Cell, Vol. 12, 901-913,
December
1977, Copyright
8 1977 by MIT
Terminal Differentiation Erythroleukemia Cells
in Cultured
Eileen A. Friedman and Carl L. Schildkraut Department of Cell Biology Albert Einstein College of Medicine 1300 Morris Park Avenue Bronx, New York 10461
Summary Two populations of differentiated, hemoglobincontaining cells have been identified in cultures of Friend murine erythroleukemia cells (Friend cells): terminally differentiated benzidine-positive (B+) cells that are no longer capable of proliferation and are arrested in the Gl phase of the cell cycle, and their precursors, traversing B+ cells which undergo two or three cell divisions before reaching their terminally differentiated state. Thus Friend cells in suspension culture retain a limited capacity to synthesize DNA and divide after commitment to erythroid differentiation. We identified terminally differentiated cells using autoradiography after benzidine staining. We also developed a quantitative flow microfluorometric assay to distinguish cells that are terminally differentiated from those cells committed to differentiation but still capable of proliferation. We developed a purification procedure to isolate terminally differentiated Friend cells. Their DNA content was the same as that of the undifferentiated cells in Gl by both the diphenylamine reaction and a fluorescence assay. No loss of DNA was detected during the differentiation of Friend cells. As many as 72% of the total cells in a culture induced with DMSO (88% B+) were differentiated cells arrested in Gl. As a control, a DMSO-resistant line derived from 745A neither differentiated nor arrested in Gl after growth in the presence of DMSO. The results of these studies were obtained using several compounds that induce differentiation and three independently isolated clones of 745A. We also observed arrest of differentiated cells in Gl with the two other well characterized, independently derived erythroleukemia cell lines, F4-1 and T3-Cl-2. Introduction Many mammalian cell types in advanced stages of differentiation are observed to have a DNA content corresponding to the Gl or GO phase of the cell cycle (for review see Prescott, 1976). For example, prior to fusion, the length of Gl increases in myoblasts (Buckley and Konigsberg, 1974). Moreover, these cells are reported to be in Gl and to have lost proliferative capacity before their fusion into myotubules (Stockdale and Holtzer, 1961; Oka-
Friend
zaki and Holtzer, 1966). Friend murine erythroleukemia cells (Friend cells) are a transformed line which can differentiate in culture, expressing erythroid characteristics such as hemoglobin synthesis, if grown for several days in the presence of certain organic compounds (Friend et al., 1971; Leder and Leder, 1975; Tanaka et al., 1975). Commitment to differentiation does not immediately cause Friend cells to lose their capacity for DNA synthesis. Instead, as shown by Gusella et al. (1976) for cells in plasma clots, and in the present studies for cells in suspension culture, they display a limited proliferative capacity, similar to untransformed murine erythroid precursor cells at the erythropoietin-sensitive stage (Cooper et al., 1974; McLeod, Shreeve and Axelrod, 1974). We have observed that in the later stages of Friend cell differentiation, even if DMSOcontaining medium is replenished daily, some benzidine-reactive cells in suspension culture lose proliferative capacity and arrest in the Gl phase of the cell cycle. Although it has undergone viral transformation, the Friend cell line is still capable of exhibiting a terminal arrest in the Gl phase similar to the arrest associated with differentiation in several types of nontransformed cells. Results Cultures of Induced Friend Cells Contain a High Proportion of Cells in Gl We grew Friend cells in the presence of 280 mM DMSO without changing the medium, and after 4 days, 62% of the cells were benzidine-reactive (B+). At this time, flow microfluorometric analysis indicated (Figure 1G) that a majority (90%) of the cells were in the Gl phase of the cell cycle. Uninduced cells in a control culture did not accumulate in Gl to the same extent (54%; Figure lE), although the culture had reached saturation density. This difference suggested that Friend cells had accumulated in Gl primarily because they had differentiated, not because they had grown to a high density. Uninduced Friend cells do not show the distinctive growth control exhibited by fibroblastic cell lines which enter a quiescent state and arrest in Gl when grown to confluence (reviewed by Halley, 1975). In contrast, uninduced Friend cells grown to saturation density were in all phases of the cell cycle (Figure lE), as were lymphoid SK-L7 cells, another cell line maintained in suspension culture (Yen, Fried and Clarkson, 1977). We performed additional controls to establish that the difference seen in the proportion of Friend cells in Gl in induced and uninduced cultures was a result of differentiation. To eliminate the possibility that cells had accumulated in Gl due to deple-
Cdl 902
MEDIUM
n
DAILY
l
v
MEDIUM
8 ac ii
CHANGED
tion of essential factors in the growth medium, we induced cells by daily resuspension in fresh medium containing inducing agent. After several days of feeding with medium containing either DMSO or sodium butyrate, the majority of cells were in Gl (83%, Flgure 1C). In contrast, if we fed cells daily with medium without inducing agent, growth continued exponentially (28% in Gl; Figure 1A). The difference between the proportion of cells in Gl in induced cultures and uninduced control cultures was even more striking in this case. Clearly the accumulation of cells in Gl in induced cultures is related to their differentiation. Possibly these Gl cells represent a discrete population in a differentiated culture. Friend cells are induced to differentiate in a stochastic manner over a period of several days (Gusella et al., 1976). After induction, therefore, cells at all stages of differentiation are present in these cultures. We wished to establish whether all or only specific classes of differentiated (hemoglobin-containing) cells were in Gl , and whether cells were in Gl because they had lost the capacity to synthesize DNA and traverse S phase.
EXPONENTIAL UNTREATED 3H-THYMIDINE
NOT
A
CHANGED
STATIONARY UNTREA’ED 3H-THYMIDINE b
5 z
fi DMSO 4DAYS
2c
4c
Figure 1. Proliferating Cells Arrested in Gi by Incubation &i/ml)
R
2c
4c
Can Be Distinguished from in the Presence of W-Thymidine
Cells (5
DS19 cells in exponential growth were diluted to 1.5 x lo5 cells per ml at the start of all experiments. Cultures 8, D, F and H had been centrifuged and resuspended in medium containing 3Hthymidine and no DMSO. Samples from cultures A-H were prepared for flow microfluorometry by methanol fixation followed by pancreatic RNAase treatment. The distributions of fluorescence (proportional to DNA content) per cell are presented above. The channel positions for cells having a DNA content of either 2C or 4C are indicated on the abscissa. The ordinates increase linearly from a baseline of zero to a maximum of about 3000 cells. Approximately 50,000 cells were analyzed for each distribution. The same intensity of fluorescence was obtained for cells in the Gl stage of the cell cycle in exponential cultures as for B+ cells which were arrested and did not synthesize DNA. If induced cells (from culture C, D, G or H) were mixed with uninduced cells (from culture A), the Gl cells from both cultures were indistinguishable by FMF analysis, appearing at identical channel positions (data not shown). Cells were treated with pancreatic RNAase before staining with propidium iodide to reduce any possible fluorescence due to the presence of RNA. (A) Cells in exponential growth. 28% of the cells were in Gl. (6) Culture A incubated for 19 hr in the presence of JH-thymidine. Identical DNA distributions were obtained after 11, 19 and 24 hr of incubation. 12% of the cells were in Gl. (C) Cells suspended daily at a density of 2-5 x 105/ml for 7 days in medium containing 210 mM DMSO. The DNA distribution profile of the culture on day 7 is shown. The culture contained 83% of the cells in Gl and 95% B+ cells by the suspension test. (D) Culture C after 24 hr of incubation in the presence of 3Hthymidine. The same DNA distributions were obtained after 18, 22 and 24 hr of incubation (61% arrested in Gl). By the suspension test, 95% of the total cells were B+. (E) Untreated cells after growth for 4 days without changing the medium. 54% of the cells were in Gl. The culture had reached stationary phase after 3 days.
Differentiated Friend Cells That Cannot Synthesize DNA Are Arrested in Gl We have identified terminally differentiated cells in suspension cultures of induced Friend cells using a flow microfluorometric assay. This assay is based on the observation that cells incubated in medium containing high concentrations of +l-thymidine attain a DNA content approaching their 4C value (C is the haploid DNA content of Friend cells in Gl) as indicated by FMF analysis (Ehmann et al., 1975; Marz et al., 1977). We incubated Friend cells (five different clones; Table 1) in exponential growth in medium containing 3H-thymidine (5 QI ml). After 12 hr, ~2% of the cells remained in Gl and early S phase as indicated by their DNA distribution profiles (Figure 1B). The majority of the cells (98%) had a DNA content of approximately 4C. These cells appeared to be in the late S or G2 phase of the cell cycle, since ~0.4% were in mitosis by microscopic examination. During incubation in medium containing 3H-thymidine, there was a 5% increase in cell number presumably due to division of cells in G2 at the time the 3H-thymidine was (F) Culture E after 27 hr of incubation with 3H-thymidine. 4% of the cells were in Gl. An identical DNA distribution profile was observed after 24 hr of incubation. (G) Cells were grown for 4 days in the presence of 280 mM DMSO without changing the medium. The cells were then centrifuged and resuspended in medium containing 3H-thymidine. At this time, 62% of the cells were B+ and 90% of the total cells were in Gl. (H) Culture G after 27 hr of incubation in 3H-thymidine. Identical DNA distribution profiles were observed after 24, 27 and 31 hr of incubation. 47% of the cells were in Gl.
Terminal
Differentiation
in Erythroleukemia
Cells
903
Table
1. Terminally
Differentiated
Friend
Cells Arrest
in Gl % Total
Cell Line
Inducing Agent
m-W
Days
0+
Arrested in Gl (FMF Analysis)
B+ and Unlabeled (Autoradiography)
DS19
DMSO
210
7
92
61”
59
DS19
DMSO
280
4
95
17
14
DS19
None
7
0
0
745A
DMSO
5
88
38
40
745A
None
5
0
0
NT
745A-11
Sodium
745A-11
None.
T3-Cl-2
DMSO
T3-Cl-2
None
520a
DMSO
520a
None
(A) Medium
(6) Medium
Changed
Concentration
Cells
Daily
280
butyrate
1.5
280
280
NTb
7
31
19
18’
7
0
0
NT
4
35
18
NT
4
3
6
NT
4
3
0
NT
4
0
0
NT
Not Changed
DS19
None
4
0
4
NT
DS19
DMSO
280
4
62
476
47’
OS19
DMSO
280
5
80
56’
NT
DS19
DMSO
280
6
88
76
70
DS19
DMSO
210
4g
72
54
NT
F4-1
None
4
0
3
NT
F4-1
DMSO
4
88
80
NT
745A-11
None
6
0
8
NT
745A-11
DMSO
112
6
20
5
NT
745A-11
DMSO
280
6
90
65
63”
745A-11
DMSO
280
8’
88
74
72
745A-11
l-methyl 2-pyrrolidinone
10
4
39
15
14
5
4
31
14
11
6
0
0
NT
6
1
0
NT
745A-11
Hypoxanthine
520a
None
520a
DMSO
280
280
a Experiment in Figure 1D. b NT = not tested. ’ Experiment in Figure 38. d Experiment in Figures 1H and 2. ’ Experiment in Figure 3C. r The percentage of cells arrested in Gl was the same after 30 hr of incubation in either 0.01 fig/ml vinblastine sulfate or 5 &i/ml JHthymidine. K Cells were induced in a different medium (Dulbecco’s) supplemented with a different lot of fetal calf serum (455630) than that used in all the other experiments in this report. ’ Experiment in Figure 3A. ’ After 6 days of induction with 280 mM DMSO, cells were resuspended in medium containing 280 mM DMSO and *H-thymidine and incubated for 2 days. A culture in exponential growth was diluted into medium containing inducing agent at 1.5 X lo5 cells per ml at the start of each experiment. In the experiments described in (B). the medium was not changed after the start of an experiment to minimize handling and loss of cells. In the experiments described in (A), cells were centrifuged and resuspended at 2-5 X lo5 cells per ml daily in medium containing inducing agent. Subsequently, cells were centrifuged and resuspended in medium containing %H-thymidine to measure the percentage of cells arrested in GI (Experimental Procedures). Cells were incubated for 20-36 hr in 3H-thymidine until DNA distribution profiles remained constant. The percentage of cells arrested in Gl was obtained by averaging data from 2-3 DNA distribution profiles. The percentage of B+ cells in each culture was determined either using the suspension test or the slide test (Experimental Procedures). Cells on slides were analyzed for total B+ cells and for B+ cells incorporating ‘H-thymidine. In other experiments, cells were not prepared on slides, and the shorter suspension test was performed. In these cases, the last column contains the entry NT. The percentage of cells arrested in Gl in induced cultures was corrected for the percentage of binucleate B+ cells which did not incorporate 3H-thymidine. These cells had a 4C DNA content. They appeared at the same position in FMF analysis as proliferating cells which had been arrested in G2 after incubation with 3H-thymidine. The percentage of unlabeled binucleate B+ cells varied with both the inducing agent and the length of induction. Cells grown in 280 mM DMSO for 6 days contained 9% of these cells, suggesting that some terminally arrested B+ cells fail to undergo cytokinesis. Cultures induced with all other agents contained ~0.6% of unlabeled binucleate B+ cells. In all experiments, the percentage of labeled binucleate cells was ~1%.
Cdl 904
added. Cells which had been arrested by incorporation of 3H-thymidine did not divide or attain a DNA content higher than 4C. After 36 to 48 hr of incubation, however, these cells enlarged and then disintegrated. In these studies, both induced cuitures and uninduced control cultures treated with 3H-thymidine were analyzed by cytofluorometry after all proliferating cells had been blocked in G2, but before their disintegration. In this way, it was possible to detect any Friend cells arrested in Gl or in S. Using this technique, we analyzed a culture of differentiated Friend cells (62% B+, 90% in Gl; Figure 1G) which had been induced by growth in 280 mM DMSO for 4 days. When we incubated this culture for an additional 24 hr in the presence of 3H-thymidine (Experimental Procedures), the DNA content increased significantly in only 53% of the cells (Figure 1 H). Thus 47% of the cells were unable to progress from Gl The percentage of cells in Gl remained constant at 47% for the next 12 hr (24-36 hr of incubation with 3H-thymidine; Figure 2 and Table 1). We observed no cellular disintegration during this interval. The fluorescence from the cells in Gl in this culture was the same as that obtained from uninduced cells having a DNA content of 2C (samples for FMF analysis were methanol-fixed, then treated with pancreatic RNAase; Figure 1). We concluded that some cells in induced cultures which were in Gl had lost the capacity to synthesize DNA and were actually arrested in the Gl stage of the cell cycle. We also analyzed a second culture, induced by daily feeding for 7 days with medium containing DMSO (95% B+, 83% in Gl; Figure lC), in the same,manner. After incubation in 3H-thymidine for 24-36 hr, 61% of the cells remained arrested in Gl (Figure 1D) and therefore had lost the capacity to traverse S phase. We obtained similar results after the induction with 280 mM DMSO of two other independently isolated erythroleukemia cell lines: F4-1 and T3-Cl-2 (Table 1). As a control, we added 3H-thymidine to uninduced control cultures of each cell line studied. We observed that
December
1977, Copyright
8 1977 by MIT
Terminal Differentiation Erythroleukemia Cells
in Cultured
Eileen A. Friedman and Carl L. Schildkraut Department of Cell Biology Albert Einstein College of Medicine 1300 Morris Park Avenue Bronx, New York 10461
Summary Two populations of differentiated, hemoglobincontaining cells have been identified in cultures of Friend murine erythroleukemia cells (Friend cells): terminally differentiated benzidine-positive (B+) cells that are no longer capable of proliferation and are arrested in the Gl phase of the cell cycle, and their precursors, traversing B+ cells which undergo two or three cell divisions before reaching their terminally differentiated state. Thus Friend cells in suspension culture retain a limited capacity to synthesize DNA and divide after commitment to erythroid differentiation. We identified terminally differentiated cells using autoradiography after benzidine staining. We also developed a quantitative flow microfluorometric assay to distinguish cells that are terminally differentiated from those cells committed to differentiation but still capable of proliferation. We developed a purification procedure to isolate terminally differentiated Friend cells. Their DNA content was the same as that of the undifferentiated cells in Gl by both the diphenylamine reaction and a fluorescence assay. No loss of DNA was detected during the differentiation of Friend cells. As many as 72% of the total cells in a culture induced with DMSO (88% B+) were differentiated cells arrested in Gl. As a control, a DMSO-resistant line derived from 745A neither differentiated nor arrested in Gl after growth in the presence of DMSO. The results of these studies were obtained using several compounds that induce differentiation and three independently isolated clones of 745A. We also observed arrest of differentiated cells in Gl with the two other well characterized, independently derived erythroleukemia cell lines, F4-1 and T3-Cl-2. Introduction Many mammalian cell types in advanced stages of differentiation are observed to have a DNA content corresponding to the Gl or GO phase of the cell cycle (for review see Prescott, 1976). For example, prior to fusion, the length of Gl increases in myoblasts (Buckley and Konigsberg, 1974). Moreover, these cells are reported to be in Gl and to have lost proliferative capacity before their fusion into myotubules (Stockdale and Holtzer, 1961; Oka-
Friend
zaki and Holtzer, 1966). Friend murine erythroleukemia cells (Friend cells) are a transformed line which can differentiate in culture, expressing erythroid characteristics such as hemoglobin synthesis, if grown for several days in the presence of certain organic compounds (Friend et al., 1971; Leder and Leder, 1975; Tanaka et al., 1975). Commitment to differentiation does not immediately cause Friend cells to lose their capacity for DNA synthesis. Instead, as shown by Gusella et al. (1976) for cells in plasma clots, and in the present studies for cells in suspension culture, they display a limited proliferative capacity, similar to untransformed murine erythroid precursor cells at the erythropoietin-sensitive stage (Cooper et al., 1974; McLeod, Shreeve and Axelrod, 1974). We have observed that in the later stages of Friend cell differentiation, even if DMSOcontaining medium is replenished daily, some benzidine-reactive cells in suspension culture lose proliferative capacity and arrest in the Gl phase of the cell cycle. Although it has undergone viral transformation, the Friend cell line is still capable of exhibiting a terminal arrest in the Gl phase similar to the arrest associated with differentiation in several types of nontransformed cells. Results Cultures of Induced Friend Cells Contain a High Proportion of Cells in Gl We grew Friend cells in the presence of 280 mM DMSO without changing the medium, and after 4 days, 62% of the cells were benzidine-reactive (B+). At this time, flow microfluorometric analysis indicated (Figure 1G) that a majority (90%) of the cells were in the Gl phase of the cell cycle. Uninduced cells in a control culture did not accumulate in Gl to the same extent (54%; Figure lE), although the culture had reached saturation density. This difference suggested that Friend cells had accumulated in Gl primarily because they had differentiated, not because they had grown to a high density. Uninduced Friend cells do not show the distinctive growth control exhibited by fibroblastic cell lines which enter a quiescent state and arrest in Gl when grown to confluence (reviewed by Halley, 1975). In contrast, uninduced Friend cells grown to saturation density were in all phases of the cell cycle (Figure lE), as were lymphoid SK-L7 cells, another cell line maintained in suspension culture (Yen, Fried and Clarkson, 1977). We performed additional controls to establish that the difference seen in the proportion of Friend cells in Gl in induced and uninduced cultures was a result of differentiation. To eliminate the possibility that cells had accumulated in Gl due to deple-
Cdl 902
MEDIUM
n
DAILY
l
v
MEDIUM
8 ac ii
CHANGED
tion of essential factors in the growth medium, we induced cells by daily resuspension in fresh medium containing inducing agent. After several days of feeding with medium containing either DMSO or sodium butyrate, the majority of cells were in Gl (83%, Flgure 1C). In contrast, if we fed cells daily with medium without inducing agent, growth continued exponentially (28% in Gl; Figure 1A). The difference between the proportion of cells in Gl in induced cultures and uninduced control cultures was even more striking in this case. Clearly the accumulation of cells in Gl in induced cultures is related to their differentiation. Possibly these Gl cells represent a discrete population in a differentiated culture. Friend cells are induced to differentiate in a stochastic manner over a period of several days (Gusella et al., 1976). After induction, therefore, cells at all stages of differentiation are present in these cultures. We wished to establish whether all or only specific classes of differentiated (hemoglobin-containing) cells were in Gl , and whether cells were in Gl because they had lost the capacity to synthesize DNA and traverse S phase.
EXPONENTIAL UNTREATED 3H-THYMIDINE
NOT
A
CHANGED
STATIONARY UNTREA’ED 3H-THYMIDINE b
5 z
fi DMSO 4DAYS
2c
4c
Figure 1. Proliferating Cells Arrested in Gi by Incubation &i/ml)
R
2c
4c
Can Be Distinguished from in the Presence of W-Thymidine
Cells (5
DS19 cells in exponential growth were diluted to 1.5 x lo5 cells per ml at the start of all experiments. Cultures 8, D, F and H had been centrifuged and resuspended in medium containing 3Hthymidine and no DMSO. Samples from cultures A-H were prepared for flow microfluorometry by methanol fixation followed by pancreatic RNAase treatment. The distributions of fluorescence (proportional to DNA content) per cell are presented above. The channel positions for cells having a DNA content of either 2C or 4C are indicated on the abscissa. The ordinates increase linearly from a baseline of zero to a maximum of about 3000 cells. Approximately 50,000 cells were analyzed for each distribution. The same intensity of fluorescence was obtained for cells in the Gl stage of the cell cycle in exponential cultures as for B+ cells which were arrested and did not synthesize DNA. If induced cells (from culture C, D, G or H) were mixed with uninduced cells (from culture A), the Gl cells from both cultures were indistinguishable by FMF analysis, appearing at identical channel positions (data not shown). Cells were treated with pancreatic RNAase before staining with propidium iodide to reduce any possible fluorescence due to the presence of RNA. (A) Cells in exponential growth. 28% of the cells were in Gl. (6) Culture A incubated for 19 hr in the presence of JH-thymidine. Identical DNA distributions were obtained after 11, 19 and 24 hr of incubation. 12% of the cells were in Gl. (C) Cells suspended daily at a density of 2-5 x 105/ml for 7 days in medium containing 210 mM DMSO. The DNA distribution profile of the culture on day 7 is shown. The culture contained 83% of the cells in Gl and 95% B+ cells by the suspension test. (D) Culture C after 24 hr of incubation in the presence of 3Hthymidine. The same DNA distributions were obtained after 18, 22 and 24 hr of incubation (61% arrested in Gl). By the suspension test, 95% of the total cells were B+. (E) Untreated cells after growth for 4 days without changing the medium. 54% of the cells were in Gl. The culture had reached stationary phase after 3 days.
Differentiated Friend Cells That Cannot Synthesize DNA Are Arrested in Gl We have identified terminally differentiated cells in suspension cultures of induced Friend cells using a flow microfluorometric assay. This assay is based on the observation that cells incubated in medium containing high concentrations of +l-thymidine attain a DNA content approaching their 4C value (C is the haploid DNA content of Friend cells in Gl) as indicated by FMF analysis (Ehmann et al., 1975; Marz et al., 1977). We incubated Friend cells (five different clones; Table 1) in exponential growth in medium containing 3H-thymidine (5 QI ml). After 12 hr, ~2% of the cells remained in Gl and early S phase as indicated by their DNA distribution profiles (Figure 1B). The majority of the cells (98%) had a DNA content of approximately 4C. These cells appeared to be in the late S or G2 phase of the cell cycle, since ~0.4% were in mitosis by microscopic examination. During incubation in medium containing 3H-thymidine, there was a 5% increase in cell number presumably due to division of cells in G2 at the time the 3H-thymidine was (F) Culture E after 27 hr of incubation with 3H-thymidine. 4% of the cells were in Gl. An identical DNA distribution profile was observed after 24 hr of incubation. (G) Cells were grown for 4 days in the presence of 280 mM DMSO without changing the medium. The cells were then centrifuged and resuspended in medium containing 3H-thymidine. At this time, 62% of the cells were B+ and 90% of the total cells were in Gl. (H) Culture G after 27 hr of incubation in 3H-thymidine. Identical DNA distribution profiles were observed after 24, 27 and 31 hr of incubation. 47% of the cells were in Gl.
Terminal
Differentiation
in Erythroleukemia
Cells
903
Table
1. Terminally
Differentiated
Friend
Cells Arrest
in Gl % Total
Cell Line
Inducing Agent
m-W
Days
0+
Arrested in Gl (FMF Analysis)
B+ and Unlabeled (Autoradiography)
DS19
DMSO
210
7
92
61”
59
DS19
DMSO
280
4
95
17
14
DS19
None
7
0
0
745A
DMSO
5
88
38
40
745A
None
5
0
0
NT
745A-11
Sodium
745A-11
None.
T3-Cl-2
DMSO
T3-Cl-2
None
520a
DMSO
520a
None
(A) Medium
(6) Medium
Changed
Concentration
Cells
Daily
280
butyrate
1.5
280
280
NTb
7
31
19
18’
7
0
0
NT
4
35
18
NT
4
3
6
NT
4
3
0
NT
4
0
0
NT
Not Changed
DS19
None
4
0
4
NT
DS19
DMSO
280
4
62
476
47’
OS19
DMSO
280
5
80
56’
NT
DS19
DMSO
280
6
88
76
70
DS19
DMSO
210
4g
72
54
NT
F4-1
None
4
0
3
NT
F4-1
DMSO
4
88
80
NT
745A-11
None
6
0
8
NT
745A-11
DMSO
112
6
20
5
NT
745A-11
DMSO
280
6
90
65
63”
745A-11
DMSO
280
8’
88
74
72
745A-11
l-methyl 2-pyrrolidinone
10
4
39
15
14
5
4
31
14
11
6
0
0
NT
6
1
0
NT
745A-11
Hypoxanthine
520a
None
520a
DMSO
280
280
a Experiment in Figure 1D. b NT = not tested. ’ Experiment in Figure 38. d Experiment in Figures 1H and 2. ’ Experiment in Figure 3C. r The percentage of cells arrested in Gl was the same after 30 hr of incubation in either 0.01 fig/ml vinblastine sulfate or 5 &i/ml JHthymidine. K Cells were induced in a different medium (Dulbecco’s) supplemented with a different lot of fetal calf serum (455630) than that used in all the other experiments in this report. ’ Experiment in Figure 3A. ’ After 6 days of induction with 280 mM DMSO, cells were resuspended in medium containing 280 mM DMSO and *H-thymidine and incubated for 2 days. A culture in exponential growth was diluted into medium containing inducing agent at 1.5 X lo5 cells per ml at the start of each experiment. In the experiments described in (B). the medium was not changed after the start of an experiment to minimize handling and loss of cells. In the experiments described in (A), cells were centrifuged and resuspended at 2-5 X lo5 cells per ml daily in medium containing inducing agent. Subsequently, cells were centrifuged and resuspended in medium containing %H-thymidine to measure the percentage of cells arrested in GI (Experimental Procedures). Cells were incubated for 20-36 hr in 3H-thymidine until DNA distribution profiles remained constant. The percentage of cells arrested in Gl was obtained by averaging data from 2-3 DNA distribution profiles. The percentage of B+ cells in each culture was determined either using the suspension test or the slide test (Experimental Procedures). Cells on slides were analyzed for total B+ cells and for B+ cells incorporating ‘H-thymidine. In other experiments, cells were not prepared on slides, and the shorter suspension test was performed. In these cases, the last column contains the entry NT. The percentage of cells arrested in Gl in induced cultures was corrected for the percentage of binucleate B+ cells which did not incorporate 3H-thymidine. These cells had a 4C DNA content. They appeared at the same position in FMF analysis as proliferating cells which had been arrested in G2 after incubation with 3H-thymidine. The percentage of unlabeled binucleate B+ cells varied with both the inducing agent and the length of induction. Cells grown in 280 mM DMSO for 6 days contained 9% of these cells, suggesting that some terminally arrested B+ cells fail to undergo cytokinesis. Cultures induced with all other agents contained ~0.6% of unlabeled binucleate B+ cells. In all experiments, the percentage of labeled binucleate cells was ~1%.
Cdl 904
added. Cells which had been arrested by incorporation of 3H-thymidine did not divide or attain a DNA content higher than 4C. After 36 to 48 hr of incubation, however, these cells enlarged and then disintegrated. In these studies, both induced cuitures and uninduced control cultures treated with 3H-thymidine were analyzed by cytofluorometry after all proliferating cells had been blocked in G2, but before their disintegration. In this way, it was possible to detect any Friend cells arrested in Gl or in S. Using this technique, we analyzed a culture of differentiated Friend cells (62% B+, 90% in Gl; Figure 1G) which had been induced by growth in 280 mM DMSO for 4 days. When we incubated this culture for an additional 24 hr in the presence of 3H-thymidine (Experimental Procedures), the DNA content increased significantly in only 53% of the cells (Figure 1 H). Thus 47% of the cells were unable to progress from Gl The percentage of cells in Gl remained constant at 47% for the next 12 hr (24-36 hr of incubation with 3H-thymidine; Figure 2 and Table 1). We observed no cellular disintegration during this interval. The fluorescence from the cells in Gl in this culture was the same as that obtained from uninduced cells having a DNA content of 2C (samples for FMF analysis were methanol-fixed, then treated with pancreatic RNAase; Figure 1). We concluded that some cells in induced cultures which were in Gl had lost the capacity to synthesize DNA and were actually arrested in the Gl stage of the cell cycle. We also analyzed a second culture, induced by daily feeding for 7 days with medium containing DMSO (95% B+, 83% in Gl; Figure lC), in the same,manner. After incubation in 3H-thymidine for 24-36 hr, 61% of the cells remained arrested in Gl (Figure 1D) and therefore had lost the capacity to traverse S phase. We obtained similar results after the induction with 280 mM DMSO of two other independently isolated erythroleukemia cell lines: F4-1 and T3-Cl-2 (Table 1). As a control, we added 3H-thymidine to uninduced control cultures of each cell line studied. We observed that
