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Probability of commitment of murine erythroleukemia cells is determined by c-myc level
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P r o b a b i l i t y of c o m m i t m e n t of m u r i n e erythroleukemia c e l l s is d e t e r m i n e d by c - m y c level. Masuo Obinata,*, Tohru Yamamoto, Yasuhisa Matsui, and Shinji Takada, Dept. Cell Biol., R e s . I n s t . T B & Cancer, Tohoku University, Sendai, 980, J a p a n During the differentiation of mouse erythroleukemia (MEL) c e l l s , c-myc mRNA levels change dramatically. To examine the involvement of c-myc in the c o m m i t m e n t of M E L cells, the rat cmyc gene under the control of metallothionein gene promoter was t r a n s f e r r e d to M E L cells. In t h e s e transformants, the elevated levels of c - m y c in t h e p r e s e n c e of Zn i o n inhibited the commitment and the differentiation of t h e M E L c e l l s . with these transformants, we d e m o n s t r a t e d t h a t the p r o b a b i l i t y of commitment of MEL cell was determined by a stoichiometric a m o u n t of c - m y c . The elevated levels of c - m y c inhibited the time-dependent e x p r e s s i o n of t w o e r y t h r o i d - s p e c i f i c genes (globin and glycophorin) in the mechanism different from the i n h i b i t i o n of c o m m i t m e n t .
ONCOGENE IMPACT ON HAEMOPOIETIC CELLS J.M. Adams, S. Cory, A. Harris, W. Langdon, P. Klinken, W. Alexander, K. Kongsuwan, E. Webb, H. Rosenbaum, D. Vaux, J. Allen. Walter and Eliza Hall Institute, Melbourne, 3050, Australia. To determine how on(:ogenes influence haemopoietic differentiation and contribute to neoplasia, we have made transgenic mice bearing different oneogenes (myc, N-rc~, v-abl bcr-abl) coupled to an immunoglobulin enhancer (E~), which is operative in lymphoid and some myeloid cells. Both the ,~c and N-,~.c transgenes cause over-production of early B lineage cells a n d , eventually, B lymphoid tumors. Tumorigenesis requires spontaneous genetic changes that can involve ,~s mutations. The other transgenes elicit tumours at other stages of B lymphoid development, or in other haemopoietic cell types, suggesting that oncogenic action is related to the differentiation programme of the cell. A striking lineage conversion could be elicited by the v-tar oncogene: B lymphoma cells acquired a complete macrophage phenotype. To gain purchase on genes that may directly govern cell phenotype, we have searched for homeobox mRNAs in haemopoietic cells and have shown that multiple homeobox genes are expressed, some in a lineagerestricted fashion. Thus, homeobox genes may contribute to cell diversification in adult animals as well as in the embryo.
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Detection of blood cell precursors in the c h i c k embryo's sequentially active hemopoietic sites. Are they seeded by a common ancestor ? F. Dieterlen-Li%vre, F. Yassine, C. Bolnet and F. C o r m i e r - Institut d'Embryologie du CNRS Nogent-sur-Marne France We have demonstrated earlier that stem cells responsible for the colonization of definitive blood forming organs are intraembryonic in origin. We are now performing an inventory in organs that carry out hemopoietic activities successively during development. Conditioned media allowing the development of G-CFCs, MG-CFCs and mixed CFCs in clonal assays, as well as a Dexter-like long term culture technique, have been developed. The clonal assays dected 3-4 times more MG-CFCs in the E3-4 aortic wall and 10 times more in the Ell-13 spleen than in the newborn bone marrow. The Eli-13 spleen synthesizes factors which promote powerfully the m u l t i p l i c a t i o n of early progenitors. Quail-chick interspecies grafting, followed by immunocytotogical analysis with mab QHI, a quail hemangioblastic lineage marker, i s applied to address the question as to whether these successive hemopoietic sites are linked to one another by virtue of p r o g e n i t o r seeding or whether the process of stem cell formation, already known to occur in the extraembryonic area and then in the aortic wall, can be reiterated in the bone marrow.
CULTURE OF PLURIPOTENT HEMOPOIETIC STEM CELLS PURIFIED FROM MOUSE BONE MARROW Visser JWM and Hogeweg-Platenburg MGC Radiobiological Institute TNO, Rijswijk, The Netherlands Pluripotent hemopoietic stem cells (PHSC) were purified from mouse bone marrow using density gradient centrifugation, and fluorescence-activated cell sorting with as selection parameters fight scatter and the binding of wheat germ agglutinin and of an anti-monocyte antibody, 151.1 (Visser and De Vries, 1988, Blood Cells 14, 369). Additional labeling with the mitochondria specific dye rhodamine 123 revealed two subsets of PHSC. Rh123 dull ones and bright ones. The Rh123 bright cells could be cultured in the presence of interleukin 3, M-CSF, hemin and erythropoietin in serum-free conditions to yield colonies of hemopoietic cells in vitro with a plating efficiency of 50%. Few of these colonies were of mixed type. The Rh123 dull sorted stem cells showed a poor plating efficiency of 10 to 20 % with these growth factors, also if interleukin 1 or 6 was added. In vivo, however, the Rh123 dull sorted stem cells provided long term restoration of the immuno-hemopoietic system in lethally irradiated mice. The Rh123 bright, sorted PHSC gave many spleen colonies, but only transient restoration of hemopoiesis. This indicates that spleen colony formation is not a reliable assay for PHSC. The data also indicate, that pluripotent hemopoietic stem cells cannot be cultured efficiently with the presently known growth factors. It can be speculated that the replication of the PHSC requires cell-cell interactions and adhesion, in addition to growth factors. S178
585
P r o b a b i l i t y of c o m m i t m e n t of m u r i n e erythroleukemia c e l l s is d e t e r m i n e d by c - m y c level. Masuo Obinata,*, Tohru Yamamoto, Yasuhisa Matsui, and Shinji Takada, Dept. Cell Biol., R e s . I n s t . T B & Cancer, Tohoku University, Sendai, 980, J a p a n During the differentiation of mouse erythroleukemia (MEL) c e l l s , c-myc mRNA levels change dramatically. To examine the involvement of c-myc in the c o m m i t m e n t of M E L cells, the rat cmyc gene under the control of metallothionein gene promoter was t r a n s f e r r e d to M E L cells. In t h e s e transformants, the elevated levels of c - m y c in t h e p r e s e n c e of Zn i o n inhibited the commitment and the differentiation of t h e M E L c e l l s . with these transformants, we d e m o n s t r a t e d t h a t the p r o b a b i l i t y of commitment of MEL cell was determined by a stoichiometric a m o u n t of c - m y c . The elevated levels of c - m y c inhibited the time-dependent e x p r e s s i o n of t w o e r y t h r o i d - s p e c i f i c genes (globin and glycophorin) in the mechanism different from the i n h i b i t i o n of c o m m i t m e n t .
ONCOGENE IMPACT ON HAEMOPOIETIC CELLS J.M. Adams, S. Cory, A. Harris, W. Langdon, P. Klinken, W. Alexander, K. Kongsuwan, E. Webb, H. Rosenbaum, D. Vaux, J. Allen. Walter and Eliza Hall Institute, Melbourne, 3050, Australia. To determine how on(:ogenes influence haemopoietic differentiation and contribute to neoplasia, we have made transgenic mice bearing different oneogenes (myc, N-rc~, v-abl bcr-abl) coupled to an immunoglobulin enhancer (E~), which is operative in lymphoid and some myeloid cells. Both the ,~c and N-,~.c transgenes cause over-production of early B lineage cells a n d , eventually, B lymphoid tumors. Tumorigenesis requires spontaneous genetic changes that can involve ,~s mutations. The other transgenes elicit tumours at other stages of B lymphoid development, or in other haemopoietic cell types, suggesting that oncogenic action is related to the differentiation programme of the cell. A striking lineage conversion could be elicited by the v-tar oncogene: B lymphoma cells acquired a complete macrophage phenotype. To gain purchase on genes that may directly govern cell phenotype, we have searched for homeobox mRNAs in haemopoietic cells and have shown that multiple homeobox genes are expressed, some in a lineagerestricted fashion. Thus, homeobox genes may contribute to cell diversification in adult animals as well as in the embryo.
586
587
Detection of blood cell precursors in the c h i c k embryo's sequentially active hemopoietic sites. Are they seeded by a common ancestor ? F. Dieterlen-Li%vre, F. Yassine, C. Bolnet and F. C o r m i e r - Institut d'Embryologie du CNRS Nogent-sur-Marne France We have demonstrated earlier that stem cells responsible for the colonization of definitive blood forming organs are intraembryonic in origin. We are now performing an inventory in organs that carry out hemopoietic activities successively during development. Conditioned media allowing the development of G-CFCs, MG-CFCs and mixed CFCs in clonal assays, as well as a Dexter-like long term culture technique, have been developed. The clonal assays dected 3-4 times more MG-CFCs in the E3-4 aortic wall and 10 times more in the Ell-13 spleen than in the newborn bone marrow. The Eli-13 spleen synthesizes factors which promote powerfully the m u l t i p l i c a t i o n of early progenitors. Quail-chick interspecies grafting, followed by immunocytotogical analysis with mab QHI, a quail hemangioblastic lineage marker, i s applied to address the question as to whether these successive hemopoietic sites are linked to one another by virtue of p r o g e n i t o r seeding or whether the process of stem cell formation, already known to occur in the extraembryonic area and then in the aortic wall, can be reiterated in the bone marrow.
CULTURE OF PLURIPOTENT HEMOPOIETIC STEM CELLS PURIFIED FROM MOUSE BONE MARROW Visser JWM and Hogeweg-Platenburg MGC Radiobiological Institute TNO, Rijswijk, The Netherlands Pluripotent hemopoietic stem cells (PHSC) were purified from mouse bone marrow using density gradient centrifugation, and fluorescence-activated cell sorting with as selection parameters fight scatter and the binding of wheat germ agglutinin and of an anti-monocyte antibody, 151.1 (Visser and De Vries, 1988, Blood Cells 14, 369). Additional labeling with the mitochondria specific dye rhodamine 123 revealed two subsets of PHSC. Rh123 dull ones and bright ones. The Rh123 bright cells could be cultured in the presence of interleukin 3, M-CSF, hemin and erythropoietin in serum-free conditions to yield colonies of hemopoietic cells in vitro with a plating efficiency of 50%. Few of these colonies were of mixed type. The Rh123 dull sorted stem cells showed a poor plating efficiency of 10 to 20 % with these growth factors, also if interleukin 1 or 6 was added. In vivo, however, the Rh123 dull sorted stem cells provided long term restoration of the immuno-hemopoietic system in lethally irradiated mice. The Rh123 bright, sorted PHSC gave many spleen colonies, but only transient restoration of hemopoiesis. This indicates that spleen colony formation is not a reliable assay for PHSC. The data also indicate, that pluripotent hemopoietic stem cells cannot be cultured efficiently with the presently known growth factors. It can be speculated that the replication of the PHSC requires cell-cell interactions and adhesion, in addition to growth factors. S178