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Correlation of differentiation state and silver staining of nucleolar organizers in the promyelocytic leukemia cell line HL-60
Correlation of Differentiation State and Silver Staining of Nucleolar Organizers in the Promyelocytic Leukemia Cell Line HL-60 B. R. Reeves, G. Casey, Juliet R. Honeycombe, and Sandra Smith
ABSTRACT: We have applied nucleolar organizer region (NOR) silver staining to the promyelocytic leukemia cell line HL-60, before and after dimethylsulfoxide (DMSO) mediated differentiation. The results demonstrated a gradual suppression of rDNA transcription during terminal maturation of these bone-marrow-derived cells and support our hypothesis [1] that there are characteristic NOR staining profiles for different bone marrow cell types. INTRODUCTION In a recent report, we described variations in silver staining of nucleolar organizer regions (AgNORs) in bone marrow cells from hematologically normal subjects and patients with leukemia [1]. In particular, we drew attention to differences in AgNOR staining profiles that a p p e a r e d to be characteristic for different cytologic types of leukemia. We suggested two explanations for these differences: either that rRNA gene activity was normal in the leukemic cells, with regulation of rDNA transcription d e p e n d i n g on cell lineage and maturation state, or that there were pathologic changes in rRNA gene expression. In order to resolve this question we have carried out studies on the cell line HL60. Under normal culture conditions, this line, w h i c h was derived from a patient with p r o m y e l o c y t i c leukemia, retains a l i m i t e d capacity for spontaneous granulocytic differentiation [2]. In addition, it has the capability for more extensive morphological and functional m y e l o i d differentiation u n d e r the influence of various agents, i n c l u d i n g d i m e t h y l s u l f o x i d e (DMSO) [3]. The results we have obtained following silver staining of metaphases from cultures i n d u c e d to differentiate with DMSO, suggest that, at least in bone-marrowderived cells, AgNOR staining is an indicator of differentiation/maturation state.
From the Section of Human Genetics, Institute of Cancer Research, Royal Marsden Hospital, London, England. Address requests for reprints to Dr. B. B. Reeves, Section of Human Genetics, Institute of Cancer Research, Royal Marsden Hospital, Fulham Road, London SW3 61J, England. Received August 8, 1983; accepted October 12, 1983.
159 © 1984 by Elsevier Science Publishing Co.. Inc. 52 Vanderbilt Ave., New York, NY 10017
Cancer Genetics and Cytogenetics13, 159-166 (1984) 0165-4808/84/$03.00
160
B.R. Reeves et al.
MATERIALS AND METHODS Cell Lines In a d d i t i o n to HL-60, three control (i.e., n o n i n d u c i b l e ) l y m p h o b l a s t o i d lines were studied: Molt 4 (T cell), Maja, and Daudi (B cell). Cell lines were m a i n t a i n e d in RPMI 1640 m e d i u m , s u p p l e m e n t e d with 10% fetal calf serum (Flow Laboratories). Experimental Procedures Duplicate 100-ml cultures were set up containing 5 x 105 cells/mE 24 hr later (day 0), DMSO was a d d e d to one culture of each pair at a final concentration of 1.25%
(v/v). T w e n t y - m i l l i l i t e r aliquots were subsequently taken from the cultures at 24-hr intervals: 2 ml being used for cytologic smears and the r e m a i n d e r for c h r o m o s o m e studies. Chromosome preparations were m a d e after further incubation at 37°C for 1.5 hr with 0.5 ixg/ml'colchicine and h y p o t o n i c treatment with 0.07 M KC1 for 10 rain at room temperature. Cells were then fixed in 3:1 m e t h a n o l : g l a c i a l acetic acid. Standard air-dried slides were aged for 24 hr and stained with 50% (w/v) AgNO3 by the m e t h o d of Bloom and Goodpasture [4]. For cell kinetic studies of HL-60, parallel cultures containing 5 ' - b r o m o d e o x y u r i dine (BrdU) at a final concentration of 10 ixM were set up with and w i t h o u t DMSO and harvested as above. Chromosomes were harlequin stained by the m e t h o d of Perry and Wolf [5]. Figure 1 Silver-stained nuclei from control HL-60 culture. Note the large intensely Ag-positive nucleoli.
161
AgNOR Staining and Differentiation in HL-60
Table 1
Differentiation of HL-60 cells following DMSO treatment Percent of cell types present
Control (uninduced) ÷DMSO day 1 ÷ DMSO day 2 ÷DMSO day 3 ÷ DMSO day 4
Myeloblasts and promyelocytes
Myelocytes
Metamyelocytes
Bands and polymorphs
98
1
1
0
97 95 91 79
1 2 3 8
2 3 6 10
0 9 0 3
RESULTS
Lymphoblastoid Cell Lines All metaphases from untreated cultures were AgNOR-positive, with m e a n n u m b e r s of AgNORs/cell of 8 (range 7-9) for Daudi and Maja, and 16 (range 15-18) for the p o l y p l o i d line Molt 4. The presence of DMSO in the cultures for up to 5 days d i d not affect either the cell m o r p h o l o g y or AgNOR staining profiles.
HL-60
Cytology. U n i n d u c e d HL-60 cultures consist p r e d o m i n a n t l y of p r o m y e l o c y t e s together with some blasts and myelocytes, whereas metamyelocytes and bands are rare. Several large nucleoli are u s u a l l y present, w h i c h are intensely silver positive in the majority of cells (Fig. 1). The proportions of metamyelocytes and bands increases following DMSO treatment. Table 1 shows the differential counts obtained in a representative experiment. With the increase in numbers of maturing forms, there is a reduction in size and silver staining intensity of nucleoli in the majority of cells (Fig. 2). Chromosomes. This cell line is h y p o d i p l o i d , but contains a full c o m p l e m e n t of Dand G-group chromosomes. All metaphases from untreated cultures were AgNORpositive, with a range of 3-7 AgNORs/cell, the majority having 5 or 6 (Fig. 3). Exposure of HL-60 cells to 1.25% DMSO for up to 5 days resulted in a r a p i d fall in both the numbers of AgNORs/cell and their staining intensity. Figure 4 shows the results from a typical experiment. Twenty-four hours after i n d u c t i o n there is a reduction in the numbers of AgNORs/cell, w h i c h becomes more marked on subsequent days, so that by days 4 or 5 the pattern resembles that of a n o r m a l bone marrow, w i t h the majority of metaphases AgNOR-negative. The pattern of loss of AgNOR staining in metaphases from DMSO-treated cultures was fairly constant from cell to cell (Table 2). The c h r o m o s o m e s w i t h the smallest AgNORs were the first to appear unstained. However, there was some variability between metaphases with fewer than 4 AgNORs. Cells w i t h 3 AgNORs had either c h r o m o s o m e s #13, #21, and #22 or #13, #15, and #21 stained, and those containing 2 AgNORs had either chromosomes #13 and #21 or #21 and #22 stained. Cells with only 1 AgNOR were equally likely to have a positive #13 or #21.
162
B.R. Reeves et al.
Figure 2 Silver-stained nuclei from an HL-60 culture grown for 4 clays in the presence of DMSO. Note the reduction in the amount of Ag stain on nucleoli.
Kinetic studies. Kinetic studies, using BrdU incorporation and harlequin staining (Table 3), s h o w e d that DMSO treatment resulted in an a c c u m u l a t i o n of cells in the third cycle: the m a x i m u m n u m b e r of divisions n e e d e d by HL-60 cultures to reach the m e t a m y e l o c y t e stage. Some preparations from these DMSO-treated cultures were counterstained with AgNO3. Data from cells collected over the first 2 days following i n d u c t i o n (Table 4) show that c o m p l e t e s u p p r e s s i o n of AgNOR staining m a y occur in only one cell cycle. By 48 hr, the majority of cells were in their second cycle, with 30% being AgNOR-negative. However, there were still some second cycle cells present with up to 4 AgNORs.
DISCUSSION In our original s t u d y [1] we were unable to detect AgNORs in up to 61% of metaphases from hematologically n o r m a l bone marrows. Of the cells that were silver positive, the majority had one AgNOR, with only occasional cells having higher numbers. This observation, and our finding of AgNOR staining profiles characteristic for different cytologic types of leukemia, suggested either that variations in rDNA transcription might characterize different cell lineages and m a t u r a t i o n states in the bone marrow, or that pathologic changes in rRNA gene expression might
AgNOR Staining and Differentiation in HL-60
163
Figure 3 Metaphase from control HL-60 culture. Note the 6 AgNOR-positive chromosomes, identified by subsequent G-banding as numbers #13, #14, #15, #21, #21, and #22.
occur in leukemic cells. The results presented here, using the cell line HL-60, which can be induced to mature in vitro, support the first suggestion. Metaphases from uninduced cultures usually had 5 or 6 AgNORs, in agreement with our published data obtained from a small group of acute nonlymphocytic leukemia marrows [1]. Some of the cells had fewer AgNORs, which we think represents a fraction of the population undergoing spontaneous maturation. Following DMSO treatment, the number of detectable AgNORs fell rapidly, reaching levels characteristic of normal bone marrows by days 4 or 5 (Fig. 4). As positive silver staining of nucleolar organizers indicates those that were transcriptionally active in the preceding interphase [6-8], the results clearly show that, in this system, suppression of rDNA transcription occurs during terminal differentiation and that Ag-staining reflects regulation of rRNA synthesis in the transition from myeloblast to metamyelocyte.
164
B.R. Reeves et al.
HL 60 SO 40
DAY 0
t~ c~ 10
~ 38
0
1
2
3
4
S
6
q
S
[---q 6 7
~20 DAY 1
lO
~
O
1
2
3
4
30
DAY 2
c~lO O
1
2
3
4
S
6
q
2
DAY 3
o-10 O
l
2
3
4
5
6
7
40
DAY 4 ,o
90
O
1
2
3
4
5
6
7
80 70
DAY S 60,
50
5C 7 - 7
40
40
~3o [3-
Normol B.M,
60
L~
o cL
i
LL~_
13
10
.-2-v O I 2
3
4
No.of A g N O R s
5' 6 ~ per cell
0
1
2
3
4
5
6
?
8
No.of AgNORs per cell
Figure 4 AgNOR staining profiles for control HL-60 culture (day 0) and cultures grown in the presence of DMSO for 1-5 days compared with results from a typical hematologically normal bone marrow. Loss of AgNOR staining is correlated with morphological maturation and is not a direct effect of DMSO, as AgNORs were unaffected in the control lymphoblastoid cell lines. We found a general correlation between the amount of silver stain on particular chromosomes in u n i n d u c e d HL-60 cultures and its gradual loss during DMSO-induced maturation. The chromosomes with least stain, and by inference, the smallest numbers of transcriptionally active genes, were the first to appear unstained after DMSO treatment (Table 2). We found some cells to be AgNOR-negative in the first
165
AgNOR Staining and Differentiation in HL-60 Table 2
Pattern of AgNOR staining in metaphase chromosomes from DMSO-treated HL-60 cells Ag-positive D- and G-group chromosomes
Number of AgNORs
13
14
15
21
21
22
22
+ -b
+ q-
÷
+
+ q-
7
+
+
+
+
6
--k
-b
÷
--k
5 4 3 (a) (b) 2 (a) (b) 1 (a) (b)
÷ + + + +
+ +
q+ + + + +
+
+
+
+ +
(a) More common pattern. (b) Less common pattern.
cycle after induction (Table 4), and these were probably already quite mature when DMSO was added. The majority of cells in the population required two or more cycles to show cytologic evidence for the complete suppression of transcription. HL-60 therefore appears to be an excellent cell line for investigating the mechanisms of activation and control of the rRNA genes. There is good cytologic and biochemical evidence, in both rodent and human systems, that suppression of rDNA transcription is correlated with methylation of rRNA genes [9, 10]. We are currently investigating whether or not changes in methylation patterns of rDNA occur during the terminal differentiation of HL-60. We thank Dr. L. Wong for the differential counts on our HL-60 preparations and Professor S. D. Lawler for her comments on the manuscript.
Table 3
Kinetics of uninduced and DMSO-treated HL-60 cells Percent cells/cycle
Control (uninduced)
+ DMSO
Day
1st
2nd
3rd
>3rd
1 2 3 4 1 2 3 4
100 10 2 0 100 14 3 0
0 82 12 8 0 86 79 48
0 8 82 42 0 0 18 52
0 0 4 50 0 0 0 0
166
B . R . R e e v e s et al.
Table 4
C o m p a r i s o n of A g N O R s t a i n i n g a n d cell k i n e t i c s of D M S O - t r e a t e d HL-60 c e l l s Number of AgNORs
Day 1 Day 2
0
1
2
3
4
5
6
7
8 ~' (30) b
8 (20)
6 (20)
17 (14)1
31 (2) 8
19 2
8 3
3 --
"Percent cells in first cycle. bNumbers in parentheses represent percent cells in second cycle.
REFERENCES 1. Reeves BR, Casey G, Harris H (1982): Variations in the activity of nucleolar organizers in different tissues, demonstrated by silver-staining of h u m a n normal and leukemic cells. Cancer Genet Cytogenet 6:223-230. 2. Collins SJ, Gallo RC, Gallagher RE (1977): Continuous growth and differentiation of human myeloid leukemic cells in suspension culture. Nature 270:347-349. 3. Collins SJ, Ruscetti FW, Gallagher RE, Gallo RC (1978): Terminal differentiation of h u m a n promyelocytic leukemia cells induced by dimethyl sulphoxide and other polar solvents. Proc Natl Acad Sci USA 75:2458-2462. 4. Bloom SE, Goodpasture C (1976): An improved technique for selective silver staining of nucleolar organiser regions in h u m a n chromosomes. Hum Genet 34:199-206. 5. Perry P, Wolff S (1974): New Giemsa method for the differential staining of sister chromatids. Nature 251:156-158. 6. Howell WM (1977): Visualization of ribosomal gene activity: Silver stains proteins associated with rRNA transcribed from oocyte chromosomes. Chromosoma 62:361-367. 7. Schwarzacher HG, Mikelsaar AV, Schnedl W (1978): The nature of the Ag-staining of the nucleolus organiser regions. Cytogenet Cell Genet 20:24-39. 8. Hubbell HR, Rothblum LI, Hsu TC (1979): Identification of a silver binding protein associated with the cytological silver staining of actively transcribing nucleolar regions. Cell Biol Int Rep 3:615-622. 9. Tantravahi U, Guntaker RV, Erlanger BF, Miller OJ, (1981): Amplified ribosomal RNA genes in a rat hepatoma cell line are enriched in 5-methylcytosine. Proc Natl Acad Sci USA 78:489-493. 10. Tantravahi U, Breg WR, Wertelecki V, Erlanger BF, Miller OJ (1981): Evidence for methylation of inactive h u m a n rRNA genes in amplified regions. Hum Genet 56:315-320.
ABSTRACT: We have applied nucleolar organizer region (NOR) silver staining to the promyelocytic leukemia cell line HL-60, before and after dimethylsulfoxide (DMSO) mediated differentiation. The results demonstrated a gradual suppression of rDNA transcription during terminal maturation of these bone-marrow-derived cells and support our hypothesis [1] that there are characteristic NOR staining profiles for different bone marrow cell types. INTRODUCTION In a recent report, we described variations in silver staining of nucleolar organizer regions (AgNORs) in bone marrow cells from hematologically normal subjects and patients with leukemia [1]. In particular, we drew attention to differences in AgNOR staining profiles that a p p e a r e d to be characteristic for different cytologic types of leukemia. We suggested two explanations for these differences: either that rRNA gene activity was normal in the leukemic cells, with regulation of rDNA transcription d e p e n d i n g on cell lineage and maturation state, or that there were pathologic changes in rRNA gene expression. In order to resolve this question we have carried out studies on the cell line HL60. Under normal culture conditions, this line, w h i c h was derived from a patient with p r o m y e l o c y t i c leukemia, retains a l i m i t e d capacity for spontaneous granulocytic differentiation [2]. In addition, it has the capability for more extensive morphological and functional m y e l o i d differentiation u n d e r the influence of various agents, i n c l u d i n g d i m e t h y l s u l f o x i d e (DMSO) [3]. The results we have obtained following silver staining of metaphases from cultures i n d u c e d to differentiate with DMSO, suggest that, at least in bone-marrowderived cells, AgNOR staining is an indicator of differentiation/maturation state.
From the Section of Human Genetics, Institute of Cancer Research, Royal Marsden Hospital, London, England. Address requests for reprints to Dr. B. B. Reeves, Section of Human Genetics, Institute of Cancer Research, Royal Marsden Hospital, Fulham Road, London SW3 61J, England. Received August 8, 1983; accepted October 12, 1983.
159 © 1984 by Elsevier Science Publishing Co.. Inc. 52 Vanderbilt Ave., New York, NY 10017
Cancer Genetics and Cytogenetics13, 159-166 (1984) 0165-4808/84/$03.00
160
B.R. Reeves et al.
MATERIALS AND METHODS Cell Lines In a d d i t i o n to HL-60, three control (i.e., n o n i n d u c i b l e ) l y m p h o b l a s t o i d lines were studied: Molt 4 (T cell), Maja, and Daudi (B cell). Cell lines were m a i n t a i n e d in RPMI 1640 m e d i u m , s u p p l e m e n t e d with 10% fetal calf serum (Flow Laboratories). Experimental Procedures Duplicate 100-ml cultures were set up containing 5 x 105 cells/mE 24 hr later (day 0), DMSO was a d d e d to one culture of each pair at a final concentration of 1.25%
(v/v). T w e n t y - m i l l i l i t e r aliquots were subsequently taken from the cultures at 24-hr intervals: 2 ml being used for cytologic smears and the r e m a i n d e r for c h r o m o s o m e studies. Chromosome preparations were m a d e after further incubation at 37°C for 1.5 hr with 0.5 ixg/ml'colchicine and h y p o t o n i c treatment with 0.07 M KC1 for 10 rain at room temperature. Cells were then fixed in 3:1 m e t h a n o l : g l a c i a l acetic acid. Standard air-dried slides were aged for 24 hr and stained with 50% (w/v) AgNO3 by the m e t h o d of Bloom and Goodpasture [4]. For cell kinetic studies of HL-60, parallel cultures containing 5 ' - b r o m o d e o x y u r i dine (BrdU) at a final concentration of 10 ixM were set up with and w i t h o u t DMSO and harvested as above. Chromosomes were harlequin stained by the m e t h o d of Perry and Wolf [5]. Figure 1 Silver-stained nuclei from control HL-60 culture. Note the large intensely Ag-positive nucleoli.
161
AgNOR Staining and Differentiation in HL-60
Table 1
Differentiation of HL-60 cells following DMSO treatment Percent of cell types present
Control (uninduced) ÷DMSO day 1 ÷ DMSO day 2 ÷DMSO day 3 ÷ DMSO day 4
Myeloblasts and promyelocytes
Myelocytes
Metamyelocytes
Bands and polymorphs
98
1
1
0
97 95 91 79
1 2 3 8
2 3 6 10
0 9 0 3
RESULTS
Lymphoblastoid Cell Lines All metaphases from untreated cultures were AgNOR-positive, with m e a n n u m b e r s of AgNORs/cell of 8 (range 7-9) for Daudi and Maja, and 16 (range 15-18) for the p o l y p l o i d line Molt 4. The presence of DMSO in the cultures for up to 5 days d i d not affect either the cell m o r p h o l o g y or AgNOR staining profiles.
HL-60
Cytology. U n i n d u c e d HL-60 cultures consist p r e d o m i n a n t l y of p r o m y e l o c y t e s together with some blasts and myelocytes, whereas metamyelocytes and bands are rare. Several large nucleoli are u s u a l l y present, w h i c h are intensely silver positive in the majority of cells (Fig. 1). The proportions of metamyelocytes and bands increases following DMSO treatment. Table 1 shows the differential counts obtained in a representative experiment. With the increase in numbers of maturing forms, there is a reduction in size and silver staining intensity of nucleoli in the majority of cells (Fig. 2). Chromosomes. This cell line is h y p o d i p l o i d , but contains a full c o m p l e m e n t of Dand G-group chromosomes. All metaphases from untreated cultures were AgNORpositive, with a range of 3-7 AgNORs/cell, the majority having 5 or 6 (Fig. 3). Exposure of HL-60 cells to 1.25% DMSO for up to 5 days resulted in a r a p i d fall in both the numbers of AgNORs/cell and their staining intensity. Figure 4 shows the results from a typical experiment. Twenty-four hours after i n d u c t i o n there is a reduction in the numbers of AgNORs/cell, w h i c h becomes more marked on subsequent days, so that by days 4 or 5 the pattern resembles that of a n o r m a l bone marrow, w i t h the majority of metaphases AgNOR-negative. The pattern of loss of AgNOR staining in metaphases from DMSO-treated cultures was fairly constant from cell to cell (Table 2). The c h r o m o s o m e s w i t h the smallest AgNORs were the first to appear unstained. However, there was some variability between metaphases with fewer than 4 AgNORs. Cells w i t h 3 AgNORs had either c h r o m o s o m e s #13, #21, and #22 or #13, #15, and #21 stained, and those containing 2 AgNORs had either chromosomes #13 and #21 or #21 and #22 stained. Cells with only 1 AgNOR were equally likely to have a positive #13 or #21.
162
B.R. Reeves et al.
Figure 2 Silver-stained nuclei from an HL-60 culture grown for 4 clays in the presence of DMSO. Note the reduction in the amount of Ag stain on nucleoli.
Kinetic studies. Kinetic studies, using BrdU incorporation and harlequin staining (Table 3), s h o w e d that DMSO treatment resulted in an a c c u m u l a t i o n of cells in the third cycle: the m a x i m u m n u m b e r of divisions n e e d e d by HL-60 cultures to reach the m e t a m y e l o c y t e stage. Some preparations from these DMSO-treated cultures were counterstained with AgNO3. Data from cells collected over the first 2 days following i n d u c t i o n (Table 4) show that c o m p l e t e s u p p r e s s i o n of AgNOR staining m a y occur in only one cell cycle. By 48 hr, the majority of cells were in their second cycle, with 30% being AgNOR-negative. However, there were still some second cycle cells present with up to 4 AgNORs.
DISCUSSION In our original s t u d y [1] we were unable to detect AgNORs in up to 61% of metaphases from hematologically n o r m a l bone marrows. Of the cells that were silver positive, the majority had one AgNOR, with only occasional cells having higher numbers. This observation, and our finding of AgNOR staining profiles characteristic for different cytologic types of leukemia, suggested either that variations in rDNA transcription might characterize different cell lineages and m a t u r a t i o n states in the bone marrow, or that pathologic changes in rRNA gene expression might
AgNOR Staining and Differentiation in HL-60
163
Figure 3 Metaphase from control HL-60 culture. Note the 6 AgNOR-positive chromosomes, identified by subsequent G-banding as numbers #13, #14, #15, #21, #21, and #22.
occur in leukemic cells. The results presented here, using the cell line HL-60, which can be induced to mature in vitro, support the first suggestion. Metaphases from uninduced cultures usually had 5 or 6 AgNORs, in agreement with our published data obtained from a small group of acute nonlymphocytic leukemia marrows [1]. Some of the cells had fewer AgNORs, which we think represents a fraction of the population undergoing spontaneous maturation. Following DMSO treatment, the number of detectable AgNORs fell rapidly, reaching levels characteristic of normal bone marrows by days 4 or 5 (Fig. 4). As positive silver staining of nucleolar organizers indicates those that were transcriptionally active in the preceding interphase [6-8], the results clearly show that, in this system, suppression of rDNA transcription occurs during terminal differentiation and that Ag-staining reflects regulation of rRNA synthesis in the transition from myeloblast to metamyelocyte.
164
B.R. Reeves et al.
HL 60 SO 40
DAY 0
t~ c~ 10
~ 38
0
1
2
3
4
S
6
q
S
[---q 6 7
~20 DAY 1
lO
~
O
1
2
3
4
30
DAY 2
c~lO O
1
2
3
4
S
6
q
2
DAY 3
o-10 O
l
2
3
4
5
6
7
40
DAY 4 ,o
90
O
1
2
3
4
5
6
7
80 70
DAY S 60,
50
5C 7 - 7
40
40
~3o [3-
Normol B.M,
60
L~
o cL
i
LL~_
13
10
.-2-v O I 2
3
4
No.of A g N O R s
5' 6 ~ per cell
0
1
2
3
4
5
6
?
8
No.of AgNORs per cell
Figure 4 AgNOR staining profiles for control HL-60 culture (day 0) and cultures grown in the presence of DMSO for 1-5 days compared with results from a typical hematologically normal bone marrow. Loss of AgNOR staining is correlated with morphological maturation and is not a direct effect of DMSO, as AgNORs were unaffected in the control lymphoblastoid cell lines. We found a general correlation between the amount of silver stain on particular chromosomes in u n i n d u c e d HL-60 cultures and its gradual loss during DMSO-induced maturation. The chromosomes with least stain, and by inference, the smallest numbers of transcriptionally active genes, were the first to appear unstained after DMSO treatment (Table 2). We found some cells to be AgNOR-negative in the first
165
AgNOR Staining and Differentiation in HL-60 Table 2
Pattern of AgNOR staining in metaphase chromosomes from DMSO-treated HL-60 cells Ag-positive D- and G-group chromosomes
Number of AgNORs
13
14
15
21
21
22
22
+ -b
+ q-
÷
+
+ q-
7
+
+
+
+
6
--k
-b
÷
--k
5 4 3 (a) (b) 2 (a) (b) 1 (a) (b)
÷ + + + +
+ +
q+ + + + +
+
+
+
+ +
(a) More common pattern. (b) Less common pattern.
cycle after induction (Table 4), and these were probably already quite mature when DMSO was added. The majority of cells in the population required two or more cycles to show cytologic evidence for the complete suppression of transcription. HL-60 therefore appears to be an excellent cell line for investigating the mechanisms of activation and control of the rRNA genes. There is good cytologic and biochemical evidence, in both rodent and human systems, that suppression of rDNA transcription is correlated with methylation of rRNA genes [9, 10]. We are currently investigating whether or not changes in methylation patterns of rDNA occur during the terminal differentiation of HL-60. We thank Dr. L. Wong for the differential counts on our HL-60 preparations and Professor S. D. Lawler for her comments on the manuscript.
Table 3
Kinetics of uninduced and DMSO-treated HL-60 cells Percent cells/cycle
Control (uninduced)
+ DMSO
Day
1st
2nd
3rd
>3rd
1 2 3 4 1 2 3 4
100 10 2 0 100 14 3 0
0 82 12 8 0 86 79 48
0 8 82 42 0 0 18 52
0 0 4 50 0 0 0 0
166
B . R . R e e v e s et al.
Table 4
C o m p a r i s o n of A g N O R s t a i n i n g a n d cell k i n e t i c s of D M S O - t r e a t e d HL-60 c e l l s Number of AgNORs
Day 1 Day 2
0
1
2
3
4
5
6
7
8 ~' (30) b
8 (20)
6 (20)
17 (14)1
31 (2) 8
19 2
8 3
3 --
"Percent cells in first cycle. bNumbers in parentheses represent percent cells in second cycle.
REFERENCES 1. Reeves BR, Casey G, Harris H (1982): Variations in the activity of nucleolar organizers in different tissues, demonstrated by silver-staining of h u m a n normal and leukemic cells. Cancer Genet Cytogenet 6:223-230. 2. Collins SJ, Gallo RC, Gallagher RE (1977): Continuous growth and differentiation of human myeloid leukemic cells in suspension culture. Nature 270:347-349. 3. Collins SJ, Ruscetti FW, Gallagher RE, Gallo RC (1978): Terminal differentiation of h u m a n promyelocytic leukemia cells induced by dimethyl sulphoxide and other polar solvents. Proc Natl Acad Sci USA 75:2458-2462. 4. Bloom SE, Goodpasture C (1976): An improved technique for selective silver staining of nucleolar organiser regions in h u m a n chromosomes. Hum Genet 34:199-206. 5. Perry P, Wolff S (1974): New Giemsa method for the differential staining of sister chromatids. Nature 251:156-158. 6. Howell WM (1977): Visualization of ribosomal gene activity: Silver stains proteins associated with rRNA transcribed from oocyte chromosomes. Chromosoma 62:361-367. 7. Schwarzacher HG, Mikelsaar AV, Schnedl W (1978): The nature of the Ag-staining of the nucleolus organiser regions. Cytogenet Cell Genet 20:24-39. 8. Hubbell HR, Rothblum LI, Hsu TC (1979): Identification of a silver binding protein associated with the cytological silver staining of actively transcribing nucleolar regions. Cell Biol Int Rep 3:615-622. 9. Tantravahi U, Guntaker RV, Erlanger BF, Miller OJ, (1981): Amplified ribosomal RNA genes in a rat hepatoma cell line are enriched in 5-methylcytosine. Proc Natl Acad Sci USA 78:489-493. 10. Tantravahi U, Breg WR, Wertelecki V, Erlanger BF, Miller OJ (1981): Evidence for methylation of inactive h u m a n rRNA genes in amplified regions. Hum Genet 56:315-320.