sites. Alternatively, in hypothesis 2, different strains vary in their integration site-specificity but are capable of integrating in cells from all lineages. In this case, only the integration at a specific site within the genome of the target cells leads to transformation whereas integration at the same site in nontarget cells does not. It is conceivable that in addition to the transformation-specific integration sites postulated in hypotheses 1 and 2, other sites exist which allow virus replication but never lead to leukemogenesis. A possible example for this situation is the amplification in target cells only of integrated virus sequences preceding the onset of leukemia in AKR mice [146]. Although the integration sites have not yet been compared in leukemic and preleukemic AKR mice this suggests that during amplification, the endogenous virus occupies a second, transformation specific site.

If it is assumed that there is a viral transformation protein, again two possibilities can be distinguished. In hypothesis 3, the viral transformation protein is synthesized in target cells only, and in 4 it is synthesized in cells from all lineages but leads to transformation in target cells only. As pointed out before, a combination of the integration hypotheses and the viral transformation protein hypotheses is also possible.

The model for the mechanism of leukemogenesis represented in Fig. 8 would be com-

295

I. Virus integrates in target cells only. Strains differ in integration specificity

2. Virus integrates in cells from all lineages. Strains differ in integration specificity

3. Viral transformation

protein is synthesized

in target cells only

4. Vlml transformation protein is synthesized in cells from all lineages

Virus a

Virus b

Virus a

Virus b

Virus a

Virus b

Virus a

Virus b

Target cell

A

o- Y _--

----*--

Q 0 --- ---

___*--_

Q 0 -- --

m __-*---

Target cell

0

-_---__

Fig. 9. Schematic representation of four hypotheses accounting for the transformation target cell specificity of leukemia viruses. ( - - - - -), chromosomes of the host cell; (X), integration site of viral genome; (0, n ), viral transforming proteins; (o), viruscell interaction leading to leukemic transformation. See text for detailed explanations.

patible with hypothesis 2 if applied to nondefective leukemia viruses and with hypothesis 4 in the case of defective leukemia viruses. It needs to be added that, regardless of the mechanism operating under in vitro conditions, it is likely that additional factors

regulate the specificity of the interaction of nondefective leukemia viruses with their target cells in vivo (see also Section IV).

E. Tools or toys? As discussed in this review, avian leukemia viruses constitute an excellent model to

analyze the interaction of oncoviruses with various types of differentiating cells and to elucidate the fascinating problem of target cell specificity. Moreover, the different types of hematopoietic cells transformed by the different strains of viruses may be used as potent tools to dissect the process of hematopoietic cell differentiation. But are these

viruses not merely toys in the hands of some curious scientists? Do similar agents cause

leukemia in man? The answer at present is more likely to be no than yes. However, there is still hope that they are more than toys. This hope is based on several ifs: if an arrest in differentiation of committed hematopoietic precursor cells is also a critical event in human leukemogenesis as has been suggested by recent studies [ 1471; if our concept is correct that avian leukemia virus infection leads to a block in the differentiation of specific target cells; and if this block is mediated by specific transformation proteins representing modified differentiation proteins of the cell - then, the elucidation of the origin and

296

function of these genes may be of crucial importance for the understanding of basic principles of hematopoietic differentiation and its aberrations which result in leukemia. In this regard, there is a good chance that the prophetic sentence of the pioneer in the field of leukemia viruses, Ellerman, will still turn out to be wrong “Die Htihnerleukose gibt also keine Beitrage zu dem Verstindnis der Genese der leukimischen Blutveranderung beim Menschen” * [ 1481.

Acknowledgments

We thank Heinz Bauer, Bob Friis, Mike Hayman, Rudolf Jaenisch, Brigitte Royer- Pokora, Ralph Smith, Werner Schafer and Charles Weissmann for critical comments on

this review. Thanks also to Lynn Graf for her help with the manuscript.

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