IN VITRO Volume14, No. 1, 1978 Allrightsreserved9
REGULATION OF DIFFERENTIATION IN NORMAL AND TRANSFORMED ERYTHROID CELLS' RICHARD A. RIFKIND, 2PAUL A. MARKS, ARTHUR BANK, MASAAIKI TERADA, ROBERTA C. REUBEN, GEORGE M. MANIATIS, EITAN FIBACH, URI NUDEL, JANE E. SALMON, ANDYAIR GAZITT
Columbia University, College o/Physicians and Surgeons, New York, New York 10032
SUMMARY Studies are described employing two erythropoietic systems to elucidate regulatory mechanisms that control both normal erythropoiesis and erythroid differentiation of transformed hemopoietic precursors. Evidence is provided suggesting that normal erythroid cell precursors require erythropoietin as a growth factor that regulates the number of precursors capable of differentiating. Murine erythroleukemia cells proliferate without need of erythropoietin; they show a variable, generally low, rate of spontaneous differentiation and a brisk rate of erythropoiesis in response to a variety of chemical agents. Present studies suggest that these chemical inducers initiate a series of events including cell surface related changes, alterations in cell cycle kinetics, and modifications of chromatin and D N A structure which result in the irreversible commitment of these leukemia cells to erythroid differentiation and the synthesis of red-cell-specific products.
K e y words: erythropoiesis; Friend cell; erythroleukemia; erythropoietin; differentiation. INTRODUCTION events involved in the induction of M E L C to difStudies in this laboratory have been designed to ferentiate to erythroid cells by exposure to dielucidate the mechanisms regulating eukaryotic methylsulfoxide IMe2SO) (2) and other chemical cell growth and differentiation, employing two inducers (3-10) may provide clues to the derangeerythropoietic model systems [one normal, one ment of normal control mechanisms in malignant malignant), erythropoietin-dependent fetal mouse cells, as well as revealing sites of regulatory activliver erythropoiesis (1), and the chemically in- ity significant to the initiation of erythroid differduced erythropoietic differentiation observed in entiation and to the expression of the complex patmurine erythroleukemia cells (MELC) trans- tern of biosynthetic and morphogenetic events formed by the Friend virus complex (2). Studies that characterize erythropoiesis. on the effects of erythropoietin, the physiological THE EFFECTS OF ERYTHROPOIETIN regulator of the rate of erythropoiesis (red-bloodcell production), can provide clues to the reguErythropoietin, prepared from the serum or lated target cell and to the nature of the cellular urine of anemic subjects and added to cultures of response to hormonal control of hematopoiesis. fetal hepatic erythroid cells ~11 ) or a purified fracThese studies are limited, however, by the rela- tion of fetal erythroid precursor cells (12), results tively short survival of normal, self-renewing pre- in the proliferation of erythroid precursors and cursor cell populations under the in vitro condi- the appearance of large numbers of differentiated tions needed for experimental study. Some aspects progeny characterized by morphological features, of this limitation are relieved by the use of trans- the accumulation of red-cell surface antigens and formed erythroid precursors. Studies elucidating hemoglobin synthesis. Both the number of precursor cells stimulated to proliferate and the number Tresented in the formal symposium on Mechanisms of Cellular Control at the 28th Annual Meeting of the of cell divisions they undergo are regulated by the Tissue Culture Association, New Orleans, Louisiana, concentration of erythropoietin provided under June 6-9, 1977. "Please address requests for reprints to Dr. Richard colony-forming in vitro conditions ~13). In the abA. Rifkind, Columbia Presbyterian Medical Center, 630 sence of erythropoietin small numbers of precurWest 168th Street, New York, N.Y. 10032. sors can differentiate, but erythropoiesis is mea155
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get and unsustained. Stimulation of RNA synthesis, in a very immature precursor resembling the proerythroblast, particularly ribosomal RNA, 4 and 5S RNA, but not, initially, globin mRNA, can be detected by 1 hr after addition of the hormone (14). Already differentiating (hemoglobincontaining) erythroblasts do not display this response to erythropoietin. Globin-specific mRNA is detected by molecular hybridization to globin cDNA (15), as well as by assay in the cell-free system (16), in erythropoietin-stimulated precursor cell cultures after about 6 to 10 hr of culture, and there is no detectable lag between the time of appearance of the m R N A and the accumulation of hemoglobin. A 500-fold increase in globin m R N A can be detected over the first 44 hr of culture with erythropoietin, accompanied by a 2- to 3-fold increase in the number of erythroid cells. D N A synthesis is not required for cells to display the early stimulation of rRNA and tRNA synthesis initiated by erythropoietin (14, 17). Nevertheless, D N A synthesis appears to be required for differentiation, that is, for transition to globin m R N A accumulation, since inhibition of D N A synthesis by over 95%, by hydroxyurea, prevents erythropoietin-mediated stimulation of globin synthesis (18). Other observations on hemoglobin synthesis, during chicken embryogenesis, for example (19), are consistent with the assumption that DNA synthesis is required for the expression of the differentiated program of transcription, but, as of this moment, the data are not sufficiently definitive. Nevertheless, a speculative model for the regulatory effects of erythropoietin can be constructed which may serve to stimulate further experimental work. In this model, erythropoietin is responsible for the proliferation of its target cell, determining, in effect, the size of the erythropoietic cell population. The higher the titer of erythropoietin, the more target cells are triggered into cell cycle. By a regulatory mechanism as yet totally undefined, a proportion of proliferating precursors are committed to express the genetic program of erythroid differentiation, including globin m R N A production. It is this process, the commitment to differentiation, which has proved relatively recalcitrant to study using normal erythroid precursors, and which, perhaps can, be elucidated more effectively with transformed erythroid cells. ~IURINE ERYTHROLEUKEMIACELLS Murine erythroleukemia cells (MELC), which are erythroid precursors transformed by the
Friend leukemia virus complex, provide several advantages for the biochemical and genetic studies required to define the regulation of induction of eukaryotic cells to differentiate. M E L C can be maintained in continuous cultures and many strains show a low level (
REGULATION OF DIFFERENTIATION IN NORMAL AND TRANSFORMED ERYTHROID CELLS' RICHARD A. RIFKIND, 2PAUL A. MARKS, ARTHUR BANK, MASAAIKI TERADA, ROBERTA C. REUBEN, GEORGE M. MANIATIS, EITAN FIBACH, URI NUDEL, JANE E. SALMON, ANDYAIR GAZITT
Columbia University, College o/Physicians and Surgeons, New York, New York 10032
SUMMARY Studies are described employing two erythropoietic systems to elucidate regulatory mechanisms that control both normal erythropoiesis and erythroid differentiation of transformed hemopoietic precursors. Evidence is provided suggesting that normal erythroid cell precursors require erythropoietin as a growth factor that regulates the number of precursors capable of differentiating. Murine erythroleukemia cells proliferate without need of erythropoietin; they show a variable, generally low, rate of spontaneous differentiation and a brisk rate of erythropoiesis in response to a variety of chemical agents. Present studies suggest that these chemical inducers initiate a series of events including cell surface related changes, alterations in cell cycle kinetics, and modifications of chromatin and D N A structure which result in the irreversible commitment of these leukemia cells to erythroid differentiation and the synthesis of red-cell-specific products.
K e y words: erythropoiesis; Friend cell; erythroleukemia; erythropoietin; differentiation. INTRODUCTION events involved in the induction of M E L C to difStudies in this laboratory have been designed to ferentiate to erythroid cells by exposure to dielucidate the mechanisms regulating eukaryotic methylsulfoxide IMe2SO) (2) and other chemical cell growth and differentiation, employing two inducers (3-10) may provide clues to the derangeerythropoietic model systems [one normal, one ment of normal control mechanisms in malignant malignant), erythropoietin-dependent fetal mouse cells, as well as revealing sites of regulatory activliver erythropoiesis (1), and the chemically in- ity significant to the initiation of erythroid differduced erythropoietic differentiation observed in entiation and to the expression of the complex patmurine erythroleukemia cells (MELC) trans- tern of biosynthetic and morphogenetic events formed by the Friend virus complex (2). Studies that characterize erythropoiesis. on the effects of erythropoietin, the physiological THE EFFECTS OF ERYTHROPOIETIN regulator of the rate of erythropoiesis (red-bloodcell production), can provide clues to the reguErythropoietin, prepared from the serum or lated target cell and to the nature of the cellular urine of anemic subjects and added to cultures of response to hormonal control of hematopoiesis. fetal hepatic erythroid cells ~11 ) or a purified fracThese studies are limited, however, by the rela- tion of fetal erythroid precursor cells (12), results tively short survival of normal, self-renewing pre- in the proliferation of erythroid precursors and cursor cell populations under the in vitro condi- the appearance of large numbers of differentiated tions needed for experimental study. Some aspects progeny characterized by morphological features, of this limitation are relieved by the use of trans- the accumulation of red-cell surface antigens and formed erythroid precursors. Studies elucidating hemoglobin synthesis. Both the number of precursor cells stimulated to proliferate and the number Tresented in the formal symposium on Mechanisms of Cellular Control at the 28th Annual Meeting of the of cell divisions they undergo are regulated by the Tissue Culture Association, New Orleans, Louisiana, concentration of erythropoietin provided under June 6-9, 1977. "Please address requests for reprints to Dr. Richard colony-forming in vitro conditions ~13). In the abA. Rifkind, Columbia Presbyterian Medical Center, 630 sence of erythropoietin small numbers of precurWest 168th Street, New York, N.Y. 10032. sors can differentiate, but erythropoiesis is mea155
156
RIFKIND ET AL.
get and unsustained. Stimulation of RNA synthesis, in a very immature precursor resembling the proerythroblast, particularly ribosomal RNA, 4 and 5S RNA, but not, initially, globin mRNA, can be detected by 1 hr after addition of the hormone (14). Already differentiating (hemoglobincontaining) erythroblasts do not display this response to erythropoietin. Globin-specific mRNA is detected by molecular hybridization to globin cDNA (15), as well as by assay in the cell-free system (16), in erythropoietin-stimulated precursor cell cultures after about 6 to 10 hr of culture, and there is no detectable lag between the time of appearance of the m R N A and the accumulation of hemoglobin. A 500-fold increase in globin m R N A can be detected over the first 44 hr of culture with erythropoietin, accompanied by a 2- to 3-fold increase in the number of erythroid cells. D N A synthesis is not required for cells to display the early stimulation of rRNA and tRNA synthesis initiated by erythropoietin (14, 17). Nevertheless, D N A synthesis appears to be required for differentiation, that is, for transition to globin m R N A accumulation, since inhibition of D N A synthesis by over 95%, by hydroxyurea, prevents erythropoietin-mediated stimulation of globin synthesis (18). Other observations on hemoglobin synthesis, during chicken embryogenesis, for example (19), are consistent with the assumption that DNA synthesis is required for the expression of the differentiated program of transcription, but, as of this moment, the data are not sufficiently definitive. Nevertheless, a speculative model for the regulatory effects of erythropoietin can be constructed which may serve to stimulate further experimental work. In this model, erythropoietin is responsible for the proliferation of its target cell, determining, in effect, the size of the erythropoietic cell population. The higher the titer of erythropoietin, the more target cells are triggered into cell cycle. By a regulatory mechanism as yet totally undefined, a proportion of proliferating precursors are committed to express the genetic program of erythroid differentiation, including globin m R N A production. It is this process, the commitment to differentiation, which has proved relatively recalcitrant to study using normal erythroid precursors, and which, perhaps can, be elucidated more effectively with transformed erythroid cells. ~IURINE ERYTHROLEUKEMIACELLS Murine erythroleukemia cells (MELC), which are erythroid precursors transformed by the
Friend leukemia virus complex, provide several advantages for the biochemical and genetic studies required to define the regulation of induction of eukaryotic cells to differentiate. M E L C can be maintained in continuous cultures and many strains show a low level (
