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A retrovirus carrying the polyomavirus middle T gene induces acute thrombocythemic myeloproliferative disease in mice

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A retrovirus carrying the polyomavirus middle T gene induces acute thrombocythemic myeloproliferative disease in mice
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  JOURNAL OF VIROLOGY, Jan. 1988, p. 361-365 0022-538X/88/010361-05 02.00/0 Copyright © 1988, American Society for Microbiology   Retrovirus Carrying the Polyomavirus Middle T Gene Induces Acute Thrombocythemic Myeloproliferative Disease in Mice  LFREDO FUSCO, * GIUSEPPE PORTELLA,1 MICHELE GRIECO, GI NFR NCO TAJANA,2 GIOV NNI DI MINNO,3 NICOLETTA POLLI,4  ND  NTONIO PINTO Centro di Endocrinologia e Oncologia Sperimentale, Consiglio Nazionale delle Ricerche, and Dipartimento di Patologia e Biologia Cellulare e Molecolare, II Facolta di Medicina e Chirurgia, University of Naples, Naples, Istituto di Anatomia2 and Istituto di SemeioticaMedica,3Facolta di Medicina e Chirurgia,Catanzaro, and Istituto di Scienze Mediche, Facolta di Medicina e Chirurgia, Milan,4 Italy Received 28 April 1987/Accepted 25 September 1987 Mice inoculated with an artificially constructed retrovirus carrying the middle T gene of polyomavirus develop acute myeloproliferative disease with severe thrombotic and hemorrhagic disorder andimpaired platelet function. The megakaryocytic lineage appears to be a target for polyoma-murine leukemia virus infection and middle T gene expression. This newly described  ise se represents a unique model system for studyingdisordersof the megakaryocytic lineage. Virus-induced leukemia is avery well-known tool to investigate the pathophysiology and the regulation of the hematopoietic system. In addition, such an approach has also provided animal models of rare human hematological diseases (13). For instance, a murine model has been re- cently described in whichAF-1, a v-ras-Ha oncogene- carrying retrovirus, causes an in vivodisease resemblingmalignant histiocytosis, an extremely rare human mononu clear phagocytic disease (4). Here we describe how inocu- lation of an artificially constructed retrovirus, the polyoma- murineleukemia virus  PyMLV), carrying the polyomavirus middle T gene (3), into NIH/OLAC adult mice results in a syndrome characterized by an acute myeloproliferative dis- ease associatedwith the proliferation of bone marrow mega- karyocytes and a severe thrombotic and hemorrhagic disor-der. PyMLV is a transforming retrovirus constructed by re- placing part of the genome of a biologicallyactive Moloney leukemia proviruswith the middle T gene of the polyoma- virus. This construct, after transfection on NIH 3T3 cells, has been rescued by using as a helper the Moloney leukemia virus, thus obtaining a transmissible virus designated PyMLV (3). This virus is able to transform fibroblasts (3, 8) and epithelial thyroid cells in vitro (A. Fusco et al. submit- ted for publication). In the present study, we have evaluated PyMLV activity in vivo by intraperitoneally injectingadult NIH/OLAC mice (8 weeks old) with 2 x 106 focus-forming units. Within the first week after inoculation, mice developed thrombi, first appearing o the tail veins and rapidly expanding to the back, ears, mouth, and mucosae. On day 15 after the inoculum, spleen enlargement up to fivefold the normal weight), petechiae, dehydrated crusts of the skin, and hair loss were found together with thrombi ofmuscles and mesentery, as well as hematomas and hemorrhagic effusions of the abdomen and chest cavities. Both hematomas and thrombi were also present in subcutaneous tissues. At later stages of the disease (3 to 4 weeks), infarctions of lung,heart,brain, and other viscera were observed. The majority of mice diedwithin 5 weeks after inoculation. This pathology cannot be ascribed to the helper virus: 20 NIH/OLAC mice   Corresponding author. were infected with 5 x 106 PFU of the helper Moloney murineleukemia virus, and the response was limited to lymphoid leukemiaobserved in some mice 2 months after inoculation. Similar observations havebeen reported by others (6). Histological examination, by glycol-methacrylate embed- ding and Giemsa staining, of tibial bones of the PyMLV- infected animals showed 10 of the marrow cells to be made up of giant multinucleated cells (Fig. 1A), identified as megakaryocytes. Such cells were also positivefor acetylcho- linesterase staining (9, 10) and showed ultrastructural local- ization of peroxidase (10) typical of megakaryocytes (data not shown). Small blasts, accounting for 20 of the total bone marrow cells, were also present and showed agranular and basophylic cytoplasm with a high nucleus/cytoplasm ratio(Fig. 1B). Cells belonging to the erythroid and lym- phoid lineages were quantitatively decreased, while the myeloid population in all stages of differentiation was abnor- mally expanded (Fig. 1B). The evaluationof peripheral blood showed anemia  hemoglobin, 9 g/dl), anisopoikilocy- tosis, mild thrombocytosis, and a huge number of large platelet clumps (Fig. 1C). Platelet counts in infected animals rose from 400 x 109/liter to more than1,000 x 109/literafter adrenalineadministration. These results suggest that the major fraction of platelets in PyMLV-infected mice were trapped eitherinto circulating clumps or into the spleen. To evaluate platelet function, platelet-rich plasma (0.5 ml)containing approximately 109 platelets per ml was stirred for 1 min at 37°C in a luminescence aggregometerwith luciferase and luciferin (50  ul . Microliter volumes of the calcium ionophore A23187 were then added, and the aggregation (Fig. 2, upper diagrams) and ATP secretion  lowerdiagrams) were recorded for 4 min. A23187 was used at concentrations of 2.5 and 5.0 p.M. The results show a marked reductionof the sensitivity of platelets from the infected animals to the aggregating effect of A23187 (Fig. 2), ADP, andcollagen (data not shown). Secretion of ATP (Fig. 2) and synthesis of thromboxane in response to thrombin (10 U/ml) (data not shown) were impaired as well. This deficient platelet re- sponse to aggregating agents in vitro represents a feature which is also peculiar to human essential thrombocytemia (5). On the other hand, no signs of the disease were observable 361 Vol. 62, No. 1  36 NOT S g¶ J VIROL  NOTES 363 CONTROL MICE INFECTED MICE 5.0 _ I 107. ab c de f   S 2.5 5,0 I_0 S oil A T ATP 1pM N 4 2,5 FIG. 2. Typical tracings ofsimultaneous measurements of aggre- gation and ATP secretion in response to ionophoreA23187 in platelets from control and infected mice. Platelet aggregation was determined in a lumiaggregometer  Mascia-Brunelli, Milano, Italy which also records the luminescence resulting from theinteraction of released ATP with firefly luciferase and luciferin  Chronolume 395; Chronolog, Havertown, Pa. . The extent of the aggregation wasmeasured as the percent of light transmittance 3 min afterthe addition of the aggregating agent A23187, which induces aggregation by mobilizing intracellular calcium. The lumiaggregometer was adjusted so thatplatelet-rich plasma and platelet-free plasma pro- duced 10 and 90 light transmittance, respectively. In each case, microliter amounts of A23187 or equ l volumes of vehicle wereadded to 0.5-ml platelet suspensions that hadbeen stirred at 1,000 rpm at 37°C for 1 min before theaddition of the agonist. Upper diagrams, Platelet aggregation expressed as percentof lighttrans- mittance  T . Lower diagrams, Extent of ATP secretion micro- molar) afterthe addition of A23187 at concentrationsof 2.5 and 5.0 ,uM. up to 3 months after PyMLV inoculum into C57BL/6 Fv-2r mice.Therefore, PyMLV replication in the target tissues seems to be genetically controlled, as observed for other retroviruses  12 . Having established that PyMLV can induce an acute myeloproliferative thrombocythemic disease, we looked for target tissues of viral replication. Total cellular RNAs from spleen, tibial bone marrow, liver, lung, andkidney were extracted from NIH/OLAC mice by the guanidium-hydro- chloride method  1 20 days after infection with PyMLV spotted onto nitrocellulose filters at decreasing concentra- tions  from the top,5.0,2.5,1.25, and 0.625 ,ug using already published methods  14 , and hybridized to 106 cpm FIG. 3. Establishment of target tissuespecificityfor PyMLV replication in infected mice. A) Dot-blot R hybridization against a polyomavirus middle T-specific probe, pMT-1. Lanes: a, PC PyMLV  rat thyroid epithelial cells infected with PyMLV [A. Fusco,submitted for publication] ; b, spleen; c, tibial bonemarrow; d, liver; e, lung; f, kidney.  B Indirect immunofluorescence of bone marrow cells with antibodies against the middle T protein of polyomavirus. of 32P-labeled  specificactivity, 2 x 108 cpm/,lg clone PyMT 1 per ml, specificfor the middle T geneof polyoma- virus  16 . Hybridization results showed that bone marrow was the only site for PyMLV replication andmiddle T R expression  Fig. 3A . Immunofluorescence studies were then performed  Fig.3B . Tibias from NIH/OLAC mice were removed 20 days after PyMLV injection, snapfrozen in liquid nitrogen, and cut at 5 to 8 ,im in a cryostat. Sections were layered onto FIG. 1. Bone marrow histology, bone marrow cytocentrifugepreparation, and peripheral bloodsmearof NIH/OLAC mice 18 days after PyMLV injection.  A) Histological appearance of bone marrow  tibia ; at the level of diaphyses a high number of megakaryocytes at different stages ofmaturation may be observed. Magnification, x230.  B Bone marrow cytocentrifuge preparation.   small proportion of blast cells  up to 20 of total bone marrow cells with agranularand basophilic cytoplasm is present  arrows ; blasts are negative for Sudan black, myeloperoxidase, and nonspecific esterase reactions while displaying acetylcholinesterase activity  data not shown). The myeloi population is abnormallyexpanded, showing cells at all stages of maturation; a high number of promyelocytes, myelocytes, and segmented forms is present. Erythroid population is quantitatively decreased. May-Grunwald-Giemsa stain; magnification, x366.  C Peripheral blood smear.   high number of large platelets, clumps, and clusters are present. May-Grunwald-Giemsa stain; magnification, x901. VOL. 62, 1988 c.n  364 NOTES V~~~~~~~~~~~~~~~~~ 9F v : l.  tt ~~~   l e s~~~~~~~~~~i : FIG In vitro culture of bone marrov injected mice. (A) Phase-contrast micrograph (B) Acetylcholinesterase staining of a 1-mon marrow cells. (C) Indirect immunofluorescer protein antiserum ofthe megakaryocyte cull (30 days of culture). The great majority of cell middle T protein. gelatinized glass slides, air dried, and fixed in cold acetone for 10 min. Sections were then rehydrated in Tris-buffered 5<> wio J>t   4 saline and incubated for 3 hwith mouse anti-middle T Jti--st t ;-^ Sfi protein antiserum (2). Sections were then washed exten- sivelyfor 30 min in Tris-buffered saline and covered with fluorescein isothiocyanate-conjugatedgoat F ab )2 anti- mouse antiserum.After 60 min of incubation, sections were extensively washed in Tris-buffered saline and mounted in glycerol-phosphate-buffered saline  9:1). Only a few cells, possibly corresponding to the blasts present in the bone marrow of infected animals, showed the presence of the -I0iW middle T protein; most of these cells appeared to be mono- nucleated and showed a high nucleus/cytoplasm ratio, while a: :R g   a few were plurinucleated. Tentatively, these middle-T- positive cells may correspond to the blasts identified bymorphology in the bone marrows of theinfected animals We also studied long-term liquid cultures established frombone marrows of infected animals and grown for 1 month in iX scovemodified Dulbecco Eagle medium supplemented with hydrocortisone (10- M), 10 fetal calf serum  Flow Labo- ratories), and 15 conditioned medium from WEHI-3B  D-) cells as a source of interleukin-3  IL-3)  15). One month after the establishmentof theculture, >90 of the replicating cells were elements identified as megakaryocytesby phase- contrast microscopy  Fig. 4A) and Giemsa and acetylcholi- nesterase staining  Fig. 4B). These cells require the addition of IL-3 to the culture medium for growth  data not shown). Phase-contrast microscopy of bone marrow cells showed plurinucleated large  >20 p,m in diameter), round megakar-yocytes representing the great majority of cells in the cul- tures  Fig. 4B, arrows). Acetylcholinesterase activity was 4> ~ 4 seen as black granular deposits of copperferrocyanide in the cytoplasm  Fig. 4B, arrows). Megakaryocytes at different * stages of maturation were evident. The vast majority of these cells expressed the middle T protein, as assessed by immunofluorescence  Fig. 4C Incontrast, bone marrow _| .~jcultures from noninfected animals, grown in the same con- B ditions, showed at this time less than 2 acetylcholineste- rase-positive cells or morphologically identifiable megakary- ocytes. These results suggest that megakaryocytes representone of the targets for PyMLV infection andmiddle T expression, even though we cannot rule out the involvement of immature blood cell precursors different from those committed to megakaryocytic differentiation. Since bone marrow cultures from infected animals con- sisted mainly of megakaryocytic cells in all stages of differ- entiation, includingplurinucleated mature elements, our results also indicate that middle T gene expression does not prevent infected cells fromundergoing terminal differentia- tion in vitro upon addition of IL-3. Thisconclusion is also supported by histological findings showing the presence of mature megakaryocytes in bone marrow in the most ad- vanced stages of the disease. Moreover, PyMLV-infected bone marrow cells require the addition of exogenous IL-3 for proliferation and maturation; IL-3has been shown to be able to sustain megakaryocytic colonygrowth from murinebone s X marrow cells (7, 11) and to promote growth and differentia- tion of isolated murine megakaryocytes  15). v cells from PyMLV- In conclusion, our data indicatethat an artificially con- of bone marrow cells. structed retrovirus carryingthe middle T gene of polyoma- ith-old culture of bone virus affects the hemopoietic system of mice in vivo causing nce with anti-middle T an acute myeloproliferative thrombocytemic disorder. Since ture shown in panel A the megakaryocytic lineage appears to be a specific target of Is show the presence of PyMLV, we believe that thisin vivo model may providea unique tool for investigating the mechanisms leading to megakaryocytic proliferation and transformation Studies on J. VIROL. AML bl, v I ...sl A * :.. 11171 : .4 0: AML jpl.  NOTES 365 the pathophysiology of rare human hematological diseases such as essential thrombocythemiaand megakaryocytic leu- kemia, hampered thus far by the lack of a suitable animal model, may take advantage of this new in vivo model system. We thank P. Kaplanand D. J. Donoghue for kindlyproviding the PyMLV J. Pierce and P. P. Di Fiore for IL-3-producing cells, and G.Vecchio and A. Balmain for critically reading the manuscript. We thankG. Sequino for providing animals. This workwas supported by the Progetto Finalizzato Oncologia of the Consiglio Nazionale delle Ricerche andby the Associazione Italiana Ricerche sul Cancro. G.P., M.G., and A.P. are recipients of agrant from the Associazione Italiana per la Ricerca sul Cancro. LITERATURE CITED   Adams, S. L., M. E. Sobel, E. H. Howard, K. Olden, K. M. Yamada, B. De Corbrugghe, and I Pastan. 1977. Levelsof translatable mRNAs for cell surface proteins, collagen precur- sors andtwo membrane properties in Rous sarcoma virus transformed chick embryo fibroblasts.  roc Nati. Acad. Sci. USA 74:3399-3403. 2. Dilworth, S. M. 1982. Protein kinase activities associated with distinct antigene forms of polyoma virus middle T antigen. EM O J. 11:1319-1326. 3. Donoghue, D. J., C. Anderson, T. Hunter, and P. Kaplan. 1984. Transmission of the polyoma virus middle T gene as the oncogene of a murine retrovirus. Nature  London 308:748-751. 4. Franz, T., J. Lohler,A. Fusco, I B. Pragnell,   Nobis, R. Padua,and W. Ostertag. 1985. Transformation of mononuclearphagocytes in vivo and malignant histiocytosis caused by a novel murine spleen focus-forming virus. Nature  London 315: 149-151. 5. Ginsburg, A. D. 1975.Platelet function in patients with high platelet counts. Ann. Intern. Med. 82:506-509. 6. Gross, L. 1970. Isolation of the mouse leukemia virus from transplanted mouse tumors, p. 470-508. InL. Gross  ed. , Oncogenic viruses. Pergamon Press, Inc., Elmsford, N.Y. 7. Ishibashi,T., and S. A. Burstein.1986. Interleukin 3 promotes the differentiation of isolatedsingle megakaryocytes.Blood 67:1512-1514. 8. Kaplan, P. L., S. Simon,and W. Eckhart. 1985. Polyomavirus middle T protein encoded by a retrovirus transformsnonestab- lished chicken embryo cells. J Virol. 56:1023-1025. 9. Long, M. W.,and N. Williams. 1981. Immature megakaryocytes in the mouse: morphologyand quantitation by acethylcholin- esterase staining. Blood 58:1032-1036. 10. Nagasawa, T., M. Nakazawa,and T. Abe. 1982.   liquidculture system for murine megakaryocyte progenitor cell. Blood 59: 250-254. 11. Quesenberry, P. J., J. N. Ihle, and E. McGrath. 1985. The effect of interleukin 3 and GM CSA 2 on megakaryocyte andmyeloid clonal colony formation. Blood 65:214-217. 12. Steeves, R.A. 1975. Spleen-focus forming virus in Friend and Rauscher leukemia virus preparation. J. Natl. Cancer Inst. 54:289-297. 13. Teich,N., J. Wyke, and P. Kaplan. 1986. Pathogenesis of retrovirus induced disease, p. 187-248. InR. Weiss,N. Teich, H. Varmus, and J Coffin  ed. , RN tumor viruses, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 14. Thomas, D. S. 1980. Hybridizationofdenatured RN and small DN fragments transferred to nitrocellulose. Proc. Nati. Acad. Sci. USA 77:5201-5204. 15. Williams, N., R. R. Eger,H. M. Jackson, and D. J. Nelson. 1982. Two-factorrequirement for murine megakaryocyte colony for- mation. J. CellPhysiol. 110:101-104. 16. Zhu, Z., G. M. Veldman, A. Cowie, A. Carr, B. Schaffhausen, and R. Kamen. 1984. Construction and functional characteriza- tion of polyomavirus genomes that separately encode the three earlyproteins. J. Virol. 51:170-182. VOL. 62, 1988
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