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26. Precommitment gene expression changes during mouse erythroleukemia cell differentiation and their relationship to c-myc
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26. Precommitment gene expression changes during mouse erythroleukemia tiation and their relationship to c-myc A SkouItchi*,
G Cheng,
D Krimer,
cell differen-
G Rao, C Austin
Department ofCell Biology, Albert Einstein College of Medicine. Bronx, NY 1046i. USA Previous studies have shown the importance of changes in the expression of the protooncogenes c-myc and c-myb during chemically-induced terminal differentiation of mouse erythroleukemia (MEL) cells. Both genes exhibit a rapid decline in expression, followed by re-expression during the precommitment period occurring in the first 24 h after exposure to inducers. High level, constitutive expression of either protooncogene blocks commitment and terminal differentiation. We have used several strategies to isolate other genes that change their expression during the precommitment phase. By direct screening methods we have isolated two Hl histone genes (HP and Hlc) that are rapidly induced in this period. Both HI genes encode cell cycle independent, polyA+ mRNAs. Using differential hybridization methods we have isolated several other cDNAs that are either induced or down-regulated in this period. Examples of known genes obtained by the latter screening method include the genes for histone H3.3, heat shock protein 86, heat shock cognate protein 73, omithine decarboxylase and a mitochondrial chaperonin protein. By examining the changes in expression of the corresponding mRNAs in transfected cell lines that overproduce c-myc or c-myb we have shown that the induction of both Hl histone mRNAs is negatively regulated, specifically by c-myc. The results indicate that the two Hi histcne genes are targets for regulation by c-myc and that they may have an important role in the events leading to the commitment to terminal differentiation. Current efforts are directed at identifying the regulatory sequences within these genes and the mechanism of the negative regulation by c-myc. We are also carrying out transfection experiments to both force expression and inactivate by homologous recombination the Hl” gene in order to determine its role in the control of cell proliferation and terminal differentiation. 1 Lachman HM, Cheng G, Skoultchi A (1986) Proc Nat1 Acad Sci USA 83, 6480 2 Cheng G, Skoultchi A (1989) Mel Cull Biol 9, 2332
27. Cell death and differentiation in human myeloid leukemia cells Y Yoshida’, N Anzai, T Hirama. H Kawatxa, H Masutani First Division: Department of Medicine, Kyoto University, Sakyo, Kyoto, Japan Apoptosis (programmed cell death) is essential in many physio!ogLal processes, such as embryonic development. adult tissue maintenance and clonal selection in the immune system. A novel human myeloid leukemia cell line, P 39, underwent profound apoptosis in vitro in the presence of rctinoic acid and vitamin Ds. Apoptosis in P 39 cells were associated with distinctive morphological changes that include chromatin condensation and degradation of DNA to oligonucleosomal fragments. In addition to differentiation agents, metabolic inhibitors such as actinomycin D and cycloheximide, and calcium ionophore A 23187 also induced large scale apoptosis in P 39 cells. Actinomycin D and A 23187 provoked spoptosis in P 39 cells to a greater extent than in other human myeloid leukemia cell lines examined (HL 60, U 937, K 562. KG-l). As assessed by the proportion of apoptotic cells, the effects of these compounds were dose- and time-dependent. When P 39 cells were preincubated with 22-oxa-la. 25 dihydroxyvitamin Ds (oxa-Ds), treatment with Actinomycin D (0.1 mg/ml) or A 23187 (1mM) resulted in a 3-5 fold increase in % apoptotic cells over controls without preincubation. Oxa-Ds is a synthetic vttamin Ds analogue with potent differentiation-inducing capacity and less effect on calcium metabolism. Thus, the preincubation exwriments suggest that apoptosis is a differentiation-related event. P 39 cell line was derived from a patient with the myelodysplastic syndrome (MDS) in leukemic phase [I]. P 39 Cells have myelomonocytoid blast cell morphology, complex karyotypic abnormalities and a wide spectrum of phenotypic expression [2]. The dysplastic hematopoiesis in MDS is characterized by abortive differentiation and ineffective hematopoiesis leading to hypercellular bone marrow and peripheral cytopenia. Apoptosis has been postulated to be responsible for the intramedullary cell death and hence in ineffective hematopoiesis. We propose that this cell line provides a useful model to study apoptosis in myeloid cell differentiation and the biologic abnormalities of dysplastic hemacopoiesis in MDS. 1 Nagai M (1984) Gann 75, 1100 2 Yoshida Y (1991) Exp Hemotol 19, 537
26. Precommitment gene expression changes during mouse erythroleukemia tiation and their relationship to c-myc A SkouItchi*,
G Cheng,
D Krimer,
cell differen-
G Rao, C Austin
Department ofCell Biology, Albert Einstein College of Medicine. Bronx, NY 1046i. USA Previous studies have shown the importance of changes in the expression of the protooncogenes c-myc and c-myb during chemically-induced terminal differentiation of mouse erythroleukemia (MEL) cells. Both genes exhibit a rapid decline in expression, followed by re-expression during the precommitment period occurring in the first 24 h after exposure to inducers. High level, constitutive expression of either protooncogene blocks commitment and terminal differentiation. We have used several strategies to isolate other genes that change their expression during the precommitment phase. By direct screening methods we have isolated two Hl histone genes (HP and Hlc) that are rapidly induced in this period. Both HI genes encode cell cycle independent, polyA+ mRNAs. Using differential hybridization methods we have isolated several other cDNAs that are either induced or down-regulated in this period. Examples of known genes obtained by the latter screening method include the genes for histone H3.3, heat shock protein 86, heat shock cognate protein 73, omithine decarboxylase and a mitochondrial chaperonin protein. By examining the changes in expression of the corresponding mRNAs in transfected cell lines that overproduce c-myc or c-myb we have shown that the induction of both Hl histone mRNAs is negatively regulated, specifically by c-myc. The results indicate that the two Hi histcne genes are targets for regulation by c-myc and that they may have an important role in the events leading to the commitment to terminal differentiation. Current efforts are directed at identifying the regulatory sequences within these genes and the mechanism of the negative regulation by c-myc. We are also carrying out transfection experiments to both force expression and inactivate by homologous recombination the Hl” gene in order to determine its role in the control of cell proliferation and terminal differentiation. 1 Lachman HM, Cheng G, Skoultchi A (1986) Proc Nat1 Acad Sci USA 83, 6480 2 Cheng G, Skoultchi A (1989) Mel Cull Biol 9, 2332
27. Cell death and differentiation in human myeloid leukemia cells Y Yoshida’, N Anzai, T Hirama. H Kawatxa, H Masutani First Division: Department of Medicine, Kyoto University, Sakyo, Kyoto, Japan Apoptosis (programmed cell death) is essential in many physio!ogLal processes, such as embryonic development. adult tissue maintenance and clonal selection in the immune system. A novel human myeloid leukemia cell line, P 39, underwent profound apoptosis in vitro in the presence of rctinoic acid and vitamin Ds. Apoptosis in P 39 cells were associated with distinctive morphological changes that include chromatin condensation and degradation of DNA to oligonucleosomal fragments. In addition to differentiation agents, metabolic inhibitors such as actinomycin D and cycloheximide, and calcium ionophore A 23187 also induced large scale apoptosis in P 39 cells. Actinomycin D and A 23187 provoked spoptosis in P 39 cells to a greater extent than in other human myeloid leukemia cell lines examined (HL 60, U 937, K 562. KG-l). As assessed by the proportion of apoptotic cells, the effects of these compounds were dose- and time-dependent. When P 39 cells were preincubated with 22-oxa-la. 25 dihydroxyvitamin Ds (oxa-Ds), treatment with Actinomycin D (0.1 mg/ml) or A 23187 (1mM) resulted in a 3-5 fold increase in % apoptotic cells over controls without preincubation. Oxa-Ds is a synthetic vttamin Ds analogue with potent differentiation-inducing capacity and less effect on calcium metabolism. Thus, the preincubation exwriments suggest that apoptosis is a differentiation-related event. P 39 cell line was derived from a patient with the myelodysplastic syndrome (MDS) in leukemic phase [I]. P 39 Cells have myelomonocytoid blast cell morphology, complex karyotypic abnormalities and a wide spectrum of phenotypic expression [2]. The dysplastic hematopoiesis in MDS is characterized by abortive differentiation and ineffective hematopoiesis leading to hypercellular bone marrow and peripheral cytopenia. Apoptosis has been postulated to be responsible for the intramedullary cell death and hence in ineffective hematopoiesis. We propose that this cell line provides a useful model to study apoptosis in myeloid cell differentiation and the biologic abnormalities of dysplastic hemacopoiesis in MDS. 1 Nagai M (1984) Gann 75, 1100 2 Yoshida Y (1991) Exp Hemotol 19, 537