Terminal Differentiationa S. FERRAR1,b A. GRANDE, R. MANFREDINI, AND U. TORELLI Experimental Hematology Center II Medical Clinic University of Modena Via Del Pozzo 71, Policlinico 41100 Modena, Italy
Cell differentiation ultimately proceeds as a response to signals from the extracellular microenvironment. If the cells are competent, that is, if they carry the corresponding receptors and signal transduction pathways, they can be triggered in the commitment state. In many cases the response includes complex gene expression programs whose progress and completion are relatively autonomous and whose outcome is determined by inherent regulatory factors of the cell type affected.' As a short introduction to the problems of differentiation and aging, we survey the main points that characterize the differentiation of hematopoietic cells. We can identify several main aspects. PROGRESSIVE GROWTH ARREST IN THE G1 PHASE OF THE CELL CYCLE
Several lines of evidence are reported to support a functionally coupled relation between cell growth and differentiation. This relation is characterized initially by the expression of cell cycle-related genes along with genes coding for cytokines and their receptors. Subsequently, down-regulation of proliferation, partially mediated by extracellular stimuli, parallels the expression of a series of genes associated with the maturation pathways. Important progress is represented by elucidation of the mechanism by which these cell-cycle associated genes are suppressed.* For instance, it is commonly accepted that leukemic blast cells are arrested, but at different stages, along the differentiation pathway and never spontaneously reach terminal differentia t i ~ nWe . ~ do not yet know the correlations between proliferation and differentiation despite an incredible amount of experimental work on the kinetics of these cells4and on the mechanisms of action of growth factors and their receptor^.^^^ In fact, several cytokines are capable of triggering the target cells simultaneously along the proliferation and differentiation pathways. However, in vitro cultured leukemic blast cells can be rescued only to proliferate when incubated with purified growth factors. On the other hand, acute promyelocytic leukemic blast cells carrying the t(15;17) in which the retinoic acid receptor (alpha type) is juxtaposed to the Mil gene are capable of differentiating in vivo to granulocytes when treated with all-trans-retinoic acid. When the differentiation commitment of a normal precursor cell occurs, the G1 aThis work was supported by a grant from the Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.). bAddress for correspondence and reprint requests: Dr. Sergio Ferrari, Experimental Hematology Center, I1 Medical Clinic, University of Modena, Via Del Pozzo 71, Policlinico, 41100 Modena, Italy. 180
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phase of the cell cycle progressively increases in length, and terminally differentiated cells are practically arrested in this phase of the cycle and cannot be triggered along the proliferative pathway. Furthermore, previous studies have shown that high levels of expression of genes involved in the proliferative pathway can inhibit cell differentiation. The effect of these genes on terminal differentiation is due not to significant alterations in normal growth control mechanisms but instead to an inability of the cells to enter a pathway leading to the terminally differentiated state. In fact, the genetic program of terminal differentiation often, if not always, requires the permanent withdrawal of several cell cycle gene-related products. Among these genes we can include c-myc, c-myb, erb-A, c-fos, and c-jun. For example, mouse erythroleukemia cells expressing a recombinant c-myc gene fail to differentiate; other examples are the suppression of erythroid differentiation by overexpression of the c-myb oncogene and suppression of myogenesis by the increased expression of erb-B. Overexpression of c-jun is capable of blocking the differentiation pathway in erythroleukemia Friend induced by DMSO. The preliminary conclusion of these experiments is that several oncogenes are involved primarily in cell proliferation and that cell differentiation requires permanent withdrawal of these gene products and the permanent exit from the cell cycle.’
SIGNAL RECEPTION AND TRANSDUCTION
Both proliferation and differentiation are controlled mainly by cytokines and their receptors, intracellular signal transduction pathways, and eventual gene activation. In the first level of control, cytokines interact with their corresponding receptors on the cell and initiate a cascade of events that eventually directs the cells to proliferate or differentiate.* Hematopoietic growth factors are mainly produced by bone marrow accessory cells. Primitive hematopoietic precursors (CD34 positive bone marrow cells) are induced to proliferate by several different cytokines which act in a synergistic and cooperative way. Among these cytokines are the stem cell factor (SCF), IL-1 alpha, IL-3, and IL-6 which affect the formation of colonies derived from more primitive hematopoietic progenitor cells in vitro, namely, the multicolonyforming cells ( C F C - m ~ l t i )The . ~ aforementioned cytokines are capable of controlling the surface expression of receptors that can bind other growth factors particularly involved in hematopoietic cell differentiation. Even if these cytokines are more important for proliferative pathway activation, they can simultaneously trigger the hematopoietic cells in the differentiation pathway, activating the differentiation genetic program. When the hematopoietic cell is in a commitment state, the action of several other cytokines is particularly important, such as EPO for erythroid differentiation, M-CSF for macrophage differentiation, G-CSF for granulocytic differentiation, IL-5 and G-CSF for eosinophil differentiation, IL-4 for basophil differentiation, and IL-2, IL-4, and IL-7 for lymphoid B or T differentiation, respectively.6 The specific metabolic pathways activated by these different ligand-receptor interactions are poorly understood, particularly as far as the correlation between the proliferation and differentiation pathways is concerned. Interesting biological behavior is described for acute leukemia blast cells which are unable to progress through the cell cycle and are arrested mainly in the G1 phase of the cycle but are simultaneously blocked in the terminal differentiation program and are arrested in different phases of the differentiation pathway. A further interesting observation is that these cells are able mainly to express several cytokine receptors but not the corresponding cytokines and are able to respond in vitro to growth factors that can induce
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ANNALS NEW YORK ACADEMY OF SCIENCES
proliferation but not to growth factors mainly involved in the differentiation pathway. Two other transduction pathways involved in differentiation are activated by retinoic acid (RA) and diidrocalciferol (vitamin D3). The retinoids comprise a group of compounds including retinoic acid, retinol (vitamin A), and a series of natural and synthetic derivatives that exert profound effects on development and differentiation in a wide variety of systems. The interaction of retinoic acid and its receptor (RAR alpha, beta, and gamma) induced the transcription of several genes. In the myeloid system, the interaction of retinoic acid with its receptor induces granulocytic differentiation.1° A major contribution toward understanding the biologic role of 1,25-(OH)*D3 was provided by elucidation of its mechanism of action. In fact, hormonal effects are mediated by specific nuclear receptors that were recently isolated and structurally determined. These receptors are present in hematopoietic cells, and several studies have shown that HL-60 cells are induced to monocytic differentiation when treated with diidrocalciferol (vitamin D3)." It is important that several specific differentiation transcriptional factors have been isolated and characterized such as the GATAl gene which plays an important role in erythroid differentiationI2 or the several DNA binding proteins involved in myeloid differentiation.I3 It was shown recently that the gene encoding for hRXR alpha interacts directly with and enhances the binding of nuclear receptors conferring responsiveness to vitamin D3 and thyroid hormone T3, suggesting that hRXR has a central role in multiple hormonal signaling pathways included in the differentiation pathways.I4 SELECTIVE MODIFICATION IN THE GENETIC MATERIAL Lymphoid B- and T-cell differentiation is characterized by DNA rearrangements in Ig and TCR gene loci, respectively, that occur in a precise order in the early lymphoid progenitor cells independent of antigen stimulation. In fact, all cells except B lymphocytes express these genes in the germline configuration, and only B lymphocytes express these genes in functionally rearranged forms, capable of giving rise to functional proteins. Stimuli that determine the commitment of a bone marrow stem cell to mature along the B-lymphocyte lineage are largely unknown. Such commitment, however, leads to somatic recombination of Ig genes. T lymphocytes, like B lymphocytes, originate from precursors in bone marrow and then migrate to the thymus. Functional TCR alpha and beta genes as well as gamma and delta chain genes are formed by somatic rearrangement of germline gene segments by a process that is very similar to Ig gene rearrangements. Rearrangement and expression of TCR genes during intrathymic maturation are the necessary first step in the development of T-cell repertoire, and all together with the expression of several other specific proteins characterize the T-cell differentiation genetic program.I5 DIFFERENTIAL GENE EXPRESSION Several genes have been isolated that are associated with cell differentiation.16 The definition of cell differentiation-related genes is simply based on the concept that mRNA and/or protein abundance of these genes increases in this functional state of the cell. It is clearly very important to distinguish between gene products that are simply associated with a particular functional state and gene products restricted and perhaps regulating that function, in other words, genes whose expression is
FERRARI er al.: TERMINAL DIFFERENTIATION
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essential for the differentiation genetic programs. In fact, these latter genes can play a regulatory role because of their interference with several signal transduction pathways underlying differentiation. That a gene product is restricted to a cell function was demonstrated using very different technical approaches based on different methods with the specific goal of selectively inactivating that gene product. Among these different methods are microinjection of cells with antibodies or synthetic antisense R N A transcripts, treatment of cell populations with specific antisense oligomers, transfection of plasmids or constructs expressing high levels of specific antisense R N A molecules under the control of strong and often inducible promoters, transgenic mice, and more recently homologous recombination. As mentioned, one of the opportunities offered by these methods is to obtain specific and possibly complete inhibition of a gene product at the level of either the genetic locus or its transcribed product (precursor mRNA, mature mRNA, and protein) and to look at the biological consequences of the spccific gene’s inactivation. As pointed out before, these experimental approaches allow the discrimination of gene products that limit and perhaps regulate cellular differentiation. In fact, inactivation of some cell differentiation-related genes inhibits in different ways the genetic program of a specific pathway, such as granulocytic differentiation by c-fes, macrophage differentiation by c-fms, erythroid differentiation by MERS, and muscular differentiation by myosin. Instead, inhibition of some other genes whose expression increases during differentiation does not interfere with the specific differentiation pathway, but may deprive the differentiated cells of one of its specific functions. This is the case, for instance, of myeloperoxidase and lactoferrin genes in granulocytic ~ ~ 1 1 s . ’ ~
REORGANIZATION OF GENE EXPRESSION PROGRAMS The cDNA-poly(A)+RNA reassociation technique has been used extensively to study the R N A sequence complexity and kinetic abundance in different stages. We studied the kinetic composition of polyadenylated RNA molecules of HL-60 (myeloid, M2 phenotype cells) using the aforementioned technique before (proliferating cells) and after induction of differentiation with retinoic acid (terminal differentiation to granulocytes). The data obtained with this investigation showed: (1) a 70% decrease in the sequence complexity (number of different transcribed sequences) in HL-60 cells treated with R A compared with proliferating cells; (2) a drastic drop in the number of abundant sequences in RA-treated HL-60 cells; and (3) a marked increase in the repetition frequency of the abundant and rare components after induction of differentiation of HL-60 with RA. Finally, the most abundant sequences in HL-60 cells after RA induction are already present before treatment. O u r data suggest that at least during myeloid differentiation, important transcriptional and posttranscriptional regulatory mechanisms of gene expression are active soon after the first commitment event of the hematopoietic undifferentiated precursor cells, leading to a reduction in the number of expressed genes together with an increased abundance of hundreds of so-called “cell differentiation associated genes.” These results are in keeping with the biological function of terminally differentiated cells whose main metabolic activity differs according to the specific cell function, such as phagocytosis for granulocytes and macrophages, and globin storage for erythrocytes.18
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ANNALS NEW YORK ACADEMY OF SCIENCES IMPORTANCE OF THE POS'ITRANSCRIPTIONAL REGULATION OF GENE EXPRESSION
An important step in the cascade of molecular events leading to the accumulation of mRNA is the efficient processing of primary transcripts. Evidence of mechanisms operating at this level has so far been obtained only for regulation of the expression of cell cycle related genes. In fact, the concentration of thymidine kinase (TK) during the G1 progression phase is strictly related to stabilization of the primary transcript and to subsequent activation of the processing mechanisms during the S phase.19 As for terminal differentiation, it is well known that this process is accompanied by a short reduction in the production of ribosomal RNA. This production is the consequence of both a reduced rate of transcription and a reduced rate of processing the primary transcript. We have evidence that during terminal differentiation of HL-60 cells the 32 S ribosomal precursor RNA accumulates, suggesting that this molecule undergoes remarkable stabilization and it is not processed further. Similar behavior seems to underly the disappearance of c-myc mRNA during granulocytic differentiation of HL-60 cells. This differentiation is conditioned by arrest of the expression of c-myc, and this arrest is obtained through a reduction in the transcription rate as well as a complete arrest of the processing of the primary transcript, so that the abundance of the precursor remains elevated?O Stabilization of a translational product is also a well-documented mechanism through which a terminally differentiated cell increases the concentration of a specific protein. For instance, during granulocytic differentiation, myeloperoxidase as well as lactoferrin remarkably increase from the promyelocyte, myelocyte stages, whereas their mature mRNA are strongly decreased so that they are completely absent in the final differentiation stages.21
TERMINAL DIFFERENTIATION AND PROGRAMMED CELL DEATH
The average life span of terminally differentiated cells can no longer be regarded as an event regulated by a stochastic model. This was well documented in a study of the fate of naturally differentiated neutrophils and HL-60 cells induced to granulocytic differentiation by RA. In these cells, programmed cell death occurs through a recently described phenomenon called apoptosis.22In this process, an endogenous suicide mechanism is triggered within the cell, leading to cell destruction in an ordered series of events. Natural death of these cells is therefore clearly different from necrosis, which is the pathological mode of cell death. It is particularly important to emphasize that programmed cell death is due not to cessation of macromolecular synthesis in condemned cells, but rather to activation of a differentiation pathway.23As for the specific genes involved in this pathway, evidence has been presented concerning some well-known oncogenes that are presumably operating through a complex pattern related on one side to cell proliferation and on the other to cell death. c-myc, for instance, is a gene-promoting apoptosis, because its inhibition prevents the endonucleolytic fragmentation of DNA.24Wild-type p53 is also promoting a p o p t o s i ~a, ~property ~ consistent with its being a tumor suppressor gene. On the other side, BCL2 is an antiapoptotic gene whose expression inhibits programmed cell death.26c-fes is also presumably an antiapoptotic gene, because its inactivation during granulocytic differentiation leads to cell death by a p o p t o s i ~It. ~ ~ was also noted that several growth factors, such as IL3, IL-6, and CSF, promote cell survival by suppressing a p o p t o s i ~ .All ~ . ~of~these observations have created entirely
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new perspectives on the potential mechanisms of oncogenesis, leading to malignant transformation by forcing cell division and causing an unbalance between cell production and cell death. In conclusion, many observations suggest that senescence may be understood by studying the genetic programs of cell differentiation and cell death rather than the genetic programs underlying cell proliferation. REFERENCES 1. NOVER,L., M. LUCKNER & B. PARTHIER.(Eds.) 1982. Cell Differentiation. Molecular Basis and Problems. Springer-Verlag. Berlin, Heidelberg, New York. R. BORTELL & A. J. VANWIJNEN. 1992. 2. STEIN,G. S., J. B. LIAN,T. A. OWEN,J. HOLTHUIS, Molecular mechanisms that mediate a functional relationship between proliferation and differentiation. In Molecular and Cellular Approaches to the Control of Proliferation and Differentiation. G. S. Stein & J. B. Lian, eds. Cell Biology, a Series of Monographs. :299-341. Academic Press Inc. New York, NY. G. CECCHERELLI, L. SELLERI,B. CALABRETTA, A. DONELLI, 3. FERRARI,S., E. TAGLIAFICO, 1989. P. TEMPERANI, M. SARTI,S. SACCHI,G. EMILIA,G. TORELLI& U. TORELLI. Myeloperoxidase gene expression in acute and chronic myeloid leukemias: Relationship to the expression of cell cycle related genes. Leukemia 3: 423-430. 4. KILLMANN, S. A. 1968.Acute leukemia: Development, remission/relapse pattern, relationship between normal and leukemic haemopoiesis, and the “sleeper to feeder” stem cell hypothesis. Ser. Haematol. 1: 103-128. 5. SACHS,L. 1987. Hematopoietic growth and differentiation factors and the reversal of malignancy. In Tumor Cell Differentiation, Biology and Pharmacology. J. Aarbakke, P. K. Chiang & H. P. Koeffler, eds. Vol. 1: 3-27. The Humana Press Inc. Clifton, NJ. D. 1989. The molecular control of cell division, differentiation commitment and 6. METCALF, maturation in haematopoietic cells. Nature 3 3 9 27-30. A. GRANDE,G. TORELLI & U. TORELLI. 1992. Proliferation, 7. FERRARI,S., R. MANFREDINI, differentiation arrest and survival in leukemic blast cells. In Aging and Cellular Defense Mechanisms. C. Franceschi, G. Crepaldi, V. J. Cristofalo, L. Masotti & J. Vijg. Ann. N.Y. Acad. Sci., This volume. 1992. Growth and differentiation of myelomonocytic cells. 8. KREIDER,B. L. & G. ROVERA. In Molecular and Cellular Approaches to the Control of Proliferation and Differentiation. G. S. Stein & J. B. Lian, eds. Cell Biology, a Series of Monographs. :223-288. Academic Press Inc. New York, NY. D. M. P. S. CROSIER & S. C. CLARK.1991. Effects of hematopoieticgrowth factors 9. BODINE, on the survival of primitive stem cells in liquid suspension culture. Blood 7 8 914-920. 10. DE LUCA,L. M. 1991. Retinoids and their receptors in differentiation, embryogenesis, and neoplasia. FASEB J. 5: 29262933. T. SUDA& Y. NISHII.1987. Control of proliferatT., Y. HONMA,M. HOZUMI, 11. KASUKABE, ing potential of myeloid leukemia cells during long-term treatment with vitamin D3 analogues and other differentiation inducers in combination with antileukemic drugs: in v i m and in vivo studies. Cancer Res. 47: 567-572. E. ROBERTSON, W. H. KLEIN,S. F. TSAI,V. D’AGATI,S. H. ORKIN 12. PEVNY,L., M. C. SIMON, & F. COSTANTINI. 1991. Erythroid differentiation in chimeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1. Nature 3 4 9 257-260. V. SHALHOUB, K. WRIGHT,U. PAULI& A. VAN 13. STEIN,G., J. LIAN,J. STEIN,R. BRIGGS, WIJNEN.1989. Altered binding of human histone gene transcription factors during the shutdown of proliferation and onset of differentiation in HL-60 cells. Proc. Natl. Acad. Sci. USA 8 6 1865-1869. D. J. MANGELSDORF & R. M. EVANS. 1992. Retinoid X 14. KLIEWER,S. A,, K. UMESONO, receptor interacts with nuclear receptors in retinoic acid, thyroid hormone and vitamin D3 signalling. Nature 355: 446449. & J. S. POBER.(Eds.) 1991. Cellular and Molecular 15. ABBAS,A. K., A. H. LICHTMAN Immunology. W. B. Saunders Co. Philadelphia, PA.
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16. SHiosaKa, T. & Y. TANAKA.1989. Expression of selected genes and oncogenes in differentiated HL-60 cells and primary cells from human leukemias. Anticancer Res. 9: 1249-1264. A. GRANDE& U. TORELLI. 1992. Antisense strategies to 17. FERRARI,S . , R. MANFREDINI, characterize the role of genes and oncogenes involved in myeloid differentiation. I n Antisense Strategies. R. Baserga & D. T. Denhardt, eds. Ann. N.Y. Acad. Sci. In press. 18. TORELLI, G., A. DONELLI,s. FERRARI,L. MORETTI,R. CADOSSI, G . CECCHERELLI, ST. FERRARI& U. TORELLI. 1984. Sequence complexity and diversity of polyadenylated RNA molecules transcribed in human myeloid cells. Differentiation 27: 133-140. 1988. Nuclear posttranscriptional process19. GUDAS,J. M., G. B. KNIGHT& A. B. PARDBE. ing of thymidine kinase mRNA at the onset of DNA synthesis. Proc. Natl. Acad. Sci. USA 85: 4705-4709. R. MANFREDINI, A. GRANDE, E. ROW, P. ZUCCHINI, G. 20. FERRARI, S., E. TAGLIAFICO, TORELLI & U. TORELLI.1992. Abundance of the primary transcript and its processed product of growth-related genes in normal and leukemic cells during proliferation and differentiation. Cancer Res. 52: 11-16. T. A. RAJIO, J. W. SCHNEIDER & R. W. 21. BENZ,E. J., K. A. HIGH,K. LQMAX,C. STOLLE, MERCER.1987. Studies of gene expression during granulocytic differentiation. In Tumor Cell Differentiation, Biology and Pharmacology. J. Aarbakke, P. K. Chiang & H. P. Koeffler, eds. :79-103. Humana Press. Clifton, NJ. & T. G . COTTER.1990. HL-60 cells induced to differentiate 22. MARTIN, S. J., J. G . BRADLEY towards neutrophils subsequently die via apoptosis. Clin. Exp. Immunol. 7 9 448-453. L. M., L. Kosz & B. K. KAY.1990. Gene activation is required for developmen23. SCHWARTZ, tally programmed cell death. Proc. Natl. Acad. Sci. USA 87: 6594-6598. 24. GREEN,D. R., H. ZHENG& Y.SHI. 1992. Antisense oligodeoxynucleotides that alter lymphocyte function. In Antisense Strategies. R. Baserga & D. T. Denhardt, eds. Ann. N.Y. Acad. Sci. In press. E., D. RESNITZKY, J. LOTEM,L. SACHS,A. KIMCHI& M. OREN.1991. 25. YONISH-ROLJACH, Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6. Nature 352: 345-347. D., G . NUNEZ,c. MILLIMAN, R. D. SCHREIBER & s. J. KORSMEYER. 1990. 26. HOCKENBERY, Bcl-2 is an inner mitochondria1 membrane protein that blocks programmed cell death. Nature 348: 334-336. S., A. DONELLI, R. MANFREDINI, M. SARTI,R.RONCAGLIA, E. TAGLIAFICO, E. 27. FERRARI, Rossi, G. TORELLI & U. TORELLI. 1990. Differential effects of c-myb and c-fes antisense oligodeoxynucleotides on granulocytic differentiation of human myeloid leukemia HL60 cells. Cell Growth Diff. 1: 543-548. G. T., C. A. SMITH,E. SPOONCER, T. M. DEXTER& D. R. TAYLOR. 1990. 28. WILLIAMS, Haemopoietic colony stimulating factors promote cell survival by suppressing apoptosis. Nature 343: 77-79. 29. LOTEM,J., E. J. CRAGOE& L. SACHS.1991. Rescue from programmed cell death in leukemic and normal myeloid cells. Blood 7 8 953-960.
Cell differentiation ultimately proceeds as a response to signals from the extracellular microenvironment. If the cells are competent, that is, if they carry the corresponding receptors and signal transduction pathways, they can be triggered in the commitment state. In many cases the response includes complex gene expression programs whose progress and completion are relatively autonomous and whose outcome is determined by inherent regulatory factors of the cell type affected.' As a short introduction to the problems of differentiation and aging, we survey the main points that characterize the differentiation of hematopoietic cells. We can identify several main aspects. PROGRESSIVE GROWTH ARREST IN THE G1 PHASE OF THE CELL CYCLE
Several lines of evidence are reported to support a functionally coupled relation between cell growth and differentiation. This relation is characterized initially by the expression of cell cycle-related genes along with genes coding for cytokines and their receptors. Subsequently, down-regulation of proliferation, partially mediated by extracellular stimuli, parallels the expression of a series of genes associated with the maturation pathways. Important progress is represented by elucidation of the mechanism by which these cell-cycle associated genes are suppressed.* For instance, it is commonly accepted that leukemic blast cells are arrested, but at different stages, along the differentiation pathway and never spontaneously reach terminal differentia t i ~ nWe . ~ do not yet know the correlations between proliferation and differentiation despite an incredible amount of experimental work on the kinetics of these cells4and on the mechanisms of action of growth factors and their receptor^.^^^ In fact, several cytokines are capable of triggering the target cells simultaneously along the proliferation and differentiation pathways. However, in vitro cultured leukemic blast cells can be rescued only to proliferate when incubated with purified growth factors. On the other hand, acute promyelocytic leukemic blast cells carrying the t(15;17) in which the retinoic acid receptor (alpha type) is juxtaposed to the Mil gene are capable of differentiating in vivo to granulocytes when treated with all-trans-retinoic acid. When the differentiation commitment of a normal precursor cell occurs, the G1 aThis work was supported by a grant from the Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.). bAddress for correspondence and reprint requests: Dr. Sergio Ferrari, Experimental Hematology Center, I1 Medical Clinic, University of Modena, Via Del Pozzo 71, Policlinico, 41100 Modena, Italy. 180
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phase of the cell cycle progressively increases in length, and terminally differentiated cells are practically arrested in this phase of the cycle and cannot be triggered along the proliferative pathway. Furthermore, previous studies have shown that high levels of expression of genes involved in the proliferative pathway can inhibit cell differentiation. The effect of these genes on terminal differentiation is due not to significant alterations in normal growth control mechanisms but instead to an inability of the cells to enter a pathway leading to the terminally differentiated state. In fact, the genetic program of terminal differentiation often, if not always, requires the permanent withdrawal of several cell cycle gene-related products. Among these genes we can include c-myc, c-myb, erb-A, c-fos, and c-jun. For example, mouse erythroleukemia cells expressing a recombinant c-myc gene fail to differentiate; other examples are the suppression of erythroid differentiation by overexpression of the c-myb oncogene and suppression of myogenesis by the increased expression of erb-B. Overexpression of c-jun is capable of blocking the differentiation pathway in erythroleukemia Friend induced by DMSO. The preliminary conclusion of these experiments is that several oncogenes are involved primarily in cell proliferation and that cell differentiation requires permanent withdrawal of these gene products and the permanent exit from the cell cycle.’
SIGNAL RECEPTION AND TRANSDUCTION
Both proliferation and differentiation are controlled mainly by cytokines and their receptors, intracellular signal transduction pathways, and eventual gene activation. In the first level of control, cytokines interact with their corresponding receptors on the cell and initiate a cascade of events that eventually directs the cells to proliferate or differentiate.* Hematopoietic growth factors are mainly produced by bone marrow accessory cells. Primitive hematopoietic precursors (CD34 positive bone marrow cells) are induced to proliferate by several different cytokines which act in a synergistic and cooperative way. Among these cytokines are the stem cell factor (SCF), IL-1 alpha, IL-3, and IL-6 which affect the formation of colonies derived from more primitive hematopoietic progenitor cells in vitro, namely, the multicolonyforming cells ( C F C - m ~ l t i )The . ~ aforementioned cytokines are capable of controlling the surface expression of receptors that can bind other growth factors particularly involved in hematopoietic cell differentiation. Even if these cytokines are more important for proliferative pathway activation, they can simultaneously trigger the hematopoietic cells in the differentiation pathway, activating the differentiation genetic program. When the hematopoietic cell is in a commitment state, the action of several other cytokines is particularly important, such as EPO for erythroid differentiation, M-CSF for macrophage differentiation, G-CSF for granulocytic differentiation, IL-5 and G-CSF for eosinophil differentiation, IL-4 for basophil differentiation, and IL-2, IL-4, and IL-7 for lymphoid B or T differentiation, respectively.6 The specific metabolic pathways activated by these different ligand-receptor interactions are poorly understood, particularly as far as the correlation between the proliferation and differentiation pathways is concerned. Interesting biological behavior is described for acute leukemia blast cells which are unable to progress through the cell cycle and are arrested mainly in the G1 phase of the cycle but are simultaneously blocked in the terminal differentiation program and are arrested in different phases of the differentiation pathway. A further interesting observation is that these cells are able mainly to express several cytokine receptors but not the corresponding cytokines and are able to respond in vitro to growth factors that can induce
182
ANNALS NEW YORK ACADEMY OF SCIENCES
proliferation but not to growth factors mainly involved in the differentiation pathway. Two other transduction pathways involved in differentiation are activated by retinoic acid (RA) and diidrocalciferol (vitamin D3). The retinoids comprise a group of compounds including retinoic acid, retinol (vitamin A), and a series of natural and synthetic derivatives that exert profound effects on development and differentiation in a wide variety of systems. The interaction of retinoic acid and its receptor (RAR alpha, beta, and gamma) induced the transcription of several genes. In the myeloid system, the interaction of retinoic acid with its receptor induces granulocytic differentiation.1° A major contribution toward understanding the biologic role of 1,25-(OH)*D3 was provided by elucidation of its mechanism of action. In fact, hormonal effects are mediated by specific nuclear receptors that were recently isolated and structurally determined. These receptors are present in hematopoietic cells, and several studies have shown that HL-60 cells are induced to monocytic differentiation when treated with diidrocalciferol (vitamin D3)." It is important that several specific differentiation transcriptional factors have been isolated and characterized such as the GATAl gene which plays an important role in erythroid differentiationI2 or the several DNA binding proteins involved in myeloid differentiation.I3 It was shown recently that the gene encoding for hRXR alpha interacts directly with and enhances the binding of nuclear receptors conferring responsiveness to vitamin D3 and thyroid hormone T3, suggesting that hRXR has a central role in multiple hormonal signaling pathways included in the differentiation pathways.I4 SELECTIVE MODIFICATION IN THE GENETIC MATERIAL Lymphoid B- and T-cell differentiation is characterized by DNA rearrangements in Ig and TCR gene loci, respectively, that occur in a precise order in the early lymphoid progenitor cells independent of antigen stimulation. In fact, all cells except B lymphocytes express these genes in the germline configuration, and only B lymphocytes express these genes in functionally rearranged forms, capable of giving rise to functional proteins. Stimuli that determine the commitment of a bone marrow stem cell to mature along the B-lymphocyte lineage are largely unknown. Such commitment, however, leads to somatic recombination of Ig genes. T lymphocytes, like B lymphocytes, originate from precursors in bone marrow and then migrate to the thymus. Functional TCR alpha and beta genes as well as gamma and delta chain genes are formed by somatic rearrangement of germline gene segments by a process that is very similar to Ig gene rearrangements. Rearrangement and expression of TCR genes during intrathymic maturation are the necessary first step in the development of T-cell repertoire, and all together with the expression of several other specific proteins characterize the T-cell differentiation genetic program.I5 DIFFERENTIAL GENE EXPRESSION Several genes have been isolated that are associated with cell differentiation.16 The definition of cell differentiation-related genes is simply based on the concept that mRNA and/or protein abundance of these genes increases in this functional state of the cell. It is clearly very important to distinguish between gene products that are simply associated with a particular functional state and gene products restricted and perhaps regulating that function, in other words, genes whose expression is
FERRARI er al.: TERMINAL DIFFERENTIATION
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essential for the differentiation genetic programs. In fact, these latter genes can play a regulatory role because of their interference with several signal transduction pathways underlying differentiation. That a gene product is restricted to a cell function was demonstrated using very different technical approaches based on different methods with the specific goal of selectively inactivating that gene product. Among these different methods are microinjection of cells with antibodies or synthetic antisense R N A transcripts, treatment of cell populations with specific antisense oligomers, transfection of plasmids or constructs expressing high levels of specific antisense R N A molecules under the control of strong and often inducible promoters, transgenic mice, and more recently homologous recombination. As mentioned, one of the opportunities offered by these methods is to obtain specific and possibly complete inhibition of a gene product at the level of either the genetic locus or its transcribed product (precursor mRNA, mature mRNA, and protein) and to look at the biological consequences of the spccific gene’s inactivation. As pointed out before, these experimental approaches allow the discrimination of gene products that limit and perhaps regulate cellular differentiation. In fact, inactivation of some cell differentiation-related genes inhibits in different ways the genetic program of a specific pathway, such as granulocytic differentiation by c-fes, macrophage differentiation by c-fms, erythroid differentiation by MERS, and muscular differentiation by myosin. Instead, inhibition of some other genes whose expression increases during differentiation does not interfere with the specific differentiation pathway, but may deprive the differentiated cells of one of its specific functions. This is the case, for instance, of myeloperoxidase and lactoferrin genes in granulocytic ~ ~ 1 1 s . ’ ~
REORGANIZATION OF GENE EXPRESSION PROGRAMS The cDNA-poly(A)+RNA reassociation technique has been used extensively to study the R N A sequence complexity and kinetic abundance in different stages. We studied the kinetic composition of polyadenylated RNA molecules of HL-60 (myeloid, M2 phenotype cells) using the aforementioned technique before (proliferating cells) and after induction of differentiation with retinoic acid (terminal differentiation to granulocytes). The data obtained with this investigation showed: (1) a 70% decrease in the sequence complexity (number of different transcribed sequences) in HL-60 cells treated with R A compared with proliferating cells; (2) a drastic drop in the number of abundant sequences in RA-treated HL-60 cells; and (3) a marked increase in the repetition frequency of the abundant and rare components after induction of differentiation of HL-60 with RA. Finally, the most abundant sequences in HL-60 cells after RA induction are already present before treatment. O u r data suggest that at least during myeloid differentiation, important transcriptional and posttranscriptional regulatory mechanisms of gene expression are active soon after the first commitment event of the hematopoietic undifferentiated precursor cells, leading to a reduction in the number of expressed genes together with an increased abundance of hundreds of so-called “cell differentiation associated genes.” These results are in keeping with the biological function of terminally differentiated cells whose main metabolic activity differs according to the specific cell function, such as phagocytosis for granulocytes and macrophages, and globin storage for erythrocytes.18
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ANNALS NEW YORK ACADEMY OF SCIENCES IMPORTANCE OF THE POS'ITRANSCRIPTIONAL REGULATION OF GENE EXPRESSION
An important step in the cascade of molecular events leading to the accumulation of mRNA is the efficient processing of primary transcripts. Evidence of mechanisms operating at this level has so far been obtained only for regulation of the expression of cell cycle related genes. In fact, the concentration of thymidine kinase (TK) during the G1 progression phase is strictly related to stabilization of the primary transcript and to subsequent activation of the processing mechanisms during the S phase.19 As for terminal differentiation, it is well known that this process is accompanied by a short reduction in the production of ribosomal RNA. This production is the consequence of both a reduced rate of transcription and a reduced rate of processing the primary transcript. We have evidence that during terminal differentiation of HL-60 cells the 32 S ribosomal precursor RNA accumulates, suggesting that this molecule undergoes remarkable stabilization and it is not processed further. Similar behavior seems to underly the disappearance of c-myc mRNA during granulocytic differentiation of HL-60 cells. This differentiation is conditioned by arrest of the expression of c-myc, and this arrest is obtained through a reduction in the transcription rate as well as a complete arrest of the processing of the primary transcript, so that the abundance of the precursor remains elevated?O Stabilization of a translational product is also a well-documented mechanism through which a terminally differentiated cell increases the concentration of a specific protein. For instance, during granulocytic differentiation, myeloperoxidase as well as lactoferrin remarkably increase from the promyelocyte, myelocyte stages, whereas their mature mRNA are strongly decreased so that they are completely absent in the final differentiation stages.21
TERMINAL DIFFERENTIATION AND PROGRAMMED CELL DEATH
The average life span of terminally differentiated cells can no longer be regarded as an event regulated by a stochastic model. This was well documented in a study of the fate of naturally differentiated neutrophils and HL-60 cells induced to granulocytic differentiation by RA. In these cells, programmed cell death occurs through a recently described phenomenon called apoptosis.22In this process, an endogenous suicide mechanism is triggered within the cell, leading to cell destruction in an ordered series of events. Natural death of these cells is therefore clearly different from necrosis, which is the pathological mode of cell death. It is particularly important to emphasize that programmed cell death is due not to cessation of macromolecular synthesis in condemned cells, but rather to activation of a differentiation pathway.23As for the specific genes involved in this pathway, evidence has been presented concerning some well-known oncogenes that are presumably operating through a complex pattern related on one side to cell proliferation and on the other to cell death. c-myc, for instance, is a gene-promoting apoptosis, because its inhibition prevents the endonucleolytic fragmentation of DNA.24Wild-type p53 is also promoting a p o p t o s i ~a, ~property ~ consistent with its being a tumor suppressor gene. On the other side, BCL2 is an antiapoptotic gene whose expression inhibits programmed cell death.26c-fes is also presumably an antiapoptotic gene, because its inactivation during granulocytic differentiation leads to cell death by a p o p t o s i ~It. ~ ~ was also noted that several growth factors, such as IL3, IL-6, and CSF, promote cell survival by suppressing a p o p t o s i ~ .All ~ . ~of~these observations have created entirely
FERRARI ef al.: TERMINAL DIFFERENTIATION
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new perspectives on the potential mechanisms of oncogenesis, leading to malignant transformation by forcing cell division and causing an unbalance between cell production and cell death. In conclusion, many observations suggest that senescence may be understood by studying the genetic programs of cell differentiation and cell death rather than the genetic programs underlying cell proliferation. REFERENCES 1. NOVER,L., M. LUCKNER & B. PARTHIER.(Eds.) 1982. Cell Differentiation. Molecular Basis and Problems. Springer-Verlag. Berlin, Heidelberg, New York. R. BORTELL & A. J. VANWIJNEN. 1992. 2. STEIN,G. S., J. B. LIAN,T. A. OWEN,J. HOLTHUIS, Molecular mechanisms that mediate a functional relationship between proliferation and differentiation. In Molecular and Cellular Approaches to the Control of Proliferation and Differentiation. G. S. Stein & J. B. Lian, eds. Cell Biology, a Series of Monographs. :299-341. Academic Press Inc. New York, NY. G. CECCHERELLI, L. SELLERI,B. CALABRETTA, A. DONELLI, 3. FERRARI,S., E. TAGLIAFICO, 1989. P. TEMPERANI, M. SARTI,S. SACCHI,G. EMILIA,G. TORELLI& U. TORELLI. Myeloperoxidase gene expression in acute and chronic myeloid leukemias: Relationship to the expression of cell cycle related genes. Leukemia 3: 423-430. 4. KILLMANN, S. A. 1968.Acute leukemia: Development, remission/relapse pattern, relationship between normal and leukemic haemopoiesis, and the “sleeper to feeder” stem cell hypothesis. Ser. Haematol. 1: 103-128. 5. SACHS,L. 1987. Hematopoietic growth and differentiation factors and the reversal of malignancy. In Tumor Cell Differentiation, Biology and Pharmacology. J. Aarbakke, P. K. Chiang & H. P. Koeffler, eds. Vol. 1: 3-27. The Humana Press Inc. Clifton, NJ. D. 1989. The molecular control of cell division, differentiation commitment and 6. METCALF, maturation in haematopoietic cells. Nature 3 3 9 27-30. A. GRANDE,G. TORELLI & U. TORELLI. 1992. Proliferation, 7. FERRARI,S., R. MANFREDINI, differentiation arrest and survival in leukemic blast cells. In Aging and Cellular Defense Mechanisms. C. Franceschi, G. Crepaldi, V. J. Cristofalo, L. Masotti & J. Vijg. Ann. N.Y. Acad. Sci., This volume. 1992. Growth and differentiation of myelomonocytic cells. 8. KREIDER,B. L. & G. ROVERA. In Molecular and Cellular Approaches to the Control of Proliferation and Differentiation. G. S. Stein & J. B. Lian, eds. Cell Biology, a Series of Monographs. :223-288. Academic Press Inc. New York, NY. D. M. P. S. CROSIER & S. C. CLARK.1991. Effects of hematopoieticgrowth factors 9. BODINE, on the survival of primitive stem cells in liquid suspension culture. Blood 7 8 914-920. 10. DE LUCA,L. M. 1991. Retinoids and their receptors in differentiation, embryogenesis, and neoplasia. FASEB J. 5: 29262933. T. SUDA& Y. NISHII.1987. Control of proliferatT., Y. HONMA,M. HOZUMI, 11. KASUKABE, ing potential of myeloid leukemia cells during long-term treatment with vitamin D3 analogues and other differentiation inducers in combination with antileukemic drugs: in v i m and in vivo studies. Cancer Res. 47: 567-572. E. ROBERTSON, W. H. KLEIN,S. F. TSAI,V. D’AGATI,S. H. ORKIN 12. PEVNY,L., M. C. SIMON, & F. COSTANTINI. 1991. Erythroid differentiation in chimeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1. Nature 3 4 9 257-260. V. SHALHOUB, K. WRIGHT,U. PAULI& A. VAN 13. STEIN,G., J. LIAN,J. STEIN,R. BRIGGS, WIJNEN.1989. Altered binding of human histone gene transcription factors during the shutdown of proliferation and onset of differentiation in HL-60 cells. Proc. Natl. Acad. Sci. USA 8 6 1865-1869. D. J. MANGELSDORF & R. M. EVANS. 1992. Retinoid X 14. KLIEWER,S. A,, K. UMESONO, receptor interacts with nuclear receptors in retinoic acid, thyroid hormone and vitamin D3 signalling. Nature 355: 446449. & J. S. POBER.(Eds.) 1991. Cellular and Molecular 15. ABBAS,A. K., A. H. LICHTMAN Immunology. W. B. Saunders Co. Philadelphia, PA.
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