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Haematology and Blood Transfusion Val. 26 <strong>Modern</strong> Trends in Human Leukemia IV Edited by Neth, Gallo, Graf, Mannweiler, Winkler © Springer-Verlag Berlin Heidelberg 1981 Enhancing Effect of Murine Leukemia Virus on Fibroblast Transformation by Normal BALB/C Mouse DNA Fragments K. J. van den Berg, V. Krump-Konvalinkova, and P. Bentvelzen A. Introduction Several rapidly transforming RNA tumor vi ru - ses contain inserts of cellular genetic material. These inserts seem to constitute the so-called onc genes of these viruses (Stephenson et al. 1979). The quest ion arises whether during the recombination process leading to the generation of highly oncogenic viruses the cellular sequences be co me altered in such a way that they will cause malignant transformation. This would then be compatible with simplistic somatic mutation theories of nonviral carcinogenesis. Alternatively, with the virus acting as a vector, the insertion of normal cellular sequences involved in control of growth and/or differentiation into other sites of the host genome may lead to overproduction of certain gene products and eventually to malignant transformation. This transposition phenomenon may have important implications for models of nonviral carcinogenesis. normal BALB/ c mice could transform NIH/3T3 as weIl as BALB/3T3 cells. In contrast to the foei of transformed cells induced by the A-MuLV pro virus which appear within 2 weeks, those induced by normal DNA were first observed after 4 weeks, corresponding with six subcultures of the recipient cell. The DNA specificity can be concluded from Table 1 in which treatment of the DNA preparation with DNase abrogated fully the transforming capacity, while RNase or pronase did not. Neither calf thymus nor E.coli DNA induced foci of transformed cells. Since DNA isolated from thymus, spleen, or liver had equal transforming capacity (see Table 2), the transforming sequences seem to be an integral part of the BALB/c genome. As DNA isolated from germfree BALB/c mice of a colony established 20 years aga is similarly transforming, this property cannot be due to re cent infection of the germ li ne with an oncogenic virus. B. Transformation by Normal Cell DNA Recently Cooper et al. (1980) reported that mechanically fragmented normal cellular DNA isolated from avian or mammalian cell lines could transform NIH/3T3 cells, albeit at a low frequency. Secondary transfections with DNA isolated from the transformed cells proved to be highly efficient. During our study on transfection with the integrated provirus of Abelson murine leukemia virus (A-MuLV) utilizing the calciumtechnique of Graham and van der Eb (1973), we observed independently of Cooper et al. in control experiments that mechanically fragmented DNA isolated from the thymus of C. Characterization of Transformed Cell Lines A battery of cell lines were developed from foci of transformants induced by normal BALB/c DNA fragments. No reverse transcriptase activity could be detected in any of the celllines, indicating that transformation occurred in the absence of replicating retroviruses. The cells grew well in soft agar, indicating anchorage independence. Upon intraperitoneal inoculation into newborn athymic BALB/ nude mice, all of six tested cell lines were oncogenic. After subcutaneous injection of 10 6 cells into 3-week-old BALB/c five of six lines produced fibrosarcomas within 3 weeks. 479
Table 1. Transformation of 3T3 cells by normal BALB/c DNA Recipient cells BALB/3T3 NIH/3T3 Fraction Average Fraction Average trans- No. trans- No. formed foci/dish formed foci/dish cultures cultures BALB/c thymus DNA 50 /-lg. ml- l 16/16 15 16/16 4 Ibid, treated with pronase" 8/8 4 7/8 1.5 BALB/c thymus DNA 50 /-lg·ml- l RNase" 8/8 2 8/8 6 BALB/c thymus DNA 50 /-lg. ml- l DNase" 0/8 0 0/8 0 BALB/c thymus DNA 50 /-lg ·ml- l heat denaturated b 3/8 0.5 3/8 0.5 Calf thymus DNA, 50 /-lg. ml- l 0/8 0 0/8 0 E. coli DNA, 50 mg· !-lI-l 0/8 0 0/8 0 " 1 hat 37°C b 5 min at 100°C, then quickly placed in ice So far, no transforming virus eould be rescued after infeetion with the Moloney strain of MuLV (Mo-MuLV) in contrast to eells transformed by A-MuLV proviral DNA. A hyperimmune antiserum, raised in C57BL against syngeneie A-MuLV induced lymphoma cells and absorbed with syngeneic Mo MuLV lymphoma cells, reaeted with two of seven testet cell lines transformed by normal DNA in the eytoplasmie immunofluoreseence test. The antiserum reaeted with a variety of A-MuLV transformed cells but not with NIH/3T3 eells infected with Mo-MuLV. D. Enhancement of Transformation by Preinfection with Leukemia Virus The effieiency of transformation of normal eell DNA is rather low. Since the nontransforming murine leukemia viruses make eells susceptible to the action of various eareinogenic chemie als (Huebner and Gilden 1972), we studied the influence of preinfeetion with MuLV -strains in this system. After incubation of NIH/3T3 cells, infeeted with either Rauscher or Moloney-MuLV, with normal eell DNA fragments, foei eould be deteeted within 2 weeks after a single passage. The transformation frequency eould be as high as 1 foeus forming unit per 1 ug DNA (see Table 3), whieh would be comparable to the results obtained with the A-MuLV provirus. A linear dose-response relationship was found in this system (Fig. 1), indicating that incorporation of a single DNA fragment would be suffieient for neoplastie conversion. Quite remarkable is that a minimal quantity of DNA is needed for transformation to occur. E. Discussion It is unlikely that the transforming capacity of normal cell DNA is due to sheer mutagenicity, since mouse DNA sequenees homologous to the src gene of Moloney murine sareoma virus cannot transform fibroblasts (Oskarsson et al. 1980). Our data and those of Cooper et al. (1980) support the notion that the eellular Table 2. Effect of origin of BALB/c DNA on transforming activity" Source of DNA Thymus 50 ug·ml- l 25 ug·ml- l Spleen 50 ug·ml- l 25 ug·ml- l Liver 50 ug·ml- 1 " N.D. =not done Recipient cells (Fraction transformed cuItures) BALB/3T3 16/16 8/8 8/8 8/8 4/4 NIH/3T3 16/16 N.D. N.D. 2/8 N.D. 480
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Haematology and Blood Transfusion 2
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Dr. Rolf Neth, Universitäts-Kinder
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Participants of the Meeting Aaronso
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Kurth, R, Friedrich-Miescher-Labora
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Wilsede Scholarship Holders (Grante
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Preface Gut ist eine Lehrart, wo ma
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Personal and scientific discussion
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Acknowledgement We should like to t
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Wilsede, June 21 1978 Memorial Trib
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limiting benign lymphoproliferative
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this most unlikely, however (for re
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longer subject to the same control
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Bregula U, Wiener F, Harris H (1971
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fresh lymph node biopsies from ten
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Fig. 3. Binucleate mitosis in an ob
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y Fig. 4. Schematic diagram of post
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than that among patients with Stage
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Tests of binding affinity, agglutin
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N Engl J Med 297: 963-968 - Green I
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Clinical Aspects
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"ring chromosome". "Mar" is marker,
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191 chromosomally abnormal patients
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tumors of both African and non-Afri
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Haematology and Blood Transfusion V
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Retrospective group 26/93 evolved l
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Treatment
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advantage in terms of duration of f
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leukaemia. An analysis of 168 patie
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11. Treatment Program Chemotherapy
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efractory congestive heart failure
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period (Sequence II) was gene rally
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children with a multiple drug proto
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DAYS 1 5 10 15 20 25 30 35 , I I I
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Biologically Drug Outcome fit" sens
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may counterbalance the potential be
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Ht (0--0) 50 40 30 20 10 0 Platelet
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10 7 ,-----------------------------
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11. Haematological and Biochemical
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0\ 0\ INTERFERON ... 10 3 rl E "'
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("pre-B" phenotype) has been descri
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human bone marrow cells exhibit the
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10 9.,0 MBKCC .. .,4 B prot:oool 1'
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demann W, Büehner T, Arlin Z, Gee
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-...l 00 a-tLDRENS CAt«:ER S TU>Y
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prognostic characteristics under in
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significantly higher in HR} (14.5%,
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normal or elevated immunoglobulin l
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References Andreeff M, Darzynkiewic
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R GROUP 1 ( 1970 - 1971 ) 17 PTS. R
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Table 3. Influence of initial featu
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LIJ (f) a.. < -1 LIJ Q: u. 0 >- I-
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me nt indicated that additional the
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42:2123-2134 - Chessells JM, Cornbl
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Baum et al. 1979). This study was b
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1. 0 '--'1--" 0.9 e: o .- VI VI E C
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e: o e: 1. 0 ,.--...-- 1'"'\ ......
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In conclusion, we can state that th
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Haematology and Blood Transfnsion V
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INDUCTION VCR + PRED + ASNASE (wk 3
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SCHED.INT 1 MTX • ! Adria MTX •
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~ looh---------ooooc>----o---c>----
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Consoll- Irra .tlon t) .tlon Indu
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Highrisk Standard risk Table 1. Cli
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Cumulo/ive comp/el9 remission 1.0 .
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10° 8 = 6 ö 4 Non T Cell .~ :ö
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% SURVIVAL 100 ~ ~II 0 80 60 40 - -
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a) Percentage of donor cell mitoses
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Table 5. Results of bone marrow tra
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...... w N Table 7. Clinical result
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agent had to be added and that in t
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C. Chronic Myelogenous Leukemia The
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Patients with a suitable donor rece
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No. Recurrent Leukaemia Other death
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followed by dialysis against isoton
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HS, HL- Heme >_ 40 0 HS I ;:. =====
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knowing at present. These genes may
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esistance to antifolates. In Vitro
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a DNA probe specific for the gene t
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translocation between chromosomes 9
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Table 1. Subdivision of 1827 Myelo-
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NORMAL CELL ®
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some change of the primary type, in
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F. Transformation of Mouse Bone Mar
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were carried out with 3H-MTX in the
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the lysozyme cDNA prepared from ovi
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et al. 1976). The BJAB cellline in
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dalton bands corresponding to light
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1979). The EBV-carrying cell lines
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producer lines (DAUDI and P3HR-1),
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G, Giovanella B, Westman A, Stehlin
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Dc;bt CT + i J.I9 Raji; 1. i .. 100
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c. Discussion During infectious mon
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C. Nonepisomal Viral DNA We have us
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A 260 3.0 A B 1670 70 N2 2.0 c: E .
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vivo. Nature 260:302-306 - Kirk JTO
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Fig. 1. a Polyacrylamide gelelectro
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1979). The lymphocyte subpopulation
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lysin 0 antigens are found, and mix
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Pope JH (1978) Long-term T cell-med
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Charaeteristie Morphologie maturati
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after TPA-induction (Table 3). The
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PA (1978) Reactivity of a rabbit an
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Blood Monocyte. B lood Monocytes 1-
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and nonhistone proteins can serve a
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The findings of the evaluation of s
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Fig. 3. Tumor grawth of L 428 cells
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Cell biological and Immunological A
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consideration. Sanel (1973) obtaine
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endotoxin serum, were used. A parti
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monocytic leukemia cell line in cul
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B. Isolation, Characterization and
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don al hemopathies generally displa
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nitors. Blood Cells 6:595-607 - Min
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\I) ....I ....I IU Jl 200 r 100 90
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their haemopoietic cells' ability t
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normal normal ALL ALL ALL persons p
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64.5 { V)
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References Biermann E, Neth R, Gros
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ehambers, the growth pattern observ
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References Anderssen LC, 10kinen M,
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REH CELL UNE TO CALLb~Ö ~q~ V.M. c
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the macrophage series in which the
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Table 1. Production of factor depen
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Table 1. Characteristics of the adh
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Fig. 3. A human marrow culture at 6
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300 250 • HYPERPROLIFERATIVE PATT
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Fig. 9. Nonadherent population from
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The cobblestone areas were often no
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L (1976) Induction of colony format
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B. Materials and Methods J. Tissue
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Fig. 2. Ultrastructural appearance
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Table 1. Monoclonal antibody reacti
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al. 1979; Janossy et al. 1979) is e
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to which this is an exact replica o
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inger JL (1976) Distribution of la-
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I region antigens were found to be
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have been reported to be present on
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V. FunctionalAssays Assays for PHA
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Functional studies of UCHT1 separat
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40 ,...... (5 L.. C o u 30 .9 ~ .~
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with immunohistochemieal methods (L
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can be dissected by cloning. Utiliz
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Table 1. Origin and marker profile
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complete identity of gp 100 from no
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Table 1. Reaction of single anti se
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media. Following 24-h incubation, c
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nonleukemic eells (Greaves 1979). L
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Fig. 2. Cytocentrifuged smears of b
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Modification of amino-groups of hum
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disturb this balance in the directi
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The majority of human blood lymphoc
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of the EBV infected B cells. Howeve
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and immunoglobulins. J Immunol 120:
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" K 10 • j ~ ALL RNlL AL wtfh les
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tion akuter Leukämiezellen in "spo
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D. Results and Discussion J. Acute
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selectively responsive to different
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l1J ~ 100 ~ Q. ::) l1J Z 0 ~ z >- .
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Table 2. Growth inhibition of S49 e
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Reverse Infectious ASC/2X1Q6 transc
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munized animals to 1316 tumor cells
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Fig. 3. Expression of retroviral gp
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pg70. J Exp Med 143: 151-166 - McGr
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Table 1. Expression of tumor antige
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Virological and Molecularbiological
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Table 1. Oncogenic properties of re
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also Fig. 1). It followed that the
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whether this sequence is related to
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zed in infected cells argue that th
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a specific viral transforming prote
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detect viral RNA as the only charac
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Eisen H (1980) Myeloproliferate vir
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p,60- -34K - 1 2 3 4 Fig. 1. In vit
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.. p - • a'T ... I .t'l Fig. 4. D
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Fig. 2. Photomicrographs of NY68 in
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DEAE (o(lJmn 'liI GI -2 2 o e s 1
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vity was deterrnined. For endogenou
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pr 180 - - - P 12/15 - A B c D E F
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85,- 66- -27 - -66 40- 27- -1'9 19-
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= 0.5 o E Q. o ILI 0.4 (J) « ILI .
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Table 1. Oncogenic potential of avi
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100 III GI c: o "8 80 > w
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Expt. No. Infecting virus Proportio
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Haematology and Blood 'fransfusion
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sequences, provided its gag portion
Page 437 and 438: 77:3009-3013 - Hu SSF, Moscoviei C,
Page 439 and 440: RI 2.2 md and Hind III 2.6 md leuke
Page 441 and 442: Haematology and Blood Transfusion V
Page 443 and 444: RELATIONSHIP OF ENDOGENOUS AND EXOG
Page 445 and 446: E ........ E Cl. U E :::l .... Q) (
Page 447 and 448: References Astrin S (1978) Endogeno
Page 449 and 450: the same animal were examined (e.g.
Page 451 and 452: I) 'IQ a \0 - CL -- Fig. 2. Restrie
Page 453 and 454: ~ ..................... ~ Cell 3' 5
Page 455 and 456: a :Sa,C! + . ~ 111 i' ( " '~ .J. b
Page 457 and 458: Table 1 Sampie Tissue TS fragment"
Page 459 and 460: a b Sac I / rep ._-_.-_ K J nl rep
Page 461 and 462: Haematology and Blood 'fiansfusion
Page 463 and 464: Osteopetrosis and osteosarcomas occ
Page 465 and 466: Lymphoma cellline Table 1. T and B
Page 467 and 468: Table 2. In vivo growth and oneogen
Page 471 and 472: Table 1. Transforming aetivity of c
Page 473 and 474: Table 3. Transformation aetivity of
Page 475 and 476: - Shoemaker C, Goff S, Gilboa E, Pa
Page 477 and 478: indieate a shifting of target eell
Page 479 and 480: Table 2. Expression of Jl and Sourc
Page 483 and 484: ~ i ,..1---------,,':::::::::::::::
Page 485 and 486: fragment of pBR322 DNA, a 3.0-kbp S
Page 487: Mak TW (1979) Proc Natl Acad Sci US
Page 491 and 492: inserts in the genome of rapidly tr
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Page 495 and 496: contains antigens which react with
Page 499 and 500: le of reacting in sensitive radioim
Page 503 and 504: gic approach. In: Hiatt HH, Watson
Page 505 and 506: 12 Eco R [ Fig. 1. Hybridization pa
Page 509 and 510: 10 - '1~ )( -~ S:! E A U I o 1 2
Page 511 and 512: Haematology and Blood 'fransfusion
Page 513 and 514: cultures. These cells did not conta
Page 515 and 516: indication of malignant T-cells in
Page 517 and 518: Fig. 3. Thin-section electron micro
Page 519 and 520: Table 3. Lack of detectable related
Page 521 and 522: J. HTLV Was Present in the Primary
Page 523 and 524: specific signals into nonspecific s
Page 525 and 526: Table 2. Distribution of HTLV-relat
Page 527 and 528: u 10 ~ 20 ~ :c 30 a 40 o Fig. 1. Ki
Page 531 and 532: SSV TrS-gp labeled effectively with
Page 535 and 536: 60 HFF/SSV cDNA CELL RNAs: 0 HF/SSV
Page 537 and 538: Antisera a Table 1. Immunofluoresce
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Haematology and Blood 'ftansfusion
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y competition RIA yielded virtually
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SiS'V' gp70: ANTIGEN, ANTI BODY, IM
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type-C virus p30 antigen in cells f
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c. Results The fractionated whole-b
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induced antibodies as well as exper
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- Fig. la-f. Retravirus particles p
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eplication of infecting murine leuk
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immunodeficiency, multic10nal lymph
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Bovine leukemia --, proteins of 499
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Human T- cell - -, characterization
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RNA -, of tumor virus 439 Rous sarc
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Haematology andBlood Transfusion Su
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