Oncogenic Properties Of The Middle T Antigens Of Polyomaviruses

ONCOGENIC PROPERTIES OF THE MIDDLE T ANTIGENS OF POLYOMAVIRUSES Friedemann Kiefer,”’ Sara A. Courtneidge,t and Erwin F. Wagner’ ‘Institute of Molecular Pathology, A-1030 Vienna, Austria tEuropean Molecular Biology Laboratory, D-69117 Heidelberg, Germany

I. Introduction A. Murine Polyomavirus B. Biochemical Properties of PymT Antigen C . Hamster Polyomavirus D. Rationale for Studying the Oncogenic Action of PymT in Vzvo 11. Consequences of PymT Expression in Vivo A. Expression of PymT under the Control of the Polyomavirus Early Region or General Promoter Elements B. Expression of PymT under the Control of Tissue-Specific Promoter Elements C. PymT Antigen Expression in Organ Reconstitution Systems 111. Expression of the Hamster Polyomavirus Middle T Antigen in Vivo IV. Analysis of PymT-Transformed Endothelial Cells V. Outlook References

I. Introduction The realization that viruses can cause cancer in humans and animals (Rous, 1911; Shope, 1932; Bittner, 1936; Zilber, 1946) clearly has been one of the biggest milestones in cancer research. Oncogenic viruses for the first time provided reagents that, in contrast to chemical carcinogens, were capable of transforming cells in a relatively small number of defined molecular events. Among the oncogenic viruses, the murine polyomavirus is outstanding in its ability to cause oncogenic transformation of a broad spectrum of cell types, which in its entirety is called the “polyomavirus tumor constellation” (Dawe, 1980). To date, two polyomaviruses have been isolated from their respective hosts, mice and hamsters. Both display a very similar genomic organization consisting of a circular genome; strands encoding early and late gene products are transcribed in opposite directions from a noncoding region that Present Address: Ontario Cancer Institute, 500 Sherbourne Street, Toronto, Ontario, Canada M4X 1K9. 125 ADVANCES IN CANCER RESEARCH, VOL. 64

Copyright 0 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.

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functions as a replication origin as well as a transcriptional control region (Delmas P t al., 1985; Fried and Prives, 1986). Polyomaviruses share this general genomic structure with other papovaviruses; therefore, the hamster polyomavirus was initially named hamster papovavirus (Graffi et ul., 1969). In this chapter, we will refer to the murine polyomavirus simply as polyomavirus, whereas w e will always use the explicit designation hamster polyomavirus for the hamster papovavirus. A. MURINEPOLYOMAVIRUS

Polyomavirus was discovered accidentally in 195 1 by Ludwik Gross in cell-free filtrates from Akr mice that had developed spontaneous leukemia (Gross, 1953a,b). When subsequently tested on newborn mice, the preparation consistently induced parotid gland carcinoma and related tumors of the salivary gland. It soon became clear that this relatively narrow oncogenic potential of the virus, which was due to low viral titers of the tissue extracts, could be dramatically enhanced when high-titer virus preparations of polyomavirus grown in tissue culture were inoculated into newborn mice. T h e virus caused, in addition to the consistent appearance of parotid neoplasms, tumors as diverse as submaxillary and sublingual gland neoplasms, thymic epitheliomas, mammary carcinomas, renal and bone sarcomas, epidermoid carcinomas, adrenal medullary tumors, and hemangiomas (Stewart et nl., 1958). However, it never caused leukemias. Because of this ability to induce a wide range of tumors, Stewart and Eddy proposed the name S. E. “polyoma” virus (Stewart and Eddy, 1959). More recently, Dawe et al. (1987) showed that four different wiid-type strains of polyomavirus show markedly different oncogenic properties. Two of the strains are highly oncogenic in vivo and two display a lower oncogenic potential, giving rise only to tumors of rnesenchymal origin that develop with prolonged latency periods. All these strains are highly transforming in zritro. High-titer virus preparations also showed a significantly expanded host range, inducing tumors not only in a variety of different mouse strains but also in Syrian (golden) hamsters, rats, ferrets, guinea pigs, and rabbits (see review by Gross, 1983). In all cases, however, tumors only arose if the host animal was inoculated immediately after birth. Under normal circumstances, polyomavirus does not behave as a tumor virus in its natural host (Rowe, 1961). T h e development of tissue culture systems that allowed the propagation of the virus and the accumulation of high amounts of virus particles led to major breakthroughs in the understanding of the structure and lytic cycle of polyomavirus. However, the basis of the transforming

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events caused by polyomavirus remained largely unclear. Polyomavirustransformed cells always contain a functional copy of the viral genome early region integrated into the host genome. However, it became clear from integration studies that the virally induced transformed phenotype does not depend on a particular integration site or integration into a specific region of the host genome, leading to the activation or inactivation of host genes. Rather, viral transformation is a consequence of the addition of a viral gene to the host genome. i n 1970, Benjamin showed that three proteins of molecular sizes 2 1,55, and 96 kDa could be immunoprecipitated by treatment of extracts from polyomavirus-infected cells with sera from animals bearing polyomavirus-induced tumors. T h e three tumor, or T antigens-which were designated “small” T antigen, “middle” T antigen, and “large” T antigen (Benjamin, 1970)-are located in the early region of the viral genome (Soeda et al., 1979). Studies with mutants of polyomavirus indicated that small and middle T or middle T alone are necessary for the initiation of transformation, whereas large T might be necessary to sustain transformation (reviewed by Topp et al., 1980). Molecular cloning of the individual cDNAs of the polyomavirus early region finally allowed the analysis of the contribution of each of the three T antigens to the transformation process. Treisman et al. (1981) could show that a cDNA encoding the polyomavirus middle T antigen (PymT) is sufficient for the transformation of established rodent cell lines. Therefore PymT was unambiguously identified as the transforming gene of polyomavirus. Although PymT suffices to transform established rodent cell lines, maintenance of the transformed state in primary cells requires the cooperation of polyomavirus large T antigen (Rassoulzadegan et al., 1982; Land et al., 1983). Large T provides an immortalizing function for the establishment of continuously growing cell lines from primary cells, whereas PymT is necessary to sustain the transformed characteristics of these cells (Rassoulzadegan et al., 1983). OF PYMTANTIGEN B. BIOCHEMICAL PROPERTIES

PymT is a membrane-bound protein (It0 et al., 1977) of -55 kDa. It does not carry any intrinsic enzymatic activity, but associates with several cellular enzymes that are crucial regulators of cell proliferation. Therefore, PymT most likely exerts it oncogenic effect by subversion of the intrinsic functions of its cellular association partners. The first activity shown to be associated with PymT was that represented by tyrosine kinases; indeed, this was one of the first descriptions of stable tyrosine phosphorylation. Since the transforming protein of Rous sarcoma virus, pp60v-~”(Brugge and Erikson, 1977), was also demonstrated to be a

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tyrosine-specific protein kinase (Collett et al., 1980; Hunter and Sefton, 1980), it was an exciting thought that these viruses transformed via similar mechanisms. However, the obsei-vation that monomers of Pyiii’l‘ were n o t kinase active raised doubts that the activity was intrinsic to the protein. Indeed, researchers subsequently showed that the cellular tyrosirie kinase c-Src (the cellular hoinolog of pp60\-”()was complexed with Pym-1. (Courtneidge and Smith, 1983); two more Src-related tyrosine kinases, Yes and Fyn, have since also beeii identified in a complex with PymT (Koi-iibluthct ~ l . 1987; , Cheng et al., 1988; Kypta et d., 1988). These three members of the SI-cfamily are thought to account for all the tyrosine kinase activity associated with PymT (Fig. 1). Although association of Src and Yes with PyrnT leads to a marked up-regulation of kinase activity. no stimulaliori of tyrosine kinase activity was detected on binding of Fvn to Yyml~.I n fibroblasts, only a portion of the tyrosine kinases Src, Fyr;, and Qes form a complex with Pyni‘I‘; similarly, only a fraction of 1’ymT is bound to tyi-osine kinases (reviewed b y Kaplan et al., 1989; Brimela ot d., 1994). High activity of the kinase iiioiety of Src is associated with dephosphorylat ion of the C-tei-niinal Tvr 327 and phosphoi-ylation of Tyr 4 16. Kinase-inactive Src molecules display the opposite phosphorylation pattern: high phosphorylat ion of Tyr 527 and low phosphorylation at ’l’yr 4 16. Current evidence suggests that phosphorylation of Tyr 527 is part o f the regulation o f Src kinase activity and results in the intracellutar association of P-’lyr 527 with the Src SH2 domain, leading to the inactivation of the kinase moiety (reviewed by Cooper and Howell, 1993; Superti-Fiirga P t (11.. 1993). Mutant versions of Src in which the tyrosine at positioii 527 is exchanged with phenylalaniiie show an elevated kinase activity and clisplap oncogenic properties in 7 d r o (Cartwright el al., 1987; Kmiecik and Shalloway, 1987; Piwiica-Wornis r t ol., 1987). The Src tiiolecules that i1i-e associated with PymT are not phosphorylated on ‘lyr 527 and therefore are constitutively active (Cartwright Pt d.,1986). Pym?‘ may cause this ef‘fect either by binding to dephosphorylated active SIC and preventing phosphorylation of- Tyi- 327 or hy exposing inactive SIT to ii tyrosine phosphatase and stabilizing this state. The atialysis of niutant versions of PymT that retain the ability to bind and fully activate Src, but are still transformation defective, suggested that additional biochemical activities are complexed to PyrnT and take part in the transformation process (reviewed by Markland and Smith, 1987; Kaplan et nl., 1989). Subsequently, a ptiosphatidylincisitol 3-kinase (PIS-K) activity was shown to associate with PymT (Whitman P t al., 1985; Kaplari P t ul., 1986). T h e presence in PymT complexes of-a protein of 85 kDa u7as shown to correlate with the detection of PIS-K activity (Court-

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NH2

FIG. 1. Schematic representation of a putative PymT protein complex in mouse fibroblasts. The PymT molecule, which is anchored to cellular membranes by its C-terminal sequence, exists as a multimeric complex with several other proteins: p85 and pl10 correspond to the regulatory and catalytic subunits of PIS-kinase; Shc denotes the adaptor protein; A and C represent the accessory and catalytic subunits of PPBA, respectively; Src, Fyn, and Yes refer to the tyrosine kinases; Y refers to tyrosine residues.

neidge and Heber, 1987; Kaplan et al., 1987). Subsequent analysis showed that this was the regulatory subunit of the enzyme. The catalytic subunit of 110 kDa is also present, although it is not phosphorylated on tyrosine. Genetic analysis shows that the ability of PymT to associate with PI3-K is closely correlated with its ability to transform fibroblasts Zn vztro. Most recently, researchers showed that the transforming protein Shc is also capable of binding PymT (Fig. 1; Dilworth et al., 1994). Again a tyrosine phosphorylation site (Tyr 250) in PymT serves as a binding partner for the Shc SH2 domain. As a result of this interaction, Shc itself becomes tyrosine phosphorylated by the kinase present in the complex,

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and is thereby capable of binding the adaptor molecule Grb2, which is likely to result in the activation of the p21?aspathway. Several years ago, biochemical analysis had shown that two other proteins of molecular weights 61 and 37 kDa were also associated with Y y m r. Subsequent cloning showed that these two proteins are the regulatory (A) and catalytic (C) subunits of PP2A, which are phosphoserineand phosphothreonine-specific phosphatases, respectively (Pallas et al., 1990; Walter et al., 1990).Whereas PP2A is normally composed of three subunits called '4,B, and C, PymT seems to compete with the B subunit of the holoenzyme. PymT as well as the C subunit of PP2A bind in a highly cooperative manner to the A subunit (Ruediger et al., 1992).T h e function of PP'LA in poiyomavirus-mediated transformation is not understood. Whether the interaction between PymT and PP2A leads to a stimulation of PP'LA activity, and whether such a stimulation results in the dephosphorylation of specific cellular proteins, is not yet clear. C. HAMSTER POLYOMAVIRUS

In 1967, Graffi and colleagues described a virus associated with skin epithelioma in a Syrian hamster (Graffi et al., 1967,1968).The virus was originally designated hamster papillomavirus, but subsequent analysis showed that the virus was in fact most closely related to the mouse poiyomavirus, so it has been designated the hamster polyomavirus (HaPV) (Delmas et al., 1985).T h e tumors seen in the Berlin-Buch colon); of Syrian hamsters involve hair root keratinocytes, primarily on the head and back. However, when tumor extracts were inoculated into newborn hamsters from a different virus-free colony (from Potsdam), epitheliomas were not detected. Rather, the hamsters developed lymphoma and leukemia with a high incidence and short latency. These tumors, which appear in the liver, thymus, and kidneys, have not been well characterized but serological analyses suggested that both T and B cells were involved (Graffi et al., 1969). The tumor profile of HaPV is very different from that of the mouse polyomavirus, which has never been reported to cause lymphoma, even when expressed under the control of a iymphoid-specific promoter (Rassoulzadegan et al., 1990). T h e restricted tumor profile may in part be dictated by the inability of the virus to replicate in all tissues. However, this does not seem to be the case since viral DNA was found in all organs tested in infected hamsters. In addition, mice transgenic for the HaPV genome have been described; the founders developed both skin epitheliomas and lymphoid tumors (de la Roche et al., 1989). However, the exact nature of the lymphoid tumors was not established.

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The differences in tumor profiles observed suggested that there might be fundamental differences between the mouse and hamster polyomaviruses. However, molecular cloning of the HaPV showed it had a high degree of homology to the mouse polyomavirus (the open reading frames have an average 50% amino acid similarity), including the ability to encode a middle T antigen (Delmas et al., 1985). HaPV is thus only the second example of a middle T antigen-encoding papovavirus. The early region of the HaPV also encodes large and small T antigens. Despite the fact that HaPV causes predominantly lymphoma and leukemia in uiuo, the early region of the virus was shown to be able to transform primary rodent fibroblasts in uitro, although with a somewhat reduced efficiency compared with the mouse polyomavirus early region (Bastien and Feunteun, 1988). Furthermore, researchers also showed that the large T antigen carried the immortalizing properties, whereas the middle T antigen was responsible for the morphological transformation. A clear role for the small T antigen has not been defined. The middle T antigen of the hamster polyomavirus was identified and characterized using NIH 3T3 cells transformed with a cDNA encoding just the middle T antigen (Courtneidge et al., 1991). The use of NIH 3T3 cells also allowed a comparison of its properties with those of the mouse PymT in the same cellular background. The hamster middle T antigen (HamT) was identified as a 45-kDa phosphoprotein conforming to the corresponding predicted molecular weight. The HamT was found to have binding properties very similar to those of PymT; it could associate with cellular tyrosine kinase activity, serine/threonine phosphatase activity, and PIS-K activity. However, a surprising result was obtained when the exact nature of the associated tyrosine kinase was examined-the HamT bound exclusively to Fyn, and was not able to associate with c-Src or c-Yes. This result is in contrast with the binding specificity of PymT, which shows preferential association with c-Src and c-Yes, and only a very low degree of association with Fyn (reviewed by Brizuela et al., 1994). The specificity of association between HamT and Fyn is clearly demonstrated by the experiment shown in Fig. 2. Here, extracts of cells transformed by HamT were depleted of either Src or Fyn using specific antibodies. Subsequent analysis demonstrated that depletion was complete. When such depleted extracts were then examined for HamT-associated kinase activity, removal of Src was found not to affect mT-associated kinase activity whereas depletion of Fyn resulted in a complete loss of kinase activity in m T immunoprecipitates. These results demonstrate that, in fibroblasts at least, no tyrosine kinases other than Fyn are associated with HamT. The Fyn that is associated with HamT is activated in its intrinsic kinase activity compared with unbound

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FIG. 2. T h e association of. Ham?’ ~ i t hFyn. A lysate of HamT-transformed NIH3T3 cells was immunodepleterl using either normal rabbit serum (NKS, lanes 1-3) or antibodies specific for St-c (lanes 4-6) or Fyn (laiies 7-9). T h e depleted extracts were then used for a second round of inimunoprecipitation with antibodies specific for Src (lanes 1, 4,and 7 ) ,Fyn (lanes 2. 5. and 8).o r HamT (TBI-I; lanes 3, 6. and 9). Following washing of the immunocompl~xes,ail zit a h kinase assay was performed. The positions of the HamT (niT), the FVII( ~ 6 0 )and . the 85-kDa subunit of the P I 3 kinase (p85) are shown.

Fyn, although the mechanism of this activation has not been explored. These data therefore provide more evidence for the association of transforming proteins of polyomaviruses with members of the Src family, but suggest that in zjizto these kinases are functionally distinct. Furthermore, the fact that the two polyomaviruses have such distinct tumor profiles leads one to suggest that the ability to bind and activate different members of the Src family leads to this difference in tumor profile. Interestingly, Fyn kinase has recently been shown to be associated with the CDS/T-cell receptor complex in T cells (Samelson et nl., 1990) and has been proposed to play an important role in signaling in lymphocytes following antigen presentation. T h e possibility therefore exists that the introduction of HamT into these cells bypasses the need for antigen presentation and allows deregulated cell growth. If this is the case, the study o f lymphocytes expressing HamT might allow one to dissect the role of Fyn in the signal transduction process.

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D. RATIONALE FOR STUDYING THE ONCOCENIC ACTIONOF PYMTI N VIVO Polyomavirus is one of the most powerful carcinogens known, causing tumors in vivo with latency periods as short as 3-4 wk. In addition, PymT interacts with only few cellular proteins. These unique features give rise to the expectation that PymT-induced tumor formation might involve only a few, if any, unknown secondary genetic alterations and therefore might be amenable to systematic molecular analysis. Studying the oncogenic effect of PymT in transgenic mice in viuo rather than the complete virus has distinct advantages because the ability of the virus to infect or replicate in particular tissues is no longer relevant. Furthermore, possible effects of PymT on cell lineages that are refractory to tissue culture can be assessed; cell lines established at different time points of tumor formation should allow investigation of cellular alterations at different stages of tumor progression. i t is crucial to realize that the synchronous initiation of tumorigenic events in a large number of cells caused by the viral oncogene is essential to these studies and makes them superior to an approach in which, for example, chemical carcinogens are utilized. II. Consequences of PymT Expression in Vivo

The first experiments that aimed to investigate the oncogenic potential of PymT in uiuo were reported by Asselin el al. (1983). Direct subcutaneous injection of DNA encoding PymT did not induce the formation of tumors in newborn Fisher rats. In contrast, co-injection of a plasmid encoding either polyomavirus small or large T antigen led to the formation of tumors. In a second study, the same authors found that in newborn hamsters the direct injection of recombinant plasmids encoding PymT invariably led to the formation of subcutaneous tumors at the site of inoculation, albeit with a 5-10 times longer latency than wild-type polyomavirus DNA (Asselin et al., 1984). However, the tumors that arose in the course of these studies were not analyzed histologically. OF PYMTUNDER THE CONTROL OF A. EXPRESSION THE POLYOMAVIRUS EARLYREGION OR GENERAL PROMOTER ELEMENTS

Several investigators aimed to express polyoma middle T antigen either under its own promoter or under the control of promoter elements that confer expression in a wide number of tissues. In most of

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these studies, PymT caused the formation of endothelial tumors or hemangiomas. Transformation of endothelial cells by PymT zn vzim was first described by Kornbluth et al. (1986). Wing webs of 1-wk-old chickens were inoculated with a replication-competent avian retrovirus that transduced PymT under the control of the Rous sarcoma virus long terminal repeat (LTR). As early as 1.5 w k after the inoculation, endothelial tumors formed that developed into massive cavernous hemangiomas of u p to 1-cm diameter within the next 10 days. Hemangiomas were detected within the skin and muscles as well as in the mesenterium and intestine. Inoculation of the chorioallantoic membrane of 4-day-old eggs led to hemangioma formation within 4 days as well as to a squamous metaplasia of the ectodermal epithelium. In all cases, the provirus was found integrated into the host genome and PymT protein was expressed. T h e study also demonstrated that PymT was capable of transforming nonestablished cell lines In uttro in the absence of complementing genes such as polyomavirus large T or SV40 large T antigen, if overexpressed from a strong retroviral promoter. Bautch ut al. (1987) generated transgenic mice to study the effect of isolated expression of PymT in the natural host of polyomavirus, using a construct in which the PyniT cDNA was under the control of a replication-defective version o f the polyomavirus regulatory early region. Two transgenic sublines that originated from the same founder animal, but differed in the copy number of the transgene, were established; in both lines, all animals that carried the transgene either died or suffered from severe anemia due to the formation of multifocal hemangiomas o f the vascular endothelium. PymT acted as a dominant oncogene leading to the transformation of vascular endothelial cells within 4-10 wk after birth, with 100% penetrance. However, expression of the transgene could only be demonstrated in testis and tumor tissues. Efficient testicular expression was also observed in other transgenic lines carrying polyomavirus large and small T antigens under the control of the virus early regulatory region (Bautch, 1989), as well as in transgenic mice carrying the entire polyomavirus early region (Wang and Bautch, 1991). Interestingly, testicular tissue seems to be refractory t o the turnor-promoting effects of the polyomavirus early gene products. In transgenic mice carrying the entire polyomavirus early region, hemangiomas were not the predominant tumor type. In addition to vascular lesions, bone tumors were observed; individual lines also developed lymphangiomas and fibrosarcomas. Specific RNAs for the small and middle T antigens were detected in tumor tissues as well as in liver, spleen, and kidney. In transgenic mice generated by pronuclear DNA injection, expres-

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sion of the transgene, in this case PymT, often only occurs after birth in various tissues depending on the promoter or regulatory elements used. To elucidate a possible action of PymT on early mouse development, we decided to employ embryonic stem (ES) cell chimeras utilizing PymT-expressing ES cells. Such ES cells were generated by infection with a replication-defective PymT-transducing recombinant retrovirus, N-TKmT, and were selected and analyzed for PymT expression in vitro (Williams et al., 1988). This experimental approach to investigating PymT-induced oncogenesis through the generation of chimeric mice by blastocyst injection of PymT-expressing ES cells was based on the assumption that expression of PymT would be maintained throughout development in a broad range of tissues (reviewed by Wagner et al., 1991). By analogy to the experiments performed by Kornbluth et al. (1986), we had demonstrated the functionality of our recombinant virus, which expressed PymT under the control of the thymidine kinase promoter, by showing that a single injection into newborn mice caused the death of these animals within 2-4 wk because of the formation of cavernous hemangiomas. Surprisingly, expression of PymT had no obvious effect on the growth characteristics of ES cells in vitro, although we noted that the requirement of PymT-expressing ES cells for leukemia inhibitory factor (LIF) was decreased (E. F. Wagner, unpublished observations). Furthermore, the differentiation potential during the implantation stage and the subsequent early stages of mouse development in chimeras in vivo was also not affected. However, at midgestation, PymT exerted a dramatic effect. All chimeric embryos were arrested in development. Vessel formation in the yolk sac was disrupted; instead of primary capillary plexae, blood-filled sac-like structures formed and frequent hemorrhaging into the amniotic fluid caused the death of embryos (Fig. 3). T h e embryos often showed expanded chest cavities, enlarged primitive hearts, and misshaped heads. Injection of chimeric material originating from the embryo proper o r from the yolk sac caused the formation of hemangiomas in syngeneic 129/Sv mice, demonstrating that endothelial cells in extraembryonic as well as in embryonic structures had been transformed. Several transformed endothelial (End) cell lines were established from midgestation embryos as well as from tumor tissue derived from neonatal mice. The analysis of these cell lines will be described later. Although we had used a replication-deficient virus for our study to avoid viremia that could complicate the analysis of an observed phenotype, Fusco et al. (1988) described the development of an acute thrombocythemic myeloproliferative disease after infection of young adult

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NIHiOLAC mice with a recombinant PymT-transducing retrovirus in the presence of helper virus. Within 1-2 wk of intraperitoneal virus injection, the animals developed a profound spleen enlargement; multiple thrombi in skin, muscles, and mesenterium; and hematomas and hemorrhagic effusions. Of the cells in the bone marrow, 10% were identified as megakaryocytes; although the erythroid and lymphoid lineages were decreased, the numbers of myeloid cells of all stages of differentiation were abnormally expanded. The authors found reduced platelet aggregation and ATP secretion in response to aggregating agents. They detected PymT RNA exclusively in the bone marrow and PymT protein by indirect immunofluorescence in only few cells of this organ. Longterm bone marrow cultures obtained from infected mice contained more than 90% megakaryocytes, most of which expressed PymT as evidenced by indirect immunofluorescence. Interestingly, C57BL/6J Fv-2r mice inoculated with the same viral preparation showed no signs of disease. The study by Fusco et al. (1988) represents the only case to date in which an effect of PymT on a tissue type is reported that is not affected by the intact virus. Although the authors detected a profound effect of retrovirally transduced PymT on megakaryocytes and myeloid progenitors, they did not observe the development of hemangiomas. Since the pathology was detected in several animals, it must have been integrationsite independent. On the other hand, we have not detected any hematological disorders caused by PymT, in transgenic mice that expressed the oncogene in myeloid cells or in a bone marrow reconstitution system (discussed subsequently). Therefore, the pathology observed by Fusco et al. was very likely to be peculiar to the combination of the retrovirus and recipient strain used. An alternative explanation is that the clotting defect observed was the consequence of massive hemorrhaging in the animals rather than a specific functional impairment of the megakaryocyte compartment. T h e transgenic and chimeric mouse model systems have clearly demonstrated that expression of PymT is sufficient to induce tumors in specific cell types in vivo (Table I). These results confirm that part of the tumorigenic potential of polyomavirus resides in the viral early region and that PymT-mediated tumorigenesis does not need viral replication or the viral life cycle. Surprisingly, in the transgenic lines harboring the FIG. 3. Hemangiomas develop on the yolk sacs of PyinT ES chimeric embryos. (A) Control day 13 embryo. (B) Multiple hemangiomas are visible in a PymT chimeric embryo. (C) Embryo died prior to analysis by hemorrhaging, presumably as a result of endothelial cell disruptions. Yolk sacs of non-chimeric control (D) and PymT chimeric (E) day 12 embryos are shown.

TABLE I MODELSYSTEMS POK PYMT-MEDIATED TUMORIGENESW Oncogene

-

PpmT

Promoter

Py-early region

Experiniental system Direct DNA injection

Expression ND

Tumor types

+ (ND)”

w

30

References Asselin et al. (1983, 1984)

PymT

KSV-LTK

Retrovirdl infection

Primary CEF

Hemangioma

Kornbluth et al. (1987)

PymT

Py-early region

Trdnsgenics

Tumor tissue, testis

Heniangioma

Bautch et al. (1987)

PymT+ PylT+ PysT

l’y-early region

Transgenics

Tumor tissue

Osteosarcoma, hemangioma, lymphangioma, tibrosarcoma

Wang (1991)

PyniT

TK

Retroviral infection

Tunior-derived cell lines

Heniangioma

Williams et al. (1988)

PymT

TK

<;himeras

ES cells, tuniorderived cell lines

Heniangioma

Williams et al. (1988)

PymT

MLV-LTR

Retroviral infection

BM, megakaryocytes

Myeloproliferative disease

Fusco et al. (1988)

PymT

IgEiPy-early region

Trdnsgenics

Tumor tissue, brain, (spleen, liver) niyeloid cells

Various carcinomas, niammary tumor hemangioma

Kdssoulzadegdn et al. (1990)

-

Ds

Guy et al. (1992)

PymT

MMTV-LTR

Transgenics

PymT

Rat insulin

Transgenics

Not detected

-

Bautch (1989)

P y m T + Pylt/SV40T

Rat insulin

Double transgenics

T u m o r tissue

p Cell t u m o r <

Bautch (1989)

Mammary gland, t u m o r tissue, salivary gland, ovary, epidid ymis

M a m m a r y adenocarcinoma

PymT

TK

Transgenics

CNS t u m o r tissue

Neuroblastoma

Aguzzi et al. (1990)

PymT

TK

Retroviral infection, neuronal transplant

Neuroectoderrnal cells, t u m o r tissue

Hemangioma

Aguzzi et al. (1991)

PymT

TK

Rertroviral infection, b o n e marrow transplant

BM, T-cells, mast cells, H e m a n g i o m a

TK

Retroviral infection

HamT

myeloid colonies

-,I

Hemangioma

Abbreviations: EM, bone marrow; CNS, central nervous system; HamT, hamster polyomavirus middle T antigen; IgE, immunoglobulin enhancer; MMTV, mouse mammary tumor virus; MLV, murine leukemia virus; ND, not determined; CEF, chicken embryo fibroblasts; Py-early region, polyomavirus early regulatory region; PylT, polyomavirus large 1 antigen; PysT, polyomavirus small T antigen; RSV, rous sarcoma virus; SV4OIT. SV40 virus large T antigen; TK, thymidine kinase. The authors have only reported the formation of tumors but not the tumor type. c p Cell tumors are caused independently of PymT expression by SV40IT and PylT under the control of the rat insulin promotor. c' Due to the presence of virus-producing cells, expression of HamT could not be detected unambiguously in infected cells. 0

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complete polyomavirus early region generated by Bautch et al., the tumor spectrum caused by PymT was not expanded beyond the tumor spectrum elicited by the intact virus, arguing that viral tropism is, at least in part, based on intracellular signal transduction pathways. Indeed, none of the transgenic models described earlier completely recapitulated the full spectrum of tumors caused by polyomavirus, that is, “the polyonia tumor constellation.” When PymT was expressed under the control of either its own promoter or broad-specificity regulatory elements, hemangiomas were the predominant tumor type observed; expression of’all three -1. antigens caused the formation of bone tumors, rather than the formation of parotid tumors as initially observed by Gross (1953a). T h e rapid kinetics by which endothelial cells are transformed by Pym‘T may account for the fact that mainly this tumor type is observed in PymT transgenic and chimeric mice. Furthermore, the occurrence of lethal hemangiomas in chimeric mice at midgestation precluded obtaining answers to sonie interesting questions. It is not clear if I’vm‘1’-expressing ES cells retain their full developmental potential including colonization of the germ line, or i f they are developmentally compromised. We have recently started to approach this question by generation of chimeric mice in which the embryo proper is completely ES cell derived. Such “ES mice” generated from aggregates of tetraploid niorulae and ES cells (described by Nag)- cf al., 1993) allow1 a precise determination of the developmental potential of the ES cells used; expression of a transgene can be monitored in different tissues throughout development. Preliminary results using PyniT-expressing ES cells indicate that embryonic development ceases as soon as vasculogenesis starts. Histological analysis suggests that the formation of the primary capillary plexus in the yolk sac as well as vessel formation in the embryo proper is substantially impaired. However, these observations have not yet been correlated with a detailed expression analysis of PymT. B.

E X P R E S S I O N OF 1’YM-i- U N D E R ‘THE CONTROL

Ok

TISSUE-SPECIFIC PROMOTER

ELEMENTS

More recently researchers have described the generation of transgenic mice that express constructs in which PymT is under the control of tissue-specific promoter elements. Rassoulzadegan et ul. ( 1990) derived a transgenic strain M‘1’-75 in which PymT was expressed from a chimeric promoter containing the IgE heavy chain enhancer instead of the polyoma enhancer. Although the founder was a phenotypically normal male, following serial breeding most of the females developed tumors, including carcinomas of the salivary and the thyroid glands, mammary tumors,

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adenocarcinomas of unknown tissue origin, and small liver hemangiomas. Several pulmonary metastases were observed. With the exception of one male that developed a salivary tumor, all males were phenotypically normal. Other than in one female that showed PymT expression in spleen and liver, PymT was only detected in tumor tissue and in brain. This expression pattern does not correspond to the pattern observed previously for genes controlled by the IgE enhancer and was most likely caused by cis-acting elements present at the integration site. Investigators had shown that the IgE enhancer can efficiently direct the synthesis of the c-myc oncogene in myeloid cells (Adams et al., 1985). Therefore, we were interested in determining whether PymT was expressed in bone marrow cells of MT-75 mice. We performed in uitro kinase assays on -50 pooled colonies, each of different myeloid lineages, and found PymT expression in erythroid, macrophage, granulocytemacrophage, and mixed colonies (Fig. 4). Despite the expression of PymT in myeloid cells, we did not find any hematological abnormalities in MT-75 mice. Transgenic mice described by Bautch (1989), which carried PymT coding sequences linked to the rat insulin promoter, did not show any tumor formation. Although expression of the transgene could not be demonstrated in this line, PymT was detected in p cell tumors of the pancreas after these mice had been crossed with transgenic strains that expressed either SV40 or polyomavirus large T antigens under the control of the rat insulin promoter. Independent of the presence of PymT, p cell tumors are caused by both SV40 and polyomavirus large T antigens. In the insSV40 x insPymT and insPylT x insPymT transgenic mice, the p cell tumor profile was not altered by the expression of PymT. However, the expansion of the target tissue allowed the detection of the PymT transgene. In contrast to numerous in uitro experiments, these

FIG. 4. PymT expression in various myeloid cells from transgenic mice MT-75. Expression was measured by an in ~ i t r oassay for PymT-associated tyrosine kinase activity. D3.3 are PymT-expressing ES cells; erythroid (Ery),mixed (Mix), macrophage (Mac),and granulocyte/macrophage (GM) colonies were picked from methyl-cellulose medium and assayed for PymT activity.

142

FRIEDEMANN KIEFER E T A L .

double-transgenic mice showed no oncogene cooperativity between the PymT and large T antigens. A transgenic line described by Aguzzi et al. (1990) contained Pym'I' under the control of the thymidine kinase promoter. These mice expressed PymT in a highly tissue-specific manner; expression was only detected in the central nervous system, leading to the development of sympathetic hyperplasia and neuroblastomas with high penetrance. The unusual pattern of transgene expression, the observation that only one of four founder animals gave rise to a line that developed a neurological disorder, and the fact that in other transgenic models PymT expression in the brain did not lead to a neuronal hyperplasia suggest that the chroniosotnal integration site may be a major component of' this malignancy. Inspired by the fact that the inoculation of newborn or immunodeficient mice with polyoniavirus can lead to mammary adenocarcinomas, Guy el nl. (1992) generated several transgenic lines that expressed a PymT fusion gene under the control of the murine mammary tumor virus ( M M T V ) LTR. In all lines and in strict correlation with the onset of transgene expression, which showed a wide variation in levels and temporal pattern, multifocal tumors of the mammary gland appeared. In one transgenic line males also developed mammary tumors, most likely because of the expression of PymT before the normal regression of the male mammary epithelium. Apart from the mammary gland, PymT was detected to a lower degree in the salivary gland, the ovary, and the epididymis. Transgene expression in the lung was due to pulmonary metastases that occurred with a very high frequency in all tumor-bearing mice. Pulmonary metastases were also observed after transplantation of' primary tumors into the mammary fat pads of syngeneic mice. The simultaneous occurrence of multifocal tumors, with a latency period as short as 30 days, suggests that PymT suffices to transform the mammary epithelium in this transgenic model without additional activating events. When the MMTVIPymT strains were crossed with a strain that carried a disrupted c-Src proto-oncogene, the rapid tumor progression was no longer observed (Guy et al., 1994). Only rarely after long latency periods did abnormal hyperplasia of the mammary gland develop. These malignancies were accompanied by elevated expression of PymT and activation of the PymT-associated Yes kinase. However, crosses between MMTVIPyml' transgenic mice and mice harboring a disrupted allele of the Yes tyrosine kinase did not display an altered pattern of tumor formation compared with the parental MMTV/PymT transgenic mice. Expression of PymT in uiuo using tissue-specific promoter elements allowed the investigation of tumor formation in a variety of tissues since

MIDDLE T ANTIGENS OF POLYOMAVIRUSES

143

the lethal phenotype due to rapid transformation of vascular endothelium was no longer observed. The detected tumor types were of mesenchymal and epithelial origin, the same target compartments of the intact virus. Exceptions to this rule are the transgenic mice described by Aguzzi et al. (1990), which developed neuroblastomas. However, in this case the phenotype was most likely caused by the integration site of the transgene, since the same author has demonstrated normal differentiation of neuroectodermal cells after infection with a PymT-transducing retrovirus (Aguzzi et al., 1991; described next). Interestingly a number of tissues including testis, ovary, neuronal cells, and bone marrow cells are apparently refractory to the action of the PymT oncogene. Therefore, they must lack important intracellular signal transduction components that participate in the PymT-mediated transformation process. In contrast, PymT seems to act on endothelial cells and mammary epithelium as a single-step oncogene. The apparent susceptibility of endothelial and epithelial cells is likely to involve specific intracellular components such as Src-related tyrosine kinases. The availability of mouse strains carrying a disrupted allele of a signal transduction molecule makes it finally possible to test for the contribution of the individual components to the transformation process. In contrast to PymT-induced mammary tumorigenesis for which the Src tyrosine kinase seems to be required, we were able to show that in the transformation of endothelial cells the Yes kinase is predominantly involved (see subsequent discussion). Interestingly, the neu oncogene, which also transforms mammary epithelium in an apparently single-step fashion, is a constitutively activated tyrosine kinase. Therefore, protein tyrosine phosphorylation of particular target molecules may be part of the specific action of PymT on endothelium and mammary epithelium. The available data suggest that these targets might be different in endothelial and epithelial cells and that the transforming complex might, at leas: in part, be directed by the tyrosine kinase toward these critical intracellular mediators. Finally, there seems to be a class of tissues in which secondary events are a prerequisite for tumor formation. This is particularly obvious in the transgenic strain generated by Rassoulzadegan et al.’ ( 1990). These mice develop tumors in only a fraction of the tissues that express PymT and show a clear progression from preneoplastic to fully malignant forms of some tumors. Secondary events are obviously necessary in some organs but not in others. A threshold of PymT expression may exist that makes additional genetic modifications necessary in the case of subthreshold expression levels (see also Fig. 6B). Surprisingly no oncogene cooperation is observed in transgenic animals containing PymT in combination with PylT or SV4OlT antigens. In

144

FRIEDEMANN KIEFER E T .*\I..

a great number of other transgenic model systems, oncogene cooperation has been demonstrated (for review, see Hunter, 1991). Cell-cell interactions in the intact tissue may represent additional elements of transformation control that are missing in primary cell cultures in which t.he cooperativity of PyniT arid PylT has mainly been demonstrated. Alternatively, expression levels of PymT may be too low to be effective since, in all cases of double-transgenic mice, expression has only been demonstrated by RKase protection analysis (Bautch, 1989). C;.

PYMTL RECONSTITLTION

4EXPRESSION ~ I N ORGAN ~ ~ SYSTEMS

~

~

~

To express PymT in a particular cell compartment or tissue, organ reconstitution systems were utilized. These systems circumvent problems of inappropriate expression or the lack of suitable tissue-specific promoters encountered during the generation of transgenic mice, by retrovii-a1infection of embryonic or adult tissue h i 7dro and reimplantation of infected selected cells into a mouse. 1. 2Veitm i u I ?inw f ~ ltitut u ion

Aguzzi et d.(1991) described a neuronal transplantation model that mimics the structural and functional properties of the normal rat brain. Embryonic neuronal cells were infected with a replication-deficient PvmT-transducing retrovirus and injected stereotactically into the caudoputamen of adult. rats. O f the recipients, 70%,died between 13 and 50 days after the transplantation from intracerebral and subarachnoidal bleedings. T h e remaining 30%.of the recipients also developed hemangiomas but did not suffer from hemorrhages. PymT R N A was detected by in situ hybridization in neuroectodernial cells with neuronal and glial morpholog);; the highest RKA levels were expressed by vascular tumor tissue. Thus, the tissue-specific transformation of vascular endothelial cells was caused by a differential susceptibility o f t h e infected cells to the action of Pym'I' rather than by selective integration of- the virus. In contrast to all other murine PymT-transformed endothelial cell lines, lines that were derived from neuronal transplants failed to induce hemangiomas in syngeneic mice.

2.Bone M u rroii~Recorzstitution We were interested in investigating possible consequences of Pym?' expression in hematcipoietic cells in more detail. Although we had demonstrated that expression of PymT in four niyeloid lineages of the transgenic line MT-'75 (Rassoulzadegan et al., 1983) did not cause overt hema-

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tological alterations, Fusco et al. (1988) had reported a profound effect of PymT expression on megakaryocytes. Previously, researchers had shown that PymT can render the hematopoietic progenitor line FDC-P 1 growth factor independent (Metcalf et al., 1987; Muser et al., 1989) and that the constitutively activated tyrosine kinase v-Src causes a myeloproliferative disease when expressed in hematopoietic cells (Keller and Wagner, 1989). To direct expression of PymT to the hematopoietic system, we chose the well-established retroviral gene transfer protocol (Wagner and Keller, 1992). Primary bone marrow originating from either CBA/J or C57BL/6J mice was exposed to a recombinant replication-defective PymT-transducing retrovirus (Williams et al., 1988), either by cocultivation with a viral producer cell line or by incubation with high-titer virus supernatant. The experimental details of several infections are summarized in Table 11. The efficiency of the retroviral infection was monitored by the growth of G4 18-resistant myeloid colonies in semisolid medium. To detect a possible effect of PymT on hematopoiesis, we performed differential colony counts and established liquid cultures from the infected bone marrow. With the exception of a single experiment (indicated by * in Table 11)that gave rise to an immortalized primitive hematopoietic cell line, we did not observe significant differences between PymT-infected bone marrow and bone marrow that was infected by a retrovirus transducing only the neomycin resistance gene. The singularity of the event as well as the molecular analysis of the immortalized hematopoietic cell line obtained suggested that the retroviral integration, possibly in combination with PymT, caused the immortalization event. Having shown that the retroviral expression system was suitable for expression of PymT in hematopoietic cells, we injected infected bone marrow cells into lethally irradiated syngeneic recipient mice, in which these cells reconstituted a functional hematopoietic system. At various time points after transplantation, individual recipients were sacrificed and their hematopoietic status was assessed. We found G4 1%resistant colony-forming cells in the bone marrow and spleens of 5 of 10 analyzed recipients (data summarized in Table 111).Note that the percentage of G4 1&resistant progenitor cells in the spleen of these mice was consistently higher than in the bone marrow. In two individuals, we demonstrated the presence of PymT-associated kinase activity in bone marrow and spleens as well as in myeloid colonies, T cells, and mast cells derived from these organs. We were not able to detect any hematopoietic alterations in these animals other than mild enlargement of the number of clonogenic progenitors in the spleens of three individuals. We assume

Donor BM 5-FU pretreatment"

Strain

Yes

CBA/J C:BA/J

Yes

CUA/J C.5 7 B IJ6J (257BLJ6J

g m

~~

~

~~~

Yes NO

Yes

C:iSUI/6J

NO

CB7BI/6J

NO

G418-resistant CFC after 24 hr. Manipulation< Infection(' None None Ery-Lys Gradient None Gradient None Ery-Lys

(37 B I /ti] ~~

~~

~

~

I

I:! 10

44 14 10 >

2 2

cocult vsup

10 0.5

vsup vsup vsup

0.5 6.5

22

(%I

ND 6.5 70 ND ND ND ND 37 48 35 53 .5 9 72

PymT-associated kinasex

Hematological alterations

Bone marrow transfer"

+ + ND +

Yes* NO NO

Yes

N O

Yes

No No No No

Yes Yes

ND ND ND ND

+ + + +

N0

ND

~~

No No No No ~

1'i.iiiiary Imie niarrow ( R h l ) o f t h c indicated n i w s e strain W;IS infccrctl with the l'yni.1-traristlucing recombinant murinc retrovirus I\j-TKniT (Williams a/ nl., 1988) the prrscnce o f intei-lrukin-l a , interleukin-3, and er) thropoietin. ND,N o t determined. Whew i ndia trd. In)ne inarrow w a s harvcstrd from niice that had I~CCIIpi-etrratetl with 9-lluoroitracil (5-FU). the niiiltiplicity of infection. in sonic experiments I)one niiiri-ow w a s fract ion;itctl using a Percoll density gradient to rcniove mature hematopoietic cells, c,xperinirnts ervthrocycs were Iysed using ;inimoniuni clhriclc. Keti-ovii-alintection \ v i i ~;icconiplishecl either b y cociiltiratii)ii with virus-p luring libroblasts (co~ult)or b) incubation in a high titer virus supernatant (vsup). Aftei- 24 h r the percentage o f successfully infected cells wits determined I) I standard nicthyl ccllulose colony asaay in the presence of 1.3 mginll G4 18. / Where indicated, 21 second tolon) assay was pcrlimmd aficr a %clay selcc n period in Ole presence of 1 mg/ml G4 18. x I ' y n i l expr-ession win cicterininetl h y an t i ! 7 r i / , o kinasc assay pel-formed o n p o d s of inyeloid colonies growth in methyl cellulose or in liquid bulk cultures. Wherc. indicated, infected I)onc inarrow w;is ti-anspl;intrcl into syngcncic, lethall) irradiated recipients. (1

in

IkS

None Ery-Lys None Ery-Lys

\.sup vsup cocult vsup cocult vsup vsup cocult

(7;

G418-resistant CFC af-ter 3d selection/

'8

I'

TABLE I11 I'YMTEXPRESSION I N BONEMAKROW RE~:ONS.~ITITI.E~ MI(:E~~

Gene transferred

Time after BM graft" (mo)

PymT

2.5

G418-resistant CFC in BM a n d spleen" UM: 65%; Spl: 69%.

DNA analysis(

PyinT expressionf'

int prov

BM, T cells, mast cells

int prov

ND

int prov

ND

(>4000<:F(:/lO(i)

PymT

3

BM: 3%; Spl: 14% (825 CFC/ 10") EM: 1%; Spl: 5%

PymT

4

BM: 23%; Spl: 34%

ND

PymT

7

BM: 10%; Spl: 17% (13.50 C:FC/IW)

int prov

BM, Spl ND

neo

2.5

BM: 65%; Spl: 69% (>200 CFCI 10")

int prov

-

neo

4

BM: ~52%;Spl: 49% (<200 C F U 10")

ND

-

PymT

3

Lethally irradiated ntire werc reconstituted with syngeneic bone marrow that had k e n infected with the PymT transducing virus N-TKniT. ND,Not clctet-mined. At the indicated time point after tile bone inarrow graft, the percentage of.(;4 I8-resist;mt colony-forming cells ( W C ) in bone marrow and splecn wis clcterniinrd. Niinibers in brackets give the trcqucncy of colony-forming cclls found in the spleen per 10" splcnocytes. c The presmcc of the intact provirus (int prov) in spleen and Ixmc inarrow was demonstrated by Southern blot analysis. d Pynil' expression was shown b y is 7 4 ~ kinase 0 assay. BM, Bone niarrow; Spl, spleen. (8

'2

148

FRIEDEMANN KIEFER E7’Al..

that this effect is the result of the disturbance of steady-state hematopoiesis in these animals rather than a speciIic effect of the oncogene. The number of’ progenitor cells in the bone marrow of the same mice was normal, and no progenitor cells were found in the peripheral blood. In this model, PymT was not able to reproducibly imniortalize hematopoietic cells o r to cause gross alterations in the hematopoietic system. However, w e detected the formation of hemangiomas in -30% of our bone marrow-reconstituted mice. Ill. Expression of the Hamster Polyomavirus Middle T Antigen in Vivo We were interested in determining whether HaniT would be capable of transforming target tissues other than endothelial cells. We therefore constructed a recombinant retrovirus containing Ham’l’ sequences, designated N-TKHamT (S. A. Courtneidge, unpublished), the structure of which is analogous to that of the PymT-transducing virus N-TKmT (Williams et d., 1988).When we inoculated newborn mice with this virus w e detected the formation of minute hemangiotnas in 60% of the animals after a latency period of 30 days or longer. An attempt to isolate transformed endothelial cell lines from these lesions was unsuccessful. It was possible to generate more pronounced lesions that developed with the same latency period by injection of virus-producing fibroblasts into newborn mice. Because of the transformed nature of the producer cell line, isolation of transformed endothelial cells was not possible from these lesions. We obtained a very surprising result when viral producer cells secreting &-TKHamT were injected into mutant mice that carried a null allele for h e tyrosine kinase Fyn (Stein et al., 1992). Newborn Fyn-deficient homozygous mice developed hemangiomas after inoculation with N-TKHamT. From these experiments we concluded that, despite their different biochemical properties, both HamT arid PymT can transform target endothelial cells. Furthermore, our results suggest that a tyrosine kinase other than Fyn may associate with HamT in endothelial cells of Fyn-deficient mice, despite the evidence that Fyn preferentially binds to Hanil-. An alternative explanation is that mT-associated kinase activity is not required for heniangioma formation and that one of the other associated proteins causes the transformation. However, this possibility seems unlikely in view of the finding that hemangiomas occur less frequently in Yes-deficient mice (Kiefer el al., 1994).

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IV. Analysis of PymT-Transformed Endothelial Cells Endothelial cells have proven notoriously difficult to culture. Chimeras and young mice provide a convenient source of PymT-transformed endothelial cells (endothelioma, End cells) which, in contrast to primary endothelial cells, grow continuously although they retain important features of differentiated endothelium (for review, see Wagner and Risau, 1994; Fig. 5). Williams et al. (1988,1989) derived highly tumorigenic End cell lines that caused the formation of hemangiomas within 18 hr to 4 days of injection into syngeneic and nonsyngeneic mice, or even into other species such as rat, chicken, and quail. Hemangioma formation was shown to be a specific property of End cells, since PymT-transformed fibroblasts or embryonic carcinoma cells were not capable of eliciting this tumor type. Although syngeneic newborn mice succumbed to the tumors caused by the End cell graft within 1-2 wk, only 50% of the injected adults died. The remaining half formed hemangiomas which, on serial transplantation, lost their potential to induce tumors rapidly. Using an isoenzyme marker as well as [3H]thymidine-labeled, mitomycinC-treated End cells, Williams et al. (1989) showed that the vast majority of tumor tissue was host derived. Some insights into a possible mechanism of hemangioma formation came from in vitro experiments in which researchers showed that all End cell lines examined formed large cystic structures, very reminiscent of hemangiomas in fibrin gels (Montesano et al., 1990). The aberrant morphogenic behavior of End cells was tightly associated with a high proteolytic activity secreted by these cells that was caused by an increased production of urokinase-type plasminogen activator (u-PA) and a reduced synthesis of the plasminogen activator inhibitor PAI-1. Most surprisingly, the addition of exogenous serine protease inhibitors to fibrin gels altered the morphogenic properties of End cells; they now formed asterisk-like structures similar to the tubes formed by primary endothelial cells (Montesano et al., 1990). Several attempts to establish a causal link between proteolysis/u-PA expression and hemangioma formation using retroviral gene transfer of u-PA and PAI-1 into primary endothelial cells and End cells have failed (F. Kiefer and E. F. Wagner, unpublished data). However, we recently succeeded in deriving additional End cell lines that show a significantly reduced net proteolysis and still are able to form hemangiomas in vivo. Independent evidence arguing that u-PA is not solely responsible for hemangioma formation comes from End cell lines that have been established from mutant mice lacking

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151

a functional u-PA or t-PA gene (in collaboration with P. Carmeliet, Leuven). Preliminary data show that these cell lines are still capable of forming hemangiomas. Analysis of PymT mutants has established the importance of the interaction between PymT and the Src-related tyrosine kinases Src, Fyn, and Yes for PymT-mediated transformation. It has recently been shown that PymT can efficiently induce hemangiomas in Src-deficient mice (Thomas et al., 1993). Although the same observation was made with Fyn-deficient mice, in Yes-deficient mice hemangiomas formed with a lower efficiency and with a significantly longer latency period, arguing that the Yes kinase is particularly important for the transformation of endothelial cells (Kiefer et al., 1994). End cell lines derived from all three types of kinase-deficient mice were indistinguishable from their wildtype counterparts. Biochemical analysis of the transforming complexes in various End cell lines suggested that Yes contributes significantly more to the PymT-associated kinase activity in this cell type than in fibroblasts (Figs. 5 and 6). The availability of PymT mutants such as HamT and of Src kinasedeficient mutant mice allows the possible identification of molecules necessary and sufficient for PymT-mediated tumorigenesis. Clearly tyrosine kinases belong to this group, although there seems to be a threshold level of kinase activity that can be reached by any two of the three ubiquitously expressed kinases Src, Fyn, and Yes (Kiefer et al., 1994; Fig. 6). More interestingly, the observation that HamT is still able to transform endothelial cells in Fyn-deficient mice suggests that a further, perhaps endothelial cell-specific, kinase is involved in this process. Although all End cell lines described here were established by culture of tumor tissue obtained from midgestation embryos or newborn mice, Dubois et al. (1991) described the isolation of one PymT-transformed endothelial cell line from adult transgenic mice of the strain Py-4 (Wang and Bautch, 1991). These mice carry the complete polyoma early region and develop multiple skin hemangiomas at 6-8 wk of age with 100% penetrance. End cells were established from these hemangiomas by labeling the cells with fluorescent acetylated low density lipoprotein (LDL) and two subsequent rounds of cell sorting. The cell lines obtained showed a typical endothelial cobblestone morphology and were tumorigenic in uiuo. In contrast to the End cell lines first isolated by Williams et al. (1988), these endothelioma cells caused hemangiomas with a much FIG. 5. Morphology of End cells (A) and a typical End cell-induced hemangioma (B). (C) PymT-associated tyrosine kinase activity in different kinase-deficient End cell lines (Src, Fyn, Yes). wt, Wild-type End cells.

152

FRIEDEMANN KIEFER E T AL.

k

[PyrnT.Fynl

\ Oncogenic Signal

PymT

3 known kinases

2 known kinases lhreshold lor translormalion

low

Iransformalion efficiency

high

FIG. 6. Proposed model of PymT activity in endothelial cells as a function of its association with the three known Src-family tyrosine kinases Src, Fyn, and Yes. The existence of an as yet unidentified tvrosine kinase (X) cannot be excluded.

longer latency period of 2-4 wk and most likely without the involvement of host cell recruitment. Similar to transformation of endothelial cells by PymT in uiuo and in uitio, which appears to occur as a rapid apparent single-step process, pancreatic ductal adenocarcinomas can be induced by retroviral transduction of Yym'l' into the islets of Langerhans (Yoshida and Hanahan, 1994). Infection of pancreatic islets from juvenile mice yielded cell lines that, after several cycles of single-cell cloning, consisted of two subtypes of cells: one that does not express specific markers and a second that expresses markers of ductal epithelial and islet cells. On re-injection into mice, these cells formed well-differentiated ductal adenocarcinomas.

MIDDLE T ANTIGENS OF POLYOMAVIRUSES

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This model system may provide insights into the naturally occurring islet progenitor cells as well as the target cells in pancreatic cancer, and the molecular changes in the progression of this cancer type.

V. Outlook Tumorigenesis is currently understood as a multistep process that can be elicited by the alteration of a number of pathways and processes. Transgenic and chimeric animals that reproducibly develop tumors as the result of a defined alteration, namely the expression of an isolated viral oncogene, are important tools in developing an understanding of the various steps involved in tumor formation. The existence of different types of tissues that display a differential susceptibility to the action of PymT will ultimately lead to the identification of particular intracellular components that are only present in susceptible cell types and are responsible for the specific action of the oncogene. PymT-transformed cell lines will be crucial tools in these studies. Endothelioma cell lines, for example, should allow the identification of unknown tyrosine kinases involved in the specific transformation of this cell compartment. In addition, the availability of novel strains of mutant mice lacking particular molecules of the PymT complex will allow a genetic analysis of the complex, and will complement the studies that have been performed to date using PymT mutants. Employing PymT mutants, it has not been possible to dissect the individual roles of PIS-K, the tyrosine kinases, or PP2A. For example, all mutant forms of PymT that have lost the capacity to bind PP2A lack associated kinase activity. The availability of mice carrying a targeted allele for the regulatory subunit of PIS-K or the A subunit of PP2A would allow us to address these questions. Substantial progress toward an understanding of how PymT subverts the growth control of cells has already been achieved. We know now that polyomavirus activates several cellular signal transduction molecules such as PIS-K and ShdGrb2, and thereby most likely causes the activation of the subsequent signal transduction pathways (Fig. 7). Therefore, PymT mimics the action of activated tyrosine kinase-associated growth factor receptors, giving us a first clue to an understanding of why certain tissues, such as testes, or cell types, such as hematopoietic cells, are refractory to the action of the oncogene. Either these cells to not contain the appropriate receptor molecules and/or their corresponding signal transduction elements, or a stimulation of these has no consequences because downstream effector molecules such as transcription factors are not active. Exhaustive knowledge of the molecules that interact with PymT will

154

FRIEDEMANN KIEFEK E T AL. PymT proteincomplex

FIG. 7. Schematic view of possible molecular niecliariisms involved in PymT-induced single-step endothelial cell transformation it/ UWO. Py V, Polyomavirus; GF-GFR, hypothetical endotlielial-specific growth factor-growth factor receptor loop. For other abbreviations, see Fig. 1 .

eventually lead to an understanding of how the proliferative signals generated by this oncogene cause the rapid transformation of cells. Most importantly, the lessons learned from studying PymT-mediated viral oncogenesis in uitro and in VZZIO have impressively enhanced our knowledge of growth regulation in normal cells, and have set a paradigm of an apparent single-step rather than multistep model of oncogenesis. ACKNOWLEDGMENTS We would like to thank Denise Barlow and Agi Grigoriadis for critical reading o f t h e manuscript, 1Iannes Tkadletz for photographic assistance, and Irene Acas for helping prepare the manuscript. REFERENCES

Adanis, J. M., Harris, A. M:, Pinkert, C. A., Corcoran, L. M., Alexander, W. S., C:ory, S., Palmiter, K. D.. and Brinster, R. L. (1'385). "Va'nlurt318, 533-538.

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