Leukemia Research Vol. 14, No. 11/12, pp. 997-1006, 1990. Printed in Great Britain.
EARLY
0145-2126/90 $3.00 + .00 Pergamon Press pie
PROTOONCOGENE
HEMIN-INDUCED
EXPRESSION
DIFFERENTIATION
ERYTHROLEUKEMIC
DURING
OF HUMAN
CELLS
MARGARET O. GRIFFIN, JOHN C. NELSON and NADER G. ABRAHAM Department of Medicine, New York Medical College, Valhalla, NY 10595, U.S.A.
(Received 14 October 1989. Revision accepted 10 March 1990) Abstract--In situ hybridization was used to study the effects of hemin on the expression of the oncogenes c-myc, c-los, and myb, and on the mRNA level of erythroid porphobilinogen deaminase (PBG-D) and te-globin in HEL cells during differentiation. The technique was effective in detecting changes in mRNA levels in small numbers of HEL cells. Hemin stimulation of HEL cells results in an early increase in myb and c-myc expression and a decrease in c-los mRNA, while increased PBGD and o:-globin expression is not seen until 8 h after hemin treatment. Blast-like cells display expression of c-myc, tr-globin and PBG-D, while the more differentiated cells give a positive response to both c-los and myb. During HEL cell differentiation, the mechanism of hemin stimulation appears to be through the up regulation of myb and c-myc mRNA and down regulation of c-los. The subsequent expression of PBG-D and a~-globinmay indicate that early increases in protooncogene expression are first required for the normal progression of erythropoiesis to occur.
Key words: Heme, protooncogene, differentiation, HEL cell, in situ hybridization, erythroid PBG-D.
INTRODUCTION
myb and c-los products are seen in differentiated MEL cells [14]; Marks et al. suggest that these onco-
HEMIN has been shown to have a regulatory effect on several events in erythropoiesis [1-6]. Hemin treatment of erythroid precursor cells in vitro brings about an increase in globin m R N A levels [7, 8]. Hemin has pleitotrophic effects on the expression of heme biosynthetic enzymes such as ALAS, A L A D , and PBG-D [7, 10] in erythroleukemic cells and in human bone marrow [2]. In addition, hemin potentiates the action of several growth factors such as Epo in increasing BFU-E and CFU-E growth in normal cells [11, 12] and in sickle-cell anemia [13]. An alteration in expression of the globin genes and of genes regulating gene replication and cell proliferation has previously been shown to accompany terminal differentiation of M E L cells [14]. Modification in protooncogene expression has also been implicated in regulation of cellular growth and differentiation, notably in leukemic cell lines [15]. Protooncogenes are normal cellular genes that are related to the transforming genes carried by retroviruses [16,17]. The protooncogenes encoding nuclear proteins (c-myc, myb, c-los) apparently act as transducers of growth factor signals [18]. C-myc,
genes may have a possible regulatory role in differentiation [19], but they are also thought to activate genes whose products are needed for cell proliferation [18]. C-fos expression has been shown to increase during macrophage and monocyte differentiation [20, 21] in the human monocytic leukemia cell line THP-1, while myb levels fall. Because the expression of myb and c-los has been shown to be the inverse of the other, they are useful models for examining the effect of various growth factors on protooncogene expression in both normal and leukemic cells. In addition to altering protooncogene expression, inducers of differentiation have been shown to act upon the expression of the globin genes and on the genes for the enzymes in the heme synthetic pathway [22-25]. When K562 cells were induced to differentiate by hemin or other agents, they showed increased synthesis of globin m R N A [24]. Accumulation of globin m R N A has also been observed in MEL cells in concert with induction of oncogene expression [14], indicating that there is a relationship between hemin stimulation of oncogenes and globin gene expression. Furthermore, hemin-increased expression of heme metabolic enzymes in both erythroleukemic cell lines and in human bone marrow progenitor cells is preceded by a latent period,
Correspondence to: Dr N. G. Abraham, Department of Medicine, New York Medical College, Valhalla, NY 10595, U.S.A. 997
M.O. GRIF~r~et al.
998
during which hemin may be required for the expression of oncogene products. In this report we investigated the expression of the oncogenes c-myc, myb and c-fos, as well as the expression of erythroid P B G - D and cr-globin, in H E L cells that were induced to differentiate with hemin, in order to determine whether there was a correlation of protooncogene expression with the onset of differentiation and with the expression of cr-globin a n d / o r P B G - D .
METHODS
Cell cultures HEL cells were cultured in RPMI 1640 medium with 10% FBS supplemented with HEPES as described previously [25]. Suspension cultures seeded at 105 cells/ml density in plastic culture flasks were incubated in a humidified atmosphere at 5% CO2/95% air. After hemin, 106M, was added (2 txl/ml) to the experimental flasks, samples were taken at specific time intervals. Aliquots of the cell cultures without the addition of hemin were used as a control. Preparation of slides Cytospin preparations were made of both control cells and hemin-treated cells using approximately 1 x 1041 × 105 cells/slide. The slides were briefly fixed in freshly made modified Carnoy's solution (ethanol/acetic acid 3 : 1), passed through an alcohol series and stored for a minimum of three days at room temperature to insure adherence of the cells to the slide. In situ hybridization The procedure used was a modification of that of Hafen et al. [26] and Wilcox et al. [27]. 35S-labelled antisense c-myc DNA was obtained from Dupont/NEN (Boston, MA); the labelled sense probe (Dupont/NEN) was used as a control. C-los and myb were obtained from Oncor (Gaithersburg, MD). An erythroid PBG-D probe (a human 1200 bp cDNA fragment) was prepared as described [28]. The o~-globin, a 10 kd cDNA fragment from mouse, was a generous gift from Dr Ethan Dmitrovski of SloanKettering Institute. A second control used was PBR 322 DNA obtained from Boehringer-Mannheim. The c-myc antisense and sense probes were obtained already labeled with [35S]dATP. The other DNA was labelled with [35S]dATP using a random primer labeling kit from Boehringer-Mannheim, to a specific activity of 1 × 105 cpm/ml; 0.5-1.0 ~tl of probe was used on each slide. Slides were rinsed for 10 min in 2 × SSC and then hybridized overnight at 37°C. They were then rinsed in increasingly stringent salt solutions, passed through a second alcohol series, covered with Kodak NTB-2 liquid emulsion and exposed for 7-10 days at 4°C. After development with Dektol developer, the slides were stained with freshly prepared Geimsa stain and examined under a Nikon light microscope. Cells were examined for morphological changes to assess the onset of differentiation [29]. The slides were evaluated using a modification of the methods of McDougall et al. [30] and Godard [31]. Background values (usually
EARLY
0145-2126/90 $3.00 + .00 Pergamon Press pie
PROTOONCOGENE
HEMIN-INDUCED
EXPRESSION
DIFFERENTIATION
ERYTHROLEUKEMIC
DURING
OF HUMAN
CELLS
MARGARET O. GRIFFIN, JOHN C. NELSON and NADER G. ABRAHAM Department of Medicine, New York Medical College, Valhalla, NY 10595, U.S.A.
(Received 14 October 1989. Revision accepted 10 March 1990) Abstract--In situ hybridization was used to study the effects of hemin on the expression of the oncogenes c-myc, c-los, and myb, and on the mRNA level of erythroid porphobilinogen deaminase (PBG-D) and te-globin in HEL cells during differentiation. The technique was effective in detecting changes in mRNA levels in small numbers of HEL cells. Hemin stimulation of HEL cells results in an early increase in myb and c-myc expression and a decrease in c-los mRNA, while increased PBGD and o:-globin expression is not seen until 8 h after hemin treatment. Blast-like cells display expression of c-myc, tr-globin and PBG-D, while the more differentiated cells give a positive response to both c-los and myb. During HEL cell differentiation, the mechanism of hemin stimulation appears to be through the up regulation of myb and c-myc mRNA and down regulation of c-los. The subsequent expression of PBG-D and a~-globinmay indicate that early increases in protooncogene expression are first required for the normal progression of erythropoiesis to occur.
Key words: Heme, protooncogene, differentiation, HEL cell, in situ hybridization, erythroid PBG-D.
INTRODUCTION
myb and c-los products are seen in differentiated MEL cells [14]; Marks et al. suggest that these onco-
HEMIN has been shown to have a regulatory effect on several events in erythropoiesis [1-6]. Hemin treatment of erythroid precursor cells in vitro brings about an increase in globin m R N A levels [7, 8]. Hemin has pleitotrophic effects on the expression of heme biosynthetic enzymes such as ALAS, A L A D , and PBG-D [7, 10] in erythroleukemic cells and in human bone marrow [2]. In addition, hemin potentiates the action of several growth factors such as Epo in increasing BFU-E and CFU-E growth in normal cells [11, 12] and in sickle-cell anemia [13]. An alteration in expression of the globin genes and of genes regulating gene replication and cell proliferation has previously been shown to accompany terminal differentiation of M E L cells [14]. Modification in protooncogene expression has also been implicated in regulation of cellular growth and differentiation, notably in leukemic cell lines [15]. Protooncogenes are normal cellular genes that are related to the transforming genes carried by retroviruses [16,17]. The protooncogenes encoding nuclear proteins (c-myc, myb, c-los) apparently act as transducers of growth factor signals [18]. C-myc,
genes may have a possible regulatory role in differentiation [19], but they are also thought to activate genes whose products are needed for cell proliferation [18]. C-fos expression has been shown to increase during macrophage and monocyte differentiation [20, 21] in the human monocytic leukemia cell line THP-1, while myb levels fall. Because the expression of myb and c-los has been shown to be the inverse of the other, they are useful models for examining the effect of various growth factors on protooncogene expression in both normal and leukemic cells. In addition to altering protooncogene expression, inducers of differentiation have been shown to act upon the expression of the globin genes and on the genes for the enzymes in the heme synthetic pathway [22-25]. When K562 cells were induced to differentiate by hemin or other agents, they showed increased synthesis of globin m R N A [24]. Accumulation of globin m R N A has also been observed in MEL cells in concert with induction of oncogene expression [14], indicating that there is a relationship between hemin stimulation of oncogenes and globin gene expression. Furthermore, hemin-increased expression of heme metabolic enzymes in both erythroleukemic cell lines and in human bone marrow progenitor cells is preceded by a latent period,
Correspondence to: Dr N. G. Abraham, Department of Medicine, New York Medical College, Valhalla, NY 10595, U.S.A. 997
M.O. GRIF~r~et al.
998
during which hemin may be required for the expression of oncogene products. In this report we investigated the expression of the oncogenes c-myc, myb and c-fos, as well as the expression of erythroid P B G - D and cr-globin, in H E L cells that were induced to differentiate with hemin, in order to determine whether there was a correlation of protooncogene expression with the onset of differentiation and with the expression of cr-globin a n d / o r P B G - D .
METHODS
Cell cultures HEL cells were cultured in RPMI 1640 medium with 10% FBS supplemented with HEPES as described previously [25]. Suspension cultures seeded at 105 cells/ml density in plastic culture flasks were incubated in a humidified atmosphere at 5% CO2/95% air. After hemin, 106M, was added (2 txl/ml) to the experimental flasks, samples were taken at specific time intervals. Aliquots of the cell cultures without the addition of hemin were used as a control. Preparation of slides Cytospin preparations were made of both control cells and hemin-treated cells using approximately 1 x 1041 × 105 cells/slide. The slides were briefly fixed in freshly made modified Carnoy's solution (ethanol/acetic acid 3 : 1), passed through an alcohol series and stored for a minimum of three days at room temperature to insure adherence of the cells to the slide. In situ hybridization The procedure used was a modification of that of Hafen et al. [26] and Wilcox et al. [27]. 35S-labelled antisense c-myc DNA was obtained from Dupont/NEN (Boston, MA); the labelled sense probe (Dupont/NEN) was used as a control. C-los and myb were obtained from Oncor (Gaithersburg, MD). An erythroid PBG-D probe (a human 1200 bp cDNA fragment) was prepared as described [28]. The o~-globin, a 10 kd cDNA fragment from mouse, was a generous gift from Dr Ethan Dmitrovski of SloanKettering Institute. A second control used was PBR 322 DNA obtained from Boehringer-Mannheim. The c-myc antisense and sense probes were obtained already labeled with [35S]dATP. The other DNA was labelled with [35S]dATP using a random primer labeling kit from Boehringer-Mannheim, to a specific activity of 1 × 105 cpm/ml; 0.5-1.0 ~tl of probe was used on each slide. Slides were rinsed for 10 min in 2 × SSC and then hybridized overnight at 37°C. They were then rinsed in increasingly stringent salt solutions, passed through a second alcohol series, covered with Kodak NTB-2 liquid emulsion and exposed for 7-10 days at 4°C. After development with Dektol developer, the slides were stained with freshly prepared Geimsa stain and examined under a Nikon light microscope. Cells were examined for morphological changes to assess the onset of differentiation [29]. The slides were evaluated using a modification of the methods of McDougall et al. [30] and Godard [31]. Background values (usually
