Immunology Letters, 33 (1992) 47 52 0165
2478 / 92 / $ 5.00 © 1992 Elsevier Science Publishers B.V. All rights reserved
IMLET 01801
Deficient transformation of murine trisomy 16 fetal liver cells by the Aigelson and J2 viruses C h r i s t o p h N . B e r g e r a a n d C h a r l e s J. E p s t e i n a'b Departments of apediatrics and bBiochemistry and Biophysics, University of California, San Francisco, CA, USA (Received 9 October 1991; revision received 20 March 1992; accepted 31 March 1992)
I. Summary Mouse trisomy 16 (Tsl6), an animal model for human Down syndrome (trisomy 21), exhibits severe abnormalities in the development of lymphoid and myeloid cells. Whereas fetal liver cells from diploid mice can be easily immortalized by retroviral transformation with A b l - M u L V or J2 virus, fetal livers from Tsl6 mice contain significantly fewer transformable cells. Infection of Tsl6 fetal liver cells by A b l - M u L V results in a 52- and 12-fold reduction in the frequency of transformation at days 17 and 18 of gestation, respectively. By contrast, the efficiency of transformation with J2 virus, another retrovirus known to transform fetal liver cells, is only mildly (factor 23) affected. The Ig gene rearrangements of Tsl6 and diploid retrovirally transformed fetal liver cell lines do not differ from one another. This suggests that there is a deficiency in the early stem cell compartment, rather than in the development of pre-B cells.
2.
Introduction
In persons with Down syndrome resulting from trisomy 21, leukemia of various types, including in particular acute megakaryoblastic leukemia, Key words: Down syndrome; Leukaemia Correspondence to." Charles J. Epstein, M.D., Department of Pediatrics, University of California, San Francisco, CA 941430748, USA. Tel.: 415-476-2981; Fax: 415-476-9976.
occurs with a 10-20-fold higher incidence than in the normal diploid population [1,2]. Moreover, trisomy 21 has been identified in cases of acute megakaryoblastic leukemia in children without Down syndrome [3,4]. These findings suggest that coding or regulatory nucleotide sequences present on chromosome 21 may be involved in the etiology of certain forms of leukemia. Previous work has shown that most, if not all, of the region of chromosome 21 that appears to be responsible for the development of the Down syndrome phenotype, bands 21q22.1 to q22.3, is homologous to a region in the distal part of mouse chromosome 16 [5]. Based on this work and on similarities of developmental deficiencies of hemopoietic, nervous and cardiac systems, Tsl6 mice are considered to constitute an animal model for Down syndrome [6,7] and are being used for investigation of the pathogenesis of the Down syndrome phenotype. As an approach to studying in this model the basis for the unusual susceptibility of persons with Down syndrome to the development of leukemia early in life, we have been using transforming retroviruses. Initial work led to the unexpected finding that Tsl6 mouse fetal liver cells can hardly, if at all, be transformed with Abelson murine leukemia virus (Abl-MuLV) [8], a retrovirus known to transform primarily pre-B cells [9,10]. To determine whether Tsl6 interferes specifically with transformation by A b l - M u L V or whether this resistance to retroviral transformation is a more general phenomenon, we tested Tsl6 fetal liver cells for transformation with another retro47
virus, the J2 virus. The J2 virus is a recombinant retrovirus containing the myc and raf/mil oncogenes and, like A b l - M u L V , is known to immortalize lymphoid cells [11]. Furthermore, to study B cell development in the Tsl6 fetuses, we established A b l - M u L V and J2 virus transformed cell lines and determined the configuration of /x-H and K-L chain genes and, accordingly, their stages of development. 3.
3.1.
Materials and Methods
Animals
Mice doubly heterozygous for the Robertsonian translocations, Rb32Lub and Rb2H, were bred in our animal facilities. To generate Tsl6 mouse fetuses, C57BL/6J females were injected with 5 U human chorionic gonadotropin (Sigma) from pregnant mare's serum, followed 2 days later by 2.5 U gonadotropin (LymphoMed Inc. Melrose, IL), and mated with male Rb32Lub/ Rb2H mice. The morning of the plug formation was considered day 0 of embryonic development. The identification of Tsl6 mouse embryos in midgestation was based on the presence of massive edema and thymic hypoplasia, and in late gestation on thymic hypoplasia, a thick neck and open eyelids.
3.2.
Viruses
Abl-MuLV-producing cells (N54) [9] were obtained from R. Grosschedl, and J2 virus producing cells (J2 leuk) were obtained from U. Rapp [10]. Virus-containing supernatants were prepared by culturing a confluent layer of infected fibroblasts for 1 day in fresh medium.
3.3.
Infection of fetal liver cells and colony assay
Fetal liver cells from Tsl 6 and diploid embryos brought into suspension by passing the livers through 60 mesh stainless steel, were washed and infected at a density of 2 x 107 cells/ml by incubation in virus-containing supernatant for 4~5 h at 37°C in the presence of 4 #g/ml polybrene (Aldrich). The cells were then cultured in Iscove's medium (supplemented with 10% FCS, 5 x 48
10 -5 M 2-ME, 1 unit penicillin/streptomycin) in 0.3% soft agar (Difco) at a density of 2 x l 0 6 cells per 35 mm dish. The colonies were scored after 6~9 days of incubation.
3.4.
Establishment of cell lines
Individual colonies of A b l - M u L V and J2 virus-transformed cells were picked and transferred to liquid cultures in supplemented D M E M . The colonies obtained after A b l - M u L V transformation could be adapted easily to the conditions of liquid culture. By contrast, none of the individually picked colonies from the J2 virus transformed cells could be established as a cell line, although J2 cell lines could be obtained by continuously passaging the cells which had been set up in 2-ml mass cultures at a density of 1-4 × 10 6 cells/ ml. The adapted cell lines were subsequently expanded in supplemented D M E M for the isolation of genomic DNA and further characterization.
3.5.
Antibodies and flow cytometry analysis
Staining for the B220 antigen was done with mAb 14.8 obtained from American Type Culture Collection (TIB 164). Biotinylated mAb anti-rat ~c-L chain Mar 18.5 (TIB 216) [12] was used as secondary reagents. Streptavidin conjugated with FITC was purchased from Southern Biotechnology. Flow cytometry was performed with a FACS 440.
3.6.
Filter hybridization
Isolated DNAs from the A b l - M u L V and J2 virus-transformed cell lines were cut with EcoRI or BamHI, and 10 pg of the cut DNA was sizefractionated in a 1% agarose gel, transferred onto Genescreen, and hybridized according to Selden [13]. As hybridization probes, the inserts of plasmids p J l l and pECK containing #-H chain sequences [14] and ~-L chain sequences [15], respectively, were gel purified and randomly labelled with Klenow polymerase (Random Primers DNA Labelling System, BRL).
4.
4.1.
Results
Tsl6 fetal liver cells are severely deficient for A b l - M u L V transformation and have a reduced efficiency for J2 virus transformation
Determination o f the frequency o f transformation for A b l - M u L V showed that the T s l 6 fetal liver contains far fewer A b I - M u L V transformable cells than the diploid fetal liver. In fetal liver at day 17 o f gestation, a mean o f 54 _+ 23 (SEM) colonies were f o u n d per 106 cells, whereas T s l 6 fetal liver contained only a mean o f 1.0 -t- 0.2 transformable cells per 106 liver cells (Table 1). The p r o p o r t i o n o f transformable cells is 52-fold lower for T s l 6 animals than for diploid animals at day 17 and 11-fold lower at day 18 o f gestation. T o determine whether the reduced transformation efficiency o f T s l 6 cells is specific for A b l M u L V , and also to examine the differences between T s l 6 and diploid B cell precursors, we transformed fetal liver cells with the J2 leukemia virus. This virus is also able to transform B lymphoid cells, but since it carries a different oncogene, it is presumed to use a different mechanism for transformation. Both T s l 6 and diploid cells were transformed with the J2 virus (Table 1). At day 17 o f gestation, an average o f 19 _+ 6 colo-
nies were obtained per 106 T s l 6 fetal liver cells, whereas diploid tissues gave an average o f 37 _+ 19 colonies per 106 cells. At day 18 o f gestation, the fetal livers o f T s l 6 and diploid animals yielded 18 and 48 colonies per 106 cells, respectively. Therefore, the p r o p o r t i o n of J2-transformed T s l 6 cells was only 2-3 fold lower than was found for diploid cells. Overall, T s l 6 fetal liver contained significantly fewer A b l - M u L V (Z2, P = 0 . 0 0 0 1 ) and fewer J2 virus ( P = 0.0001) transformable cells than did diploid fetal liver. However, on day 17 o f gestation, T s l 6 animals contained significantly more J2 virus transformable cells than A b l - M u L V transformable cells whereas the control animals had more A b l - M u L V than J2 virus transformable cells (P = 0.0001).
4.2.
Rearrangements of l~-H- and K-L chain genes in Tsl6 and diploid transformed fetal liver cells
To study the development o f the pre-B cells in T s l 6 fetuses, we established retrovirally transformed cell lines and determined the configuration (rearranged/germline) o f the i m m u n o g l o b u lin genes. We isolated a total o f 14 A b l - M u L V or J2 virus transformed cell lines and found that
TABLE 1 Retroviral transformation of Tsl6 and diploid fetal liver cells Experiment Virus number
Age of fetus
Length Total coloniesb of incubation
CFU fromb retroviral transformation
Diploid
(d.g.)a
(days)
Tsl6
Tsl6
Number of fetuses analyzed Tsl6
Diploid
Ratio of CFU diploid/Ts 16
Diploid
1
Abl-MuLV 5 J2 5
4 4
17 17
6 6
3.2_+0.6 101.0_+2.8 1.3_+0.6 100.5_+2.8 77.8 31.3±3.4 74.7_+1.7 29.5_+3.4 74.2_+1.7 2.5
2
Abl-MuLV 4 J2 4
4 4
17 17
7 7
2.7±0.7 10.1_+1.0
24.5_+7.2 0.8±0.7 24.0_+7.2 30.3 13.0_+2.3 8.3_+1.0 12.5_+2.3 1.5
3
Abl-MuLV 4 J2 4
4 4
17 17
9 9
2.1_+0.6 21.5±2.2
54.6±4.5 0.8±0.6 39.4±1.7 20.3_+2.2
38.5_+4.5 47.4 23.3±1.7 1.1
4
Abl-MuLV 4 J2 4
4 4
18 18
7 7
3.2±0.5 19.9_+0.8
23.0_+2.9 1.7_+0.5 52.5±10.517.5_+0.8
18.3_+2.9 10.7 47.8_+10.5 2.7
aDays of gestation; bValues are colonies per 106 cells + SEM. 49
TABLE 2 Ig rearrangements in retrovirally transformed cell lines derived from T s l 6 and diploid fetal liver cells. Configuration of lg loci of #-H and ~c-L chain genes
H and L germline H rearranged, L germline H and L rearranged
Day 17 FL cells transformed with A b l - M u L V
Day 17 FL cells transformed with J2 virus
Day 18 FL cells transformed with AbI-MuLV
Total
Diploid
Ts 16
Diploid
Ts 16
Diploid
Ts 16
Diploid
Ts 16
1 2 0
0 1 2
0 1 0
0 0 2
0
0
l
0
1 1
1 0
4 1
2 4
the Tsl6 cell lines readily rearrange their #-H chain and ~-L chain loci (Table 2). To confirm that the A b l - M u L V and J2 viruses preferentially transformed pre-B lymphoid cells, we tested 2 Tsl6 derived lines (one A b l - M u L V and one J2 virus transformed cell line) and 4 A b l - M u L V lines derived from diploid fetal liver for the expression of B220, a pre-B cell-specific glycoprotein. All of these cell lines, which grew in culture as single cell suspensions, stained positively for B220. As controls, we established two cell lines by transformation of Tsl6 embryonic fibroblasts. These two lines were negative for the B220 expression and did not show any rearrangement of the #-H chain and ~c-L chain locus (data not shown). 5.
Discussion
The results presented above confirm that Tsl6 fetal liver cells are severely defective for transformation by Abl-MuLV. By contrast, J2 virustransformed cells can easily be obtained, although compared to diploid littermates, the efficiency of J2 virus transformation is still somewhat reduced. These data suggest that the trisomic state affects transformation by more than one retrovirus, but certainly not to the same degree and probably not by the same mechanism. There are several possible explanations for the very low efficiency of A b l - M u L V transformation of Tsl6 cells. One possibility is that in the Ts16 fetuses most of the A b l - M u L V transformable target cells are developmentally absent. It is already known that several other stem cell populations, including CFU-C, CFU-S, BFU-E, CFU-E, are reduced in Tsl6 livers [8]. Furthermore, there 50
also appears to be a deficiency of thymocyte precursors [16]. Other investigators have shown that in the bone marrow of normal mice, A b l - M u L V transformable cells are highly enriched within the subpopulation of Thy-1J°, B220 + cells. This subpopulation does not contain the myeloid-erythroid precursor cells (measured as CFU-S) or the pluripotent hemopoietic stem cells, as shown by its failure to reconstitute lethally irradiated animals [17,18]. It is possible that in fetal liver the same subpopulation also contains the preferential target cells for A b l - M u L V transformation. Although it remains to be determined whether the Thy-1 l°, B220 + subpopulation of fetal liver cells is deficient in Tsl6 fetuses, our preliminary data indicate that proportion of B220 + cells is not significantly diminished in Tsl6 fetal liver. At day 15 of gestation, there were 6.2% B220 + cells in the Tsl6 liver, as compared to 5.1% in the controls. Another possibility is that the target cell population is not developmentally absent but is defective for virus infection or propagation. A defect in virus infectivity might be due to a deficiency in the receptor for the virus. The receptor for murine ecotropic retrovirus infection has been molecularly identified from a BALB/c fibroblast cDNA library. If transfected and expressed in a virus resistant human cell line, it confers infectivity for MuLV [23]. This receptor molecule is encoded by the rec-1 locus on chromosome 5. Although not all the subgroups of type C retroviruses have been tested for the use of this receptor, it is possible that it or another member of the retrovirus receptor multigene family is used by AblMuLV and the J2 virus [19]. If so, the fact that the J2 virus does transform Tsl6 cells indicates that retrovirus receptor molecules are expressed
on T s l 6 fetal liver cells, and, therefore, that o t h e r defects have to be i n v o k e d to explain the greatly r e d u c e d efficiency for A b l - M u L V t r a n s f o r m a t i o n o f Ts 16 fetal liver cells. Yet a n o t h e r possibility is that retroviruses are able to infect a n d p r o p a g a t e equally well in T s l 6 a n d in d i p l o i d cells, b u t t h a t T s l 6 cells are m u c h less likely to u n d e r g o the s e c o n d a r y events necessary for i m m o r t a l i z a t i o n . W i t h the use o f r e c o m b i n a n t i n b r e d m o u s e strains, it has been determ i n e d t h a t susceptibility for A b l - M u L V a n d J2 v i r u s - i n d u c e d t u m o r i g e n e s i s differ f r o m one ano t h e r a n d t h a t a locus on c h r o m o s o m e 2 or 17 is responsible for the genetic c o n t r o l o f J2 virus-ind u c e d t u m o r i g e n e s i s [20]. O u r results indicate that t r i s o m y for m o u s e c h r o m o s o m e 16 also reveals differences between A b l - M u L V a n d the J2 virus in the m e c h a n i s m s o f fetal liver cell t r a n s f o r mation. D e s p i t e the low efficiencies for t r a n s f o r m a t i o n o f T s l 6 fetal liver cells, we could establish a n u m ber o f A b l - M u L V a n d J2 virus i m m o r t a l i z e d cell lines a n d d e t e r m i n e the d e v e l o p m e n t a l stage in which these cell lines h a d been i m m o r t a l i z e d . T h e finding t h a t T s l 6 a n d d i p l o i d fetal liver derived cell lines show similar p a t t e r n s for the r e a r r a n g e m e n t o f # - H a n d ~c-L chains genes indicates that, a l t h o u g h the n u m b e r o f cells which can be successfully t r a n s f o r m e d is d r a s t i c a l l y lower, there is nevertheless a low p e r c e n t a g e o f d e v e l o p m e n t a l l y n o r m a l pre-B cells in T s l 6 fetal liver. These results are in a g r e e m e n t with p r e v i o u s studies which have s h o w n t h a t the T s l 6 fetal liver cells c o n t a i n only slightly r e d u c e d n u m b e r s ( 8 5 - 9 5 % o f n o r m a l ) o f c y t o p l a s m i c # - H chain a n d s I g M positive cells [8].
Acknowledgements This w o r k was s u p p o r t e d by g r a n t s f r o m the N a t i o n a l Institutes o f H e a l t h (AG-08938) a n d the Swiss N a t i o n a l F o u n d a t i o n (Nr. 83.622.0.88). W e t h a n k T e o d o s i a Z a m o r a for her assistance in the b r e e d i n g o f mice, Elaine C a r l s o n for s u p p o r t , a n d Bill H y u n o f the L a b o r a t o r y for Cell Analysis, U n i v e r s i t y o f C a l i f o r n i a , San F r a n c i s c o , for his help with the F A C S .
References [1] Fong, C. and Brodeur, G.M. (1987) Cancer Genet. Cytogenet. 28, 55. [2] Zipurski, A., Peeters, M. and Pooh, A. (1987) in: Oncology and Immunology of Down Syndrome (E.E. McCoy and C.J. Epstein, Eds.), pp. 33 56, A.R. kiss, New York. [3] Pui, C.-H., Williams, D.L., Scarborough, V., Jackson, C.W., Price, R. and Murphy, S. (1982) Br. J. Haematol. 50, 191. [4] Cairney, A.E.I., McKenna, R., Arthur, D.C., Nesbit, M.E. Jr. and Woods, W.G. (1986) Br. J. Haematol. 63, 541. [5] Searle, A.G., Peters, J., Lyon, M.F., Hall, J.G., Evans, E.P., Edwards, J.H. and Buckle, V.J. (1989) Ann. Hum. Genet. 53, 89. [6] Cox, D.R., Smith, S.A., Epstein, L.B. and Epstein, C.J. (1984) Dev. Biol. 101,416. [7] Epstein, C.J. (1986) The Consequences of Chromosome Imbalance: Principles, Mechanisms and Models. Cambridge University Press, Cambridge and New York. [8] Epstein, C.J., Hofmeister, B.G., Yee, D., Smith, S.A., Philip, R., Cox, D.R. and Epstein, L.B. (1985) J. Exp. Med. 162, 695. [9] Rosenberg, N. and Baltimore, D. (1976) J. Exp. Med. 143, 1453. [10] Alt, F., Rosenberg, N., Lewis, S., Thomas, E. and Baltimore, D. (198l) Cell 27, 381. [11] Rapp, U.R., Cleveland, J.L., Frederickson, T.N., Holmes, K.L., Morse, H.C.III, Jansen, H.W., Patschinsky, T. and Bister, K. (1985) J. Virol. 55, 23. [12] Lanier, L.L., Gutman, C.A., Lewis, D.E., Griswold, S.T. and Warner N.L. (1982) Hybridoma 1, t25. [13] Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. (Eds.) (1989) Current Protocols in Molecular Biology, Sect. IV, Suppl. 13, 2.9.1-2.9.17. Wiley, New York. [14] Marcu, K.B., Banerji, J., Penncavage, N.H., Lang, R. and Arnheim, N. (1980) Cell 22, 187. [15] Coleclough, C., Perry, R.P., Karjalainen, K. and Weigert, M. (1981) Nature 290, 372. [16] Berger, C.N. and Epstein, C.J. (1989) J. Immunol. 143, 389. [17] Tidmarsh, G.R., Heimfeld, S., Whitlock, C.A., Weissman, I.L. and Mtil[er-Sieburg, C.E. (1989) Mol. Cell. Biol. 9, 2665. [18] Miiller-Sieburg, C.E., Townsend, K., Weissman, I.L. and Rennick, D. (1988) J. Exp. Med. 167, 1825. [19] Albritton, L.M., Tseng, L., Scadden, D. and Cunningham, J.M. (1989) Cell 57, 659. [20] Klinken, S.P., Hartley, J.W., Frederickson, T.N., Rapp, U.R. and Morse, H.C. (1989) J. Virol. 63, 2411.
51
2478 / 92 / $ 5.00 © 1992 Elsevier Science Publishers B.V. All rights reserved
IMLET 01801
Deficient transformation of murine trisomy 16 fetal liver cells by the Aigelson and J2 viruses C h r i s t o p h N . B e r g e r a a n d C h a r l e s J. E p s t e i n a'b Departments of apediatrics and bBiochemistry and Biophysics, University of California, San Francisco, CA, USA (Received 9 October 1991; revision received 20 March 1992; accepted 31 March 1992)
I. Summary Mouse trisomy 16 (Tsl6), an animal model for human Down syndrome (trisomy 21), exhibits severe abnormalities in the development of lymphoid and myeloid cells. Whereas fetal liver cells from diploid mice can be easily immortalized by retroviral transformation with A b l - M u L V or J2 virus, fetal livers from Tsl6 mice contain significantly fewer transformable cells. Infection of Tsl6 fetal liver cells by A b l - M u L V results in a 52- and 12-fold reduction in the frequency of transformation at days 17 and 18 of gestation, respectively. By contrast, the efficiency of transformation with J2 virus, another retrovirus known to transform fetal liver cells, is only mildly (factor 23) affected. The Ig gene rearrangements of Tsl6 and diploid retrovirally transformed fetal liver cell lines do not differ from one another. This suggests that there is a deficiency in the early stem cell compartment, rather than in the development of pre-B cells.
2.
Introduction
In persons with Down syndrome resulting from trisomy 21, leukemia of various types, including in particular acute megakaryoblastic leukemia, Key words: Down syndrome; Leukaemia Correspondence to." Charles J. Epstein, M.D., Department of Pediatrics, University of California, San Francisco, CA 941430748, USA. Tel.: 415-476-2981; Fax: 415-476-9976.
occurs with a 10-20-fold higher incidence than in the normal diploid population [1,2]. Moreover, trisomy 21 has been identified in cases of acute megakaryoblastic leukemia in children without Down syndrome [3,4]. These findings suggest that coding or regulatory nucleotide sequences present on chromosome 21 may be involved in the etiology of certain forms of leukemia. Previous work has shown that most, if not all, of the region of chromosome 21 that appears to be responsible for the development of the Down syndrome phenotype, bands 21q22.1 to q22.3, is homologous to a region in the distal part of mouse chromosome 16 [5]. Based on this work and on similarities of developmental deficiencies of hemopoietic, nervous and cardiac systems, Tsl6 mice are considered to constitute an animal model for Down syndrome [6,7] and are being used for investigation of the pathogenesis of the Down syndrome phenotype. As an approach to studying in this model the basis for the unusual susceptibility of persons with Down syndrome to the development of leukemia early in life, we have been using transforming retroviruses. Initial work led to the unexpected finding that Tsl6 mouse fetal liver cells can hardly, if at all, be transformed with Abelson murine leukemia virus (Abl-MuLV) [8], a retrovirus known to transform primarily pre-B cells [9,10]. To determine whether Tsl6 interferes specifically with transformation by A b l - M u L V or whether this resistance to retroviral transformation is a more general phenomenon, we tested Tsl6 fetal liver cells for transformation with another retro47
virus, the J2 virus. The J2 virus is a recombinant retrovirus containing the myc and raf/mil oncogenes and, like A b l - M u L V , is known to immortalize lymphoid cells [11]. Furthermore, to study B cell development in the Tsl6 fetuses, we established A b l - M u L V and J2 virus transformed cell lines and determined the configuration of /x-H and K-L chain genes and, accordingly, their stages of development. 3.
3.1.
Materials and Methods
Animals
Mice doubly heterozygous for the Robertsonian translocations, Rb32Lub and Rb2H, were bred in our animal facilities. To generate Tsl6 mouse fetuses, C57BL/6J females were injected with 5 U human chorionic gonadotropin (Sigma) from pregnant mare's serum, followed 2 days later by 2.5 U gonadotropin (LymphoMed Inc. Melrose, IL), and mated with male Rb32Lub/ Rb2H mice. The morning of the plug formation was considered day 0 of embryonic development. The identification of Tsl6 mouse embryos in midgestation was based on the presence of massive edema and thymic hypoplasia, and in late gestation on thymic hypoplasia, a thick neck and open eyelids.
3.2.
Viruses
Abl-MuLV-producing cells (N54) [9] were obtained from R. Grosschedl, and J2 virus producing cells (J2 leuk) were obtained from U. Rapp [10]. Virus-containing supernatants were prepared by culturing a confluent layer of infected fibroblasts for 1 day in fresh medium.
3.3.
Infection of fetal liver cells and colony assay
Fetal liver cells from Tsl 6 and diploid embryos brought into suspension by passing the livers through 60 mesh stainless steel, were washed and infected at a density of 2 x 107 cells/ml by incubation in virus-containing supernatant for 4~5 h at 37°C in the presence of 4 #g/ml polybrene (Aldrich). The cells were then cultured in Iscove's medium (supplemented with 10% FCS, 5 x 48
10 -5 M 2-ME, 1 unit penicillin/streptomycin) in 0.3% soft agar (Difco) at a density of 2 x l 0 6 cells per 35 mm dish. The colonies were scored after 6~9 days of incubation.
3.4.
Establishment of cell lines
Individual colonies of A b l - M u L V and J2 virus-transformed cells were picked and transferred to liquid cultures in supplemented D M E M . The colonies obtained after A b l - M u L V transformation could be adapted easily to the conditions of liquid culture. By contrast, none of the individually picked colonies from the J2 virus transformed cells could be established as a cell line, although J2 cell lines could be obtained by continuously passaging the cells which had been set up in 2-ml mass cultures at a density of 1-4 × 10 6 cells/ ml. The adapted cell lines were subsequently expanded in supplemented D M E M for the isolation of genomic DNA and further characterization.
3.5.
Antibodies and flow cytometry analysis
Staining for the B220 antigen was done with mAb 14.8 obtained from American Type Culture Collection (TIB 164). Biotinylated mAb anti-rat ~c-L chain Mar 18.5 (TIB 216) [12] was used as secondary reagents. Streptavidin conjugated with FITC was purchased from Southern Biotechnology. Flow cytometry was performed with a FACS 440.
3.6.
Filter hybridization
Isolated DNAs from the A b l - M u L V and J2 virus-transformed cell lines were cut with EcoRI or BamHI, and 10 pg of the cut DNA was sizefractionated in a 1% agarose gel, transferred onto Genescreen, and hybridized according to Selden [13]. As hybridization probes, the inserts of plasmids p J l l and pECK containing #-H chain sequences [14] and ~-L chain sequences [15], respectively, were gel purified and randomly labelled with Klenow polymerase (Random Primers DNA Labelling System, BRL).
4.
4.1.
Results
Tsl6 fetal liver cells are severely deficient for A b l - M u L V transformation and have a reduced efficiency for J2 virus transformation
Determination o f the frequency o f transformation for A b l - M u L V showed that the T s l 6 fetal liver contains far fewer A b I - M u L V transformable cells than the diploid fetal liver. In fetal liver at day 17 o f gestation, a mean o f 54 _+ 23 (SEM) colonies were f o u n d per 106 cells, whereas T s l 6 fetal liver contained only a mean o f 1.0 -t- 0.2 transformable cells per 106 liver cells (Table 1). The p r o p o r t i o n o f transformable cells is 52-fold lower for T s l 6 animals than for diploid animals at day 17 and 11-fold lower at day 18 o f gestation. T o determine whether the reduced transformation efficiency o f T s l 6 cells is specific for A b l M u L V , and also to examine the differences between T s l 6 and diploid B cell precursors, we transformed fetal liver cells with the J2 leukemia virus. This virus is also able to transform B lymphoid cells, but since it carries a different oncogene, it is presumed to use a different mechanism for transformation. Both T s l 6 and diploid cells were transformed with the J2 virus (Table 1). At day 17 o f gestation, an average o f 19 _+ 6 colo-
nies were obtained per 106 T s l 6 fetal liver cells, whereas diploid tissues gave an average o f 37 _+ 19 colonies per 106 cells. At day 18 o f gestation, the fetal livers o f T s l 6 and diploid animals yielded 18 and 48 colonies per 106 cells, respectively. Therefore, the p r o p o r t i o n of J2-transformed T s l 6 cells was only 2-3 fold lower than was found for diploid cells. Overall, T s l 6 fetal liver contained significantly fewer A b l - M u L V (Z2, P = 0 . 0 0 0 1 ) and fewer J2 virus ( P = 0.0001) transformable cells than did diploid fetal liver. However, on day 17 o f gestation, T s l 6 animals contained significantly more J2 virus transformable cells than A b l - M u L V transformable cells whereas the control animals had more A b l - M u L V than J2 virus transformable cells (P = 0.0001).
4.2.
Rearrangements of l~-H- and K-L chain genes in Tsl6 and diploid transformed fetal liver cells
To study the development o f the pre-B cells in T s l 6 fetuses, we established retrovirally transformed cell lines and determined the configuration (rearranged/germline) o f the i m m u n o g l o b u lin genes. We isolated a total o f 14 A b l - M u L V or J2 virus transformed cell lines and found that
TABLE 1 Retroviral transformation of Tsl6 and diploid fetal liver cells Experiment Virus number
Age of fetus
Length Total coloniesb of incubation
CFU fromb retroviral transformation
Diploid
(d.g.)a
(days)
Tsl6
Tsl6
Number of fetuses analyzed Tsl6
Diploid
Ratio of CFU diploid/Ts 16
Diploid
1
Abl-MuLV 5 J2 5
4 4
17 17
6 6
3.2_+0.6 101.0_+2.8 1.3_+0.6 100.5_+2.8 77.8 31.3±3.4 74.7_+1.7 29.5_+3.4 74.2_+1.7 2.5
2
Abl-MuLV 4 J2 4
4 4
17 17
7 7
2.7±0.7 10.1_+1.0
24.5_+7.2 0.8±0.7 24.0_+7.2 30.3 13.0_+2.3 8.3_+1.0 12.5_+2.3 1.5
3
Abl-MuLV 4 J2 4
4 4
17 17
9 9
2.1_+0.6 21.5±2.2
54.6±4.5 0.8±0.6 39.4±1.7 20.3_+2.2
38.5_+4.5 47.4 23.3±1.7 1.1
4
Abl-MuLV 4 J2 4
4 4
18 18
7 7
3.2±0.5 19.9_+0.8
23.0_+2.9 1.7_+0.5 52.5±10.517.5_+0.8
18.3_+2.9 10.7 47.8_+10.5 2.7
aDays of gestation; bValues are colonies per 106 cells + SEM. 49
TABLE 2 Ig rearrangements in retrovirally transformed cell lines derived from T s l 6 and diploid fetal liver cells. Configuration of lg loci of #-H and ~c-L chain genes
H and L germline H rearranged, L germline H and L rearranged
Day 17 FL cells transformed with A b l - M u L V
Day 17 FL cells transformed with J2 virus
Day 18 FL cells transformed with AbI-MuLV
Total
Diploid
Ts 16
Diploid
Ts 16
Diploid
Ts 16
Diploid
Ts 16
1 2 0
0 1 2
0 1 0
0 0 2
0
0
l
0
1 1
1 0
4 1
2 4
the Tsl6 cell lines readily rearrange their #-H chain and ~-L chain loci (Table 2). To confirm that the A b l - M u L V and J2 viruses preferentially transformed pre-B lymphoid cells, we tested 2 Tsl6 derived lines (one A b l - M u L V and one J2 virus transformed cell line) and 4 A b l - M u L V lines derived from diploid fetal liver for the expression of B220, a pre-B cell-specific glycoprotein. All of these cell lines, which grew in culture as single cell suspensions, stained positively for B220. As controls, we established two cell lines by transformation of Tsl6 embryonic fibroblasts. These two lines were negative for the B220 expression and did not show any rearrangement of the #-H chain and ~c-L chain locus (data not shown). 5.
Discussion
The results presented above confirm that Tsl6 fetal liver cells are severely defective for transformation by Abl-MuLV. By contrast, J2 virustransformed cells can easily be obtained, although compared to diploid littermates, the efficiency of J2 virus transformation is still somewhat reduced. These data suggest that the trisomic state affects transformation by more than one retrovirus, but certainly not to the same degree and probably not by the same mechanism. There are several possible explanations for the very low efficiency of A b l - M u L V transformation of Tsl6 cells. One possibility is that in the Ts16 fetuses most of the A b l - M u L V transformable target cells are developmentally absent. It is already known that several other stem cell populations, including CFU-C, CFU-S, BFU-E, CFU-E, are reduced in Tsl6 livers [8]. Furthermore, there 50
also appears to be a deficiency of thymocyte precursors [16]. Other investigators have shown that in the bone marrow of normal mice, A b l - M u L V transformable cells are highly enriched within the subpopulation of Thy-1J°, B220 + cells. This subpopulation does not contain the myeloid-erythroid precursor cells (measured as CFU-S) or the pluripotent hemopoietic stem cells, as shown by its failure to reconstitute lethally irradiated animals [17,18]. It is possible that in fetal liver the same subpopulation also contains the preferential target cells for A b l - M u L V transformation. Although it remains to be determined whether the Thy-1 l°, B220 + subpopulation of fetal liver cells is deficient in Tsl6 fetuses, our preliminary data indicate that proportion of B220 + cells is not significantly diminished in Tsl6 fetal liver. At day 15 of gestation, there were 6.2% B220 + cells in the Tsl6 liver, as compared to 5.1% in the controls. Another possibility is that the target cell population is not developmentally absent but is defective for virus infection or propagation. A defect in virus infectivity might be due to a deficiency in the receptor for the virus. The receptor for murine ecotropic retrovirus infection has been molecularly identified from a BALB/c fibroblast cDNA library. If transfected and expressed in a virus resistant human cell line, it confers infectivity for MuLV [23]. This receptor molecule is encoded by the rec-1 locus on chromosome 5. Although not all the subgroups of type C retroviruses have been tested for the use of this receptor, it is possible that it or another member of the retrovirus receptor multigene family is used by AblMuLV and the J2 virus [19]. If so, the fact that the J2 virus does transform Tsl6 cells indicates that retrovirus receptor molecules are expressed
on T s l 6 fetal liver cells, and, therefore, that o t h e r defects have to be i n v o k e d to explain the greatly r e d u c e d efficiency for A b l - M u L V t r a n s f o r m a t i o n o f Ts 16 fetal liver cells. Yet a n o t h e r possibility is that retroviruses are able to infect a n d p r o p a g a t e equally well in T s l 6 a n d in d i p l o i d cells, b u t t h a t T s l 6 cells are m u c h less likely to u n d e r g o the s e c o n d a r y events necessary for i m m o r t a l i z a t i o n . W i t h the use o f r e c o m b i n a n t i n b r e d m o u s e strains, it has been determ i n e d t h a t susceptibility for A b l - M u L V a n d J2 v i r u s - i n d u c e d t u m o r i g e n e s i s differ f r o m one ano t h e r a n d t h a t a locus on c h r o m o s o m e 2 or 17 is responsible for the genetic c o n t r o l o f J2 virus-ind u c e d t u m o r i g e n e s i s [20]. O u r results indicate that t r i s o m y for m o u s e c h r o m o s o m e 16 also reveals differences between A b l - M u L V a n d the J2 virus in the m e c h a n i s m s o f fetal liver cell t r a n s f o r mation. D e s p i t e the low efficiencies for t r a n s f o r m a t i o n o f T s l 6 fetal liver cells, we could establish a n u m ber o f A b l - M u L V a n d J2 virus i m m o r t a l i z e d cell lines a n d d e t e r m i n e the d e v e l o p m e n t a l stage in which these cell lines h a d been i m m o r t a l i z e d . T h e finding t h a t T s l 6 a n d d i p l o i d fetal liver derived cell lines show similar p a t t e r n s for the r e a r r a n g e m e n t o f # - H a n d ~c-L chains genes indicates that, a l t h o u g h the n u m b e r o f cells which can be successfully t r a n s f o r m e d is d r a s t i c a l l y lower, there is nevertheless a low p e r c e n t a g e o f d e v e l o p m e n t a l l y n o r m a l pre-B cells in T s l 6 fetal liver. These results are in a g r e e m e n t with p r e v i o u s studies which have s h o w n t h a t the T s l 6 fetal liver cells c o n t a i n only slightly r e d u c e d n u m b e r s ( 8 5 - 9 5 % o f n o r m a l ) o f c y t o p l a s m i c # - H chain a n d s I g M positive cells [8].
Acknowledgements This w o r k was s u p p o r t e d by g r a n t s f r o m the N a t i o n a l Institutes o f H e a l t h (AG-08938) a n d the Swiss N a t i o n a l F o u n d a t i o n (Nr. 83.622.0.88). W e t h a n k T e o d o s i a Z a m o r a for her assistance in the b r e e d i n g o f mice, Elaine C a r l s o n for s u p p o r t , a n d Bill H y u n o f the L a b o r a t o r y for Cell Analysis, U n i v e r s i t y o f C a l i f o r n i a , San F r a n c i s c o , for his help with the F A C S .
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