Terminal differentiation in cultured friend erythroleukemia cells

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 <4% of the cells in each culture remained in Gl (Figures 1 B and 1 F; Tables 1A and 1B). Were all the cells which had arrested in Gl in induced cultures in a differentiated state (hemoglobin-containing and therefore benzidine-reactive)? We first used autoradiographic techniques to identify cells in induced cultures which could not synthesize DNA. We prepared cytocentrifuge slides from aliquots of the DS19 culture induced with DMSO for 4 days (Figures 1G and 1 H), as indicated in Figure 2. We took samples after cells had been incubated in medium containing 3H-thymidine for 24-36 hr, before any cellular disintegration had taken place. Then after processing for autoradio-

12 Lid uu A

3

I--I

0

” o” 10

20

30

40

TIME IN 3H-THYMIDINE(Hours) Figure 2. All Induced Cells Synthesize DNA Are Arrested

Which Have Lost the Capacity in the Gl Stage of the Cell Cycle

to

DS19 cells in exponential growth were diluted to 1.5 x lo5 per ml and grown for 4days in medium containing 280 mM DMSO. Cells were centrifuged and resuspended in medium containing 3Hthymidine and no DMSO (Experimental Procedures). (A) Samples were taken for autoradiographic and FMF analysis at intervals during the incubation. Cells were prepared for FMF analysis at each time point in two ways: suspension in hypotonic citrate buffer or suspension in hypotonic Tris-HCI buffer (Table 3). The percentage of cells arrested in Gl at each time point was obtained by averaging values from two or three DNA distribution profiles and from both methods of sample preparation. (6) No increase in cell density was observed in this experiment, since only 2% of the cells were in G2 when 5H-thymidine was added. The percentage of B+ cells was determined by the slide test. Results identical to those described in (A) were also obtained with samples prepared by methanol fixation followed by pancreatic RNAase treatment (Figures 1G and 1H).

graphy, we stained the slides with benzidine for the identification of cells containing heme. The only cells which did not incorporate 3H-thymidine were benzidine-reactive cells. The percentage of total cells that were unlabeled B+ cells equalled the percentage of cells arrested in Gl as determined by FMF analysis (Figure 2). The correlation of these data established that all cells which remained in Gl under conditions of the assay were a distinct class of differentiated Friend cells. These were

Terminal

Differentiation

in Erythroleukemia

Cells

905

hemoglobin-containing cells which had lost the ability to synthesize DNA and traverse S phase. We observed that B+ cells were arrested in Gl under many conditions of induction. We induced three independently derived clones of the Friend cell line with members of three classes of inducing agents: polar solvents, purines and short-chain organic acids. We induced cells with DMSO over a range of concentrations (Figures 1 C and 1 G; Table 1) in Eagle’s medium or Dulbecco’s medium using two different lots of fetal calf serum. Because it might be thought that cells grown in 280 mM DMSO were arrested in Gl due to toxicity, we also induced cells using lower concentrations of DMSO (210 and 112 mM; Figure 1C and Table 1). In all these experiments, the percentage of total cells which were B+ and did not synthesize DNA was equal (SE = 3%) to the percentage of cells arrested in Gl (Table 1).

Cells Nonreactive to Benzidine (B-) Do Not Arrest in Gl in induced Cultures We performed

several

control

experiments

to es-

tablish that the cells identified by FMF analysis as arrested in Gl were the same cells as those identified by autoradiographic analysis as unlabeled B+ cells. It was necessary to show that all B- cells in induced cultures were capable of proliferation in medium containing 3H-thymidine. This was demonstrated by autoradiography using two different cell lines induced separately with several different compounds. We treated the cells for as long as 7 days (Figure 3 and Table 1); all B- cells, however, continued to incorporate 3H-thymidine. We examined in greater detail the capacity of B- cells in one culture (Figure 3C) to carry out extensive DNA synthesis. Autoradiography indicated that all the B- cells in this culture exhibited intense uniform nuclear labeling after 24 hr of incubation in 3Hthymidine. DNA repair might have led to the incorporation of small amounts of 3H-thymidine and resulted in lower grain counts for some of the nuclei. Although grain counts in B- cells varied over a wide range, (50-150% of the mean), however, there were no B- cells with fewer grains than 50% of the mean. The ability of these B-

C

100

80 60

TIME IN 3H-THYMIDINE Figure

3. All B-

Cells in Induced

Cultures

Incorporated

(Hours)

V-l-Thymidine

An exponential culture was diluted to 1.5 x lo5 ceils per ml. (A) For line 745A-11, DMSO was added to a final concentration of 260 mM and growth continued for 6 days, or (B) sodium butyrate was added to a final concentration of 1.5 mM and growth continued for 7 days. This culture was resuspended daily at 3 x lo5 cells per ml in medium containing 1.5 mM sodium butyrate. (C) For line DS19. DMSO was added to a final concentration of 260 mM and growth continued for 4 days. Cytocentrifuge slides were prepared, and the percentage of benzidine-reactive cells was determined for each culture: 90% for A, 31% for B and 62% for C. The cells were then centrifuged and resuspended in the absence of the inducer at 5 x lo5 cells per ml in medium containing 3H-thymidine. After autoradiography, at least 500 cells were counted for each time indicated. The results of two independent observers agreed to within 4%. Autoradiographs exposed for 24 hr gave identical results. Identical results were also obtained for experiment (A) when duplicate slides were washed for 10 min with 5% trichloroacetic acid to remove unincorporated W-thymidine.

Cell 906

cells to carry out net DNA synthesis was confirmed by FMF analysis. After resuspension of the induced culture in medium containing 3H-thymidine, 38% of the cells were B-, and 90% of the total cells were in Gl (Figure 1G). Thus a minimum of 74% of the B- ceils were in Gl at this time. After 24 hr of incubation, these B- cells traversed most of S (Figure 1H). Additional evidence supported the conclusion that all the B- cells traversed S: there was a sufficient number of unlabeled B+ cells to account for all the cells arrested in Gl in induced cultures (compare the last two columns of Table 1). Induced Cells Do Not Arrest in Gl Because of the Labeling Procedure or a General Cytotoxic Effect of the Inducing Agent Additional control experiments were performed to show that cells were arrested in Gl as a result of differentiation, not because of the assay conditions. Cells in induced cultures were not arrested in Gl due to growth in the presence of 3H-thymidine, since identical results were obtained when induced cultures were incubated with vinblastine sulfate, another agent which prevented proliferating cells from dividing and appearing in Gl. As was the case for 3H-thymidine, vinblastine sulfate (0.01 pg/ml), when added to exponential cultures for 8 hr, arrested all the cells at stages after they had traversed most of S. No cells were observed at the Gl position by FMF analysis. When vinblastine sulfate was added to an induced culture, the percentage of nonproliferating Gl cells observed was the same as that obtained using 3H-thymidine (Table 1B). Because FMF analysis indicates that the percentage of cells in S phase is ~5% after both procedures, it is improbable that the large percentage (as high as 72%) of nonlabeled B+ cells oberved on autoradiography (Table 1) are S phase cells unable to synthesize DNA. In the experiments described above, we added 3H-thymidine or vinblastine immediately after we removed the induced cells from medium containing inducing agent. The possibility remained that the intracellular concentrations of the inducing agent in combination with either arrest agent was cytotoxic to some cells. In other experiments, however, we incubated the induced cells for as long as 48 hr in medium without inducing agent before we added 3H-thymidine. These cultures still contained cells arrested in Gl (Table 2). Arrest in Gl is not due to general cytotoxic effects of the inducers. A variant cell line (520a) selected after mutagenesis of line 745A did not differentiate after 6 days of growth in 280 mM DMSO and did not arrest in Gl (Table 1B). It should also be noted that B- cells do not arrest in Gl even when they are present in differentiating cultures. Thus they serve as an internal control.

Table 2. Traversing DMSO

Cell Line

B+ Cells Arrest

Days of Growth in DMSO

Days of Growth after Removal of DMSO

in Gl after

Removal

% Total

Traversing B+

of

Cells

Arrested Bi

DS19

2

0

IO

0

DS19

2

2

10

29

745A-11

4

0

49

11

745A-11

4

2

25

35

An exponential culture was diluted to 1.5 X IO5 cells per ml and grown in the presence of 280 mM DMSO for either 2 or 4 days. Then cells were centrifuged and resuspended in an equal volume of medium without DMSO. At the time of resuspension and after 2 additional days of growth, the cultures were assayed for the percentage of B+ cells by the suspension test and for the percentage of total cells arrested in Gl by FMF analysis (Experimental Procedures). The percentage of traversing B+ cells equals the percentage of total B+ cells minus the percentage of arrested B+ cells.

There Is No Preferential Loss of Either B+ or BCells during the Measurement of Gl Arrested Cells The identification of Gl arrested cells as B+ depended upon an indirect assay, the comparison of FMF and autoradiographic data. It was necessary therefore to show that any cell loss which occurred during either analysis was nonspecific, and that B+ and B- cells were lost with equal probability. The possibility was considered that B+ cells were arrested in phases of the cell cycle other than Gl, but were selectively destroyed and not observed. Accordingly, the percentage of B+ cells was monitored during the incubation with 3H-thymidine. In addition, the number of cells was measured during the incubation of differentiated cultures with 3Hthymidine. There was a 25% increase in cell number during the first 8 hr due to the division of G2 cells. This was expected from the results with control cultures described earlier. All traversing cells had arrested in G2 by 24 hr, and during the next 12 hr, there was less than a 5% decrease in cell number (Figure 2). In eight experiments, the cell number decreased by < 5% during 36 hr of incubation with 3H-thymidine (Table 3A). The percentage of B+ cells decreased by <2%. Thus in these experiments, preferential disintegration of S phase and G2 phase cells did not occur because ~5% of the total cells were lost. No preferential loss of B+ or B- cells occurred during the preparation of slides for autoradiographic analysis. The percentage of benzidinereactive cells in highly induced cultures was identical when measured in two ways: directly in suspension and after the cells had been deposited on slides by cytocentrifugation and processed for au-

Terminal

Differentiation

in Erythroleukemia

Cells

907

Table

3. Quantitative

(A) Recovery

Recovery

of Cells from

of Induced

Induced

Cells

Cultures

Analyzed

for B+ Cells Arrested

in Gl

Procedure

Cells

(1) Resuspension

of induced

(2) Incubation (3) Preparation (a) Nuclear (b)

culture

for 32 hr in medium of samples

for measurement

preparation

Methanol-fixed

(4) Cytofluorograph (B) Comparison

before containing

by suspension

addition

96

3H-thymidine

95

in hypotonic

citrate

buffer

or hypotonic

73

Tris buffer

cells

54

analysis

95

of Methods

for FMF Sample

Preparation

(Hr)

in the Gl Phase

In Nuclear

of the Cell Cycle

(%) In Methanol-Fixed

Preparations

0

71

65

24

35

32

27

33

31

(C) Comparison

of Methods

for Autoradiography

Sample

Transfer

of cells

to slide

by pipette

Cells

Preparation Benzidine-Reactive

Cytocentrifugation

(%)

of DNA content

Cells Time in 3H-Thymidine

of JH-thymidine

5 &i/ml

Recovered

Cells

(%)

Total

‘H-Thymidine-Labeled

62

24

60

22

Exponential cultures of 745A-11 or DS19 cells were diluted to 1.5 X lo5 cells per ml. Cultures were incubated in the presence of 280 mM DMSO for 4, 5 or 6 days. Then cells were centrifuged and resuspended in medium, containing 3H-thymidine and no DMSO. Cells were incubated for 24-32 hr until all cells capable of DNA synthesis were arrested in G2. allowing cells arrested in Gl to be distinguished. (A) Recovery in steps 1-3 was determined from the average of 6-6 hemacytometer measurements. Nuclear preparations were counted using phase-contrast microscopy at 300X with the hemacytometer. The values listed are the average of results from seven separate experiments (standard error = 2.6%). For nuclear preparations, cells were centrifuged and stored at O@ for not longer than 1 hr before resuspension in hypotonic buffer which ruptured the plasma membrane (Experimental Procedures). Analysis was within 10 min of resuspension, since longer periods led to significant loss of nuclei. For cell preparations, cells were centrifuged in plastic tubes, since methanol-fixed cells tended to adhere to glass. Analysis in the cytofluorograph was performed on the same day as rehydration and incubation with pancreatic RNAase. Fluorescence microscopy was used to show that all nuclei in nuclear preparations and in methanol-fixed cells were stained with propidium iodide. Fluorescent cells in a 0.1 ml sample were counted in the cytofluorograph. The percentage of propidium iodide stained cells which were detected by the cytofluorograph was determined by comparison with the number of cells per ml determined with the hemacytometer. Higher readings (12%) were obtained using the hemacytometer compared with either the Coulter counter or the cytofluorograph. This correction factor was used in the calculations for step 4. Cytofluorograph settings were selected for maximal sensitivity in this analysis. The scatter sensor was set to compressed gain to analyze all particules between 4 and 40 ,J in diameter. No more than 5 X lo* cells or nuclei per ml were introduced into the instrument to prevent coincidence error. The value listed in step 4 is the average from five separate experiments. (B) FMF samples were prepared by suspending cells in hypotonic citrate buffer or in isotonic buffer containing 50% methanol (Experimental Procedures). The values shown are the average of two or three measurements of each sample. This experiment was repeated twice with identical results. (C) In one protocol, cells were pipetted onto slides, air-dried and then fixed in methanol. Alternatively, cells were deposited on slides using the cytocentrifuge. An average of 700 cells (six separate slides) prepared by either protocol was analyzed after benzidine staining, followed by autoradiography after exposure for 24 hr.

toradiography (data shown in Experiment Procedures section on determination of benzidine-reactive cells and Table 3). Another potential source of error could occur during FMF analysis. Cells in one stage of the cycle might be particularly susceptible to disintegration during passage through the cytofluorograph. To eliminate this possibility, we determined the proportion of the cells contributing to the FMF profile. We first determined the cell concentration

using a hemocytometer. We then determined the number of cells contributing to the FMF profile by counting a fixed volume determined electronically (see legend to Table 3). This demonstrated that under optimal conditions, fluorescence from >95% of the cells could be detected. These conditions were used for the experiments summarized in Figure 2 and Table 38, in which <5% of the cell population was excluded. We prepared samples for FMF analysis by meth-

Cell 908

anol fixation and RNAase treatment in all but one of the experiments described in this study (Table 1). Only 54% of the cells were recovered because of the large number of steps in sample preparation (Table 3A). Possibly cells in S or G2 were preferentially lost during this procedure, producing an apparent enrichment of Gl cells. To test this possibility, a method of FMF sample preparation was used which gave a better recovery of ceils. We suspended cells in hypotonic (0.1%) sodium citrate buffer containing propidium iodide. Of the 3H-thymidine-labeled, induced cells, we recovered 73% upon suspension in citrate buffer, and 95% of these cells contributed to the DNA distribution pattern. Both methods gave identical DNA distribution patterns when we analyzed the following

(*

B

MEDIUM

CHANGED DAILY

TRAV ‘ERSING

- dI

ARRESTED

i - /I! -/

I 1( -2 TIME IN DMSO Figure 4. Kinetics B+ cells

of Appearance

of Traversing

I 4 (Days)

I 6

B+ and Arrested

(A) DS19 cells in exponential growth were diluted to 1.5 x lo5 cells per ml in medium containing 260 mM DMSO. (B) DS19 cells in exponential growth were diluted to 1.5 x lo5 cells per ml in medium containing 210 mM DMSO. Cells were centrifuged and resuspended daily at between 2 and 6 x lo5 cells per ml. The percentage of B+ cells varled by an average of 1% before and after resuspension. Approximately 92% of cells were recovered upon resuspension. Cells were removed from each culture daily and incubated in the presence of 3H-thymidine for 24-30 hr. This was sufficient time to allow all proliferating cells to move out of the Gl stage of the cell cycle and to be arrested in G2. At this time, one aliquot was taken for FMF analysis to identify the percentage of cells arrested in Gi Another aliquot was taken to measure B+ cells as a percentage of the total B+ cells by the suspension test. The percentage of traversing B+ cells was the percentage of total B+ cells minus the percentage of cells arrested in Gl as measured by FMF analysis. This calculation could be made because all cells arrested in Gl were B+ under the experimental conditions used in these studies, as discussed in Results.

samples: uninduced cells (data not shown), cells induced with DMSO and DMSO-induced cultures incubated with 3H-thymidine to distinguish cells capable of DNA synthesis from cells arrested in Gl (Table 38). These results strongly suggest that the appearance of nonproliferating cells in Gl in induced cultures was not an artifact of sample preparation. We did not use suspension of cells in hypotonic citrate buffer extensively in these studies for two reasons. Cell recoveries of 70-75% required a very rapid analysis of samples, as discussed further in Table 3. In addition, RNA may remain associated with the nuclear preparations made by the citrate procedure and contribute to their fluorescence. Differentiated Friend Cells Which Had Lost Proliferative Capacity Had a DNA Content per Cell Consistent with Arrest in the Gl Phase of the Cell Cycle FMA analysis indicates that terminally differentiated cells arrest in Gl (Figure 1). These assays, however, are based on the binding of a fluorescent dye to DNA. We considered the possibility that differentiated cells bind less fluorescent dye than undifferentiated cells and may be at the same channel position as cells in Gl as a result of an artifact. We therefore compared the DNA content and fluorescence of induced cultures with a series of standards. The cultures assayed consisted of DS19 cells in exponential growth (DS19), arrested predominantly in Gl by growth in the absence of an essential amino acid (Leu-), arrested predominantly in G2 (3H-T), arrested in Gl and G2 (25°C) and arrested with an average cellular DNA content of 3.6C (Vinb). In addition, we analyzed a triploid Friend cell line (520a). We determined the DNA content of the cells in each of these six cultures by the diphenylamine test, and also calculated it using FMF data as described in the legend to Figure 5. There was a linear relationship between DNA content measured by FMF analysis or by biochemical determination (Figure 5). This correspondence between the intensity of fluorescence and DNA content was also valid for induced cells. Two cultures of cells induced by DMSO were prepared. Differentiated cells arrested in Gl were isolated from one culture as described in the legend to Figure 5. After growth in the presence of 3H-thymidine, at least 75% of the cells which had proliferated and were in G2 were removed from the culture by filtration. By this method, we purified a population consisting predominantly of terminally differentiated cells (DMSO-F). As a control, we did not fractionate the second DMSO-induced culture (DMSO). The unfractionated culture contained B- and traversing B+ cells in addition to terminally arrested B+

Terminal 909

Differentiation

in Etythroleukemia

Cells

linear relationship established for the noninduced cells (Figure 5). Direct chemical analysis therefore confirmed the FMF assignment of the Gl phase of the cycle for suspension cultures of induced Friend cells at their terminal stage of differentiation.

DNACONTENTBY DIPHENYLAMINE REACTION(pg/CELL) Figure 5. DNA Content as Measured by Flow Content as Determined

of Both Induced and Noninduced Microfluorometry Is Proportional by the Diphenylamine Reaction

Cells to DNA

The DNA content per cell was determined using FMF analysis (ordinate) and using the diphenylamine reaction (abscissa). FMF samples had been prepared by suspension in isotonic buffer containing 50% methanol, followed by incubation with pancreatic RNAase (Experimental Procedures). Because no culture contained cells in only one stage of the cell cycle, the proportion of cells in Gl, S and G2 + M was determined from their DNA distribution profiles using a modification of the computer-fitted curve method of Dean and Jett (1974) (Experimental Procedures). The average DNA content per cell in each culture was expressed as a function of C. the haploid DNA content of DS19 cells in Gl. While cells in Gl and G2 had DNA contents of 2C and 4C, respectively, cells in S phase were assigned a value of 3C. With the exception of one measurement made with the triploid Friend cell line 520a in exponential growth (520a). all experiments were performed on cultures of DS19 as follows: (leu-) after washing once with an equal volume of leucine-deficient medium, cells were grown for 46 hr in leucine-deficient medium supplemented with 15% dialyzed fetal calf serum and 30 mg/l glutamine (Experimental Procedures); (3H-T) growth for 21 hr in 5 &i/ml JHthymidine; (25”) incubation for 3 days at 25°C; (Vinb) growth for 8 hr in the presence of 0.01 pg/ml vinblastine sulfate; (DS19) in exponential growth; (DMSO) growth for 4 days in the presence of 280 mM DMSO (73% B+); (DMSO-F) growth for 5 days in 280 mM DMSO (72% B+) and then treated to isolate a culture of terminally differentiated cells arrested in Gi. First cells were incubated with ‘H-thymidine to arrest all proliferating cells in G2 (Experimental Procedures). Then the cells blocked in G2 (48% of the total cells) were allowed to lyse by continued incubation (48 hr). Finally, cells from the culture (4.5 x 105/ml) were filtered (recovery = 36%) through 4.5-4.7 F polycarbonate filters (lot 5484A132, 142 mm, Nucleopore Corp., Pleasanton, California) to remove aggregated and disintegrating cells, and to leave a population predominately (80%) in Gl and 99% B+ by the suspension test. Cells subjected to leucine deprivation were similarly filtered to isolate a population of uninduced cells predominately (86%) in Gl. Unaggregated cell suspensions were used to count the cell number, and the DNA content per cell was determined by the diphenylamine reaction on cell lysates (Experimental Procedures). The standard error in the diphenylamine test for samples below 8 pg of DNA per cell was 0.35 pg per cell, and 0.7 pg per cell for samples with a DNA content above 8 pg per cell. The average variation between four to six measurements of cell number was 2.9%.

cells; 75% of the total cells were in Gl . For these induced cultures, the average DNA content determined by both methods of analysis followed the

Friend Cells Do Not Have to Cease Division to Differentiate The number of B+ cells always exceeded the number of cells arrested in Gl (Table 1). Some of the B+ cells must therefore retain the capacity to synthesize DNA and traverse S phase. Cultures of either 745A-11 cells or DS19 cells grown in 280 mM DMSO contained B+ cells which incorporated 3H-thymidine (Figures 3A and 3C). We considered the possibility that B- cells incorporated 3H-thymidine and then differentiated and became benzidine-reactive during the incubation with 3H-thymidine. We ruled out this alternative because the number of B+ cells did not increase during incubation of cells from these induced cultures with 3H-thymidine (Figure 2; experiment also shown in Figure 3C). Identical results were found for 745A-llinduced cultures. It should be noted that we used highly induced cultures grown to saturation density and containing few B- cells to demonstrate this point. Incubation of committed cells in 3H-thymidine does not inhibit the synthesis of hemoglobin. We also found B+ cells capable of DNA synthesis (traversing B+ cells) in cultures induced with different compounds. In three similar experiments, we grew cells (745A-11) for 4 days in either 10 mM l-methyl 2-pyrrolidinone or 5 mM hypoxanthine, or for 7 days in 1.5 mM sodium butyrate. We measured the percentage of labeled B+ and Bcells by autoradiography and benzidine-staining after 24-36 hr of incubation in medium containing 3H-thymidine. In each culture, all the B- cells, but only a portion of the B+ cells, became labeled: 40% in the culture induced with sodium butyrate (Figure 3B), 60% in the culture induced with lmethyl 2-pyrrolidinone and 50% in the culture induced with hypoxanthine. B+ Cells Arrested in Gl Arise from the Further Differentiation of Traversing B+ Cells Arrested B+ cells arise from the further differentiation of traversing B+ cells and do not arise directly from B- cells, as shown by the following experiments. We measured the kinetics of appearance of traversing B+ cells and arrested B+ cells during the differentiation of DMSO-induced cultures (Figure 4), demonstrating that during differentiation, traversing B+ cells always appear at earlier times than arrested B+ cells; that l-2 days after addition of DMSO, all the B+ cells are traversing the cell cycle and none are arrested in Gl; and that 4 days after addition of DMSO, the proportion of arrested

Cell 910

B+ cells rises as the proportion of traversing B+ cells decreases. We obtained similar results with either DMSO or sodium butyrate as the inducing agent, with either DS19 or 745-11 cells, or under different conditions of induction (Figure 4). In addition, DMSO was removed from cultures which contained few arrested B+ cells (Table 2). We incubated the cultures for an additional 2 days and then assayed them for the presence of arrested B+ cells. Possibly, traversing B+ cells in each culture would continue to differentiate and become arrested or, alternatively, removal of DMSO would prevent further differentiation. To distinguish between these possibilities, we performed the following experiments. In one culture of DS19 ceils, no arrested B+ cells were present when DMSO was removed, but after 2 days of incubation, 28% of the total cells were arrested B+ cells. We performed a similar experiment with a highly differentiated culture of 745-l 1 cells. Again, the percentage of arrested B+ cells increased during incubation without DMSO, while the percentage of traversing B+ cells decreased (Table 2). Taking into account the increase in cell density (40%) which occurred during incubation without DMSO, there was an increase in the number of arrested B+ cells of about 4 x lo5 cells per ml. The most probable source for these cells was the population of traversing B+ cells, which decreased by about 2 x lo5 cells per ml. (Note that there is not a complete conversion of traversing B+ cells to arrested B+ cells, since traversing B+ cells can divide and give rise to other traversing B+ cells.) These data again suggest that traversing B+ cells and not B- cells are the direct precursors of arrested B+ cells. The traversing B+ cells in these cultures continued to differentiate and produced arrested B+ cells in the absence of DMSO. Discussion Many biochemical and morphological changes characteristic of erythroid differentiation occur during the differentiation of Friend cells: synthesis of heme and globin (Friend et al., 1971; Boyer et al., 1972), increased levels of globin mRNAs (Ross, lkawa and Leder, 1972; Preisler et al., 1973), increased levels of heme biosynthetic enzymes (Sassa, 1976) and acetylcholinesterase (Conscience et al., 1977), and decreased activities of purine biosynthetic enzymes (Reem and Friend, 1975). We wished to compare differentiating Friend cells to normal erythroblasts by another criterion. Erythroid precursors are known to divide a limited number of times as they accumulate hemoglobin, as shown by both in vivo and in vitro studies (Weintraub, Campbell and Holtzer, 1971; Cooper et al., 1974; McLeod et al., 1974; lscoveand Sieber,

1975). Do the growth regulatory mechanisms limiting the proliferation of erythroblasts in vivo function normally in cells transformed by the Friend virus complex? We questioned whether Friend cells continue to divide in suspension culture after they are committed to differentiation, and whether differentiating Friend cells arrest in a specific phase of the cell cycle as do some types of untransformed, terminally differentiated cells. While these studies were in progress, Gusella et al. (1976) reported that Friend cells exposed for 24-72 hr to DMSO divided at most 4 times after plating in plasma clots in the absence of an inducing agent. Our studies with flow microfluorometric techniques show that differentiating Friend cells have a limited proliferative capacity when tested directly in suspension culture. We extended this analysis to four different inducing agents, three different cloned lines derived from 745A and two additional erythroleukemia cell lines. All benzidinereactive cells traverse at least one S phase, and we estimate that they complete no more than three divisions in suspension culture before losing the capacity for DNA synthesis. This was estimated from the generation time (14 hr) of a culture consisting of three types of cells: B+ arrested cells (15%) which do not divide, B- cells (35%) which have a generation time of 11 hr when grown in DMSO and traversing B+ cells (50%) whose generation time was calculated from the above information to be about 16 hr. From their generation time of 16 hr and the 36 hr interval after which they give rise to terminally arrested cells, we estimate that most B+ cells traverse the cell cycle no more than 3 times. Terminally differentiated Friend cells arrest only in the Gl phase of the cell cycle. There was no evidence for loss of DNA in these cells. Terminally differentiated cells had the same DNA content as uninduced cells in Gl when compared by two criteria: by direct measurement using the diphenylamine reaction and by equal binding of propidium iodide determined from flow microfluorometry on cells fixed with methanol and treated with pancreatic RNAase. When cells are suspended directly in propidium iodide and not incubated with RNAase, and cells in the Gl phase of the cycle are compared, induced cells exhibit less fluorescence than uninduced cells (Terada et al., 1977; E. A. Friedman and C. L. Schildkraut, manuscript in preparation). Since propidium iodide binds to RNA as well as DNA, a possible explanation for this decrease in fluorescence is the decrease in cellular RNA content after induction (Sherton and Kabat, 1976). In this study, we developed techniques for the isolation of terminally differentiated cells arrested in Gl. Cells in the culture capable of proliferation

Terminal 911

Differentiation

in Erythroleukemia

Cells

were blocked by G2 by incorporation of 3H-thymidine. We allowed these cells to lyse by continued incubation at 37C, and removed the swollen and disintegrating cells by filtration. The DNA content of terminally differentiated cells isolated in this manner was 2C (C is the haploid DNA content of DS19 cells in the Gl phase of the cell cycle), indicating that no significant DNA loss (< 5%) occurred during differentiation of Friend cells. These purified populations of terminally differentiated Friend cells *are also being used to study chromatin condensation. In addition to their common biochemical characteristics, Friend cells behave like untransformed murine erythroblasts in their limited proliferative capacity after commitment to differentiation. Differentiated Friend cells do not (< 1%) extrude their nucleus, in contrast to murine erythroblasts. One line of Friend virus-transformed mouse cells, however, has been reported to extrude its nucleus when maintained in a diffusion chamber in the peritoneal cavity of a mouse (Furusawa, lkawa and Sugano, 1972). Erythrocytes of species which do not extrude their nucleus are considered from their DNA content (Mirsky and Ris, 1949) to be in the Gl phase of the cell cycle. Friend cells are observed to be arrested in Gl at a stage prior to nuclear extrusion. Thus it is possible to speculate that one stage of murine erythropoiesis in vivo may involve arrest in the Gl phase of the cell cycle. The observations presented in this report are consistent with the following model. Friend erythroleukemia cells proceed through several steps during differentiation. In the initial stages, cells committed to differentiation have begun to synthesize heme (cells are Et+) but still retain the ability to synthesize DNA. When cells have begun to differentiate in suspension culture, they no longer require the continued presence of the inducing agent to progress to the terminal stage. At this stage, their ability to traverse the cell cycle is lost, and these cells arrest in Gl without detectable loss of DNA. Experimental

Procedures

Cell Cultures The Friend leukemia cell lines used in most of these studies were 745A (obtained from Dr. C. Friend) and three of its subclonesDS19 (from Dr. P. Marks), 745-11 and 520a (from Dr. A. Skoultchi). Line 520a is a variant erythroleukemia cell line (derived by mutagenesis of line 745A) which is unable to synthesize hemoglobin in response to DMSO (P. Lin and A. Skoultchi. manuscript in preparation). Friend erythroleukemia lines F4-1 and T3-Cl-2 were obtained from Dr. W. Ostertag and Dr. Y. Ikawa, respectively. All cell growth and incubations were at 37°C in a CO* incubator. Stock cultures were usually fed daily and always maintained in exponential growth. Tests for mycoplasma were consistently negative. Cultures were replaced about every 6 months from frozen stocks.

Media Cells were grown in a modified Eagle’s spinner medium lacking calcium and containing 10 mM sodium phosphate and nonessential amino acids, formulated by this laboratory and prepared by Schwarz/Mann. Dulbecco’s modified Eagle’s medium (Gibco) was also used for some experiments. Medium was supplemented with 7.5% fetal calf serum, lot C260619 (Microbiological Associates) or lot 455630 (Flow Laboratories). Leucine-deficient modified Eagle’s medium formulated by this laboratory was prepared by Schwarz/Mann. Fetal calf serum (200 ml) was dialyzed against five changes of 2 liters each of Earle’s balanced salts solution and used only in leucine deprivation experiments. Chemicals Dimethyl sulfoxide was obtained from Mallinckrodt: l-methyl 2pyrrolidinone, acetaldehyde, dephenylamine and 3,3-dimethoxybenzidine from Eastman: benzidine dihydrochloride from Sigma; Wright-Giemsa stain from Harleco; hypoxanthine from Mann; propidium iodide and pronase from Calbiochem. A 150 mM stock of sodium butyrate was prepared by neutralizing butyric acid (Fisher). The ‘H-thymidine was obtained from Schwarz/Mann (11 Ci/mM) and New England Nuclear (20 Ci/mM), and supplied in aqueous ethanol. The ethanol in the 3H-thymidine preparation did not by itself affect cell growth. Incubation of Cells in 3H-Thymidine For all the labeling experiments described in this study, cells were centrifuged, then gently resuspended in modified Eagle’s medium containing 5 pCi/ml +l-thymidine and 15% fetal calf serum. The medium was equilibrated in the CO, incubator to 37°C (pH 7.4) before the addition of cells. Cells were centrifuged at 160 x g for 3 min and resuspended at approximately 5 x 105/ ml. These conditions were chosen to allow DNA synthesis in all proliferating cells. When cells from induced cultures were incubated with 3H-thymidine. the inducing agents were omitted from the resuspension medium. Benzidine-reactive (B+) cells, however, could synthesize DNA even when 260 mM DMSO was present in the resuspension medium (Table 1). Cells were not treated with inducing agents long enough to cause cell disintegration. Determination of Benzidine-Reactive Cells Suapenslon Test Aliquots (0.5 ml) of cultures containing from 2 x lo5 to 1.2 x IO6 cells were pipetted into 35 mm tissue culture dishes (Falcon). Samples were taken in duplicate, and the dishes were coded so that the identities of the samples were only known after the benzidine-reactive cells had been counted. Benzidine dihydrochloride (0.1 g) was dissolved in 50 ml of dilute acetic acid (1.5 ml glacial acetic acid diluted to 50 ml with water) and stored at 4°C in a brown bottle. Just before use, a 0.5 ml portion of the benzidine solution was dispensed into a test tube at 4°C and 5 ~1 of 30% H& were added followed by immediate mixing. This mixture was immediately added to the dish of cells which was swirled rapidly during the addition. After 25 min at room temperature, the percentage of blue (benzidine-reactive) cells reached a constant value and was determined at a magnification of 300 with the aid of an eyepiece micrometer grid. About 200-300 cells were counted per dish with a standard deviation of 4% between duplicate samples. As a control, it was determined that the percentage of benzidine-reactive cells (B+) was additive in a mixture of cells prepared from induced and uninduced (1% B+) cultures. This procedure has been modified from that of Orkin. Harosi and Leder (1975). Slide Test Aliquots (0.5-1.0 ml) containing 2-3 x lo5 cells per ml were introduced into each chamber of the cytocentrifuge (Shandon Eliott Cytospin). Cells were deposited on acid-washed slides by centrifugation at 400 rpm for 5 min. After air-drying, the cells were fixed in methanol for 3 min, then stained 5 min in a solution of 2% 3,3’-dimethoxybenzidine in methanol. Slides were then

Cell 912

transferred without washing into a 3% t-&O, solution in 50% ethanol made immediately before use. After 3 min. slides were removed, washed thoroughly in distilled water and counterstained for 5 min with 2% Wrights-Giemsa stain in 10 mM sodium phosphate buffer (pH 5.9). The cytoplasm of cells that were B+ was gold or bronze, while that of B- cells was blue. This test was only used for autoradiography studies. Because of its greater sensitivity, the suspension test was used routinely instead of the slide test to measure the total percentage of benzidine-reactive cells. Both tests gave similar results only after cells were grown for 4 or more days in the presence of DMSO. After 745A-11 cells were grown for 6 days in 260 mM DMSO, the percentage of B+ cells on slides or in suspension was, respectively, 93% and 93%, and 66% and 90%, for two separate experiments. The respective data for DS19 cells grown for 4 days In 260 mM DMSO were 60% B+ and 62% B+. and in a second experiment, 62% B+ and 66% B+. Slide Test for Autoradiography After the benzidine reaction, but before counter-staining, the slides were coated with Kodak NTB-2 nuclear track emulsion and stored in light-tight boxes at 4°C. Slides were exposed for 24 hr unless otherwise indicated and developed in 50% Dektol (Eastman Kodak) for 4 min at room temperature. The slides were then counter-stained as described above. Labeled cells exhibited at least twice as many grains over their nuclei as were counted in an equal area of emulsion which did not cover any cells. Cells were analyzed by two observers with a standard deviation of 3% between identical samples. The data shown (Figure 2 and Table 1) are an average of the analysis of at least 500 cells. We are grateful to Dr. Paul Marks for the details of this method. Flow Microfluorometry instrumentation Fluorescence from individual cells was measured by a cytofluorograph (Model 4602A, Ortho Instruments, Westwood, Massachusetts) using an argon ion laser at 466 nm. Data were stored in a multichannel distribution analyzer (Model 2100). Polaroid photographs were taken of an oscilliscope display on the cytograph (Model 6300A). The percentage of cells in each stage of the cell cycle was calculated using a modification of the computer-fitted curve method of Dean and Jett (1974). The percentage of cells in S phase by this method (67%) agreed with the percentage (66%) measured after autoradiography of a culture labeled for 15 min with ‘H-thymidine (5 &i/ml). Fluorescent Staining Procedure All steps were at 4°C except where indicated. Cells were centrifuged, then suspended at 5 x lo6 cells per ml in 2 ml of Earle’s buffered salts solution lacking calcium and magnesium (ESS) and containing 50% methanol. The cells were stored at this stage for at least 30 min until the remainder of the procedure and the FMF analysis could be completed on the same day. The cells were rehydrated by centrifugation and resuspension in ESS solutions containing decreasing concentrations of methanol (25 and 12%) and resuspended at 2 x lo6 cells per ml in 0.2 M sodium phosphate buffer (pH 7). Pancreatic RNAase (RASE. Worthington; preincubated for 10 min at 90°C to inactivate DNAase) was added to a final concentration of 1 mg/ml, and incubation was at 37°C for 30 min. The reaction was complete after 6 min, and further incubation (for as long as 2 hr) did not result in a change in the fluorescence per cell or in the DNA distribution profile, suggesting that no significant degradation of DNA occurred during the incubation. When cells were resuspended in a buffer of lower ionic strength [3 mM sodium citrate (pH S)] instead of 0.2 M sodium phosphate buffer and incubated with RNAase, identical results were obtained. After centrifugation and resuspension at 5 x lo5 cells per ml in ESS, propidium iodide (2 mg/ml in aqueous solution) was added to a final concentration of 50 pg/ml. Concentrations of propidium iodide between 25 and 100 pg/ml gave identical DNA distribution patterns, as also reported by Fried, Perez and Clarkson (1976). This procedure is as described by Crissman and Steinkamp (1973) and Krishan (1975).

Alternatively, propidium iodide was introduced into the nucleus by rupturing the plasma membrane. Cells were suspended in a hypotonic buffer. These ruptured cells are designated as nuclear preparations. After centrifugation out of the culture medium, 5 x IO5 cells were resuspended in 1 ml of 0.1% sodium citrate buffer (pH 6) containing 50 pg/ml of propidium iodide (Krishan. 1975). In an alternative procedure, cells were resuspended in IO mM Tris-HCI buffer (pH 7.4) containing 3 mM CaCI,. NaCl was then added to give a final concentration of 0.15 M to restore isotonicity. Propidium iodide was added to a final concentration of 50 pg/ml. Measurement of DNA Concentration Cells were centrifuged, washed once with an equal volume of ESS and then resuspended at approximately 5 x lo6 cells per ml. Several determinations of the cell number were made using a hemacytometer (to check for aggregation) and a Coulter counter (agreement within 10%). The cell suspension was made 0.1% in sodium lauryl sulfate, pronase was added (0.5 mglml) and the lysates were incubated at 37°C for 1 hr. The DNA concentration in lysates was determined using the diphenylamine reaction as modified by Burton (1956). After 16 hr of incubation at 33°C. the difference in optical density at 595 nm and 650 nm was measured in a Zeiss PMQII spectrophotometer. The assay was proportional between 6 and 60 nmole of deoxyadenosine monophosphate (used as a standard) per ml of reaction mixture. All data were the average of at least four duplicate measurements using two or three different sample volumes. The absence of interfering compounds in the cell lysates was determined in two ways. First, when dAMP was mixed with aliquots from each cell lysate. the resulting absorbance (after the diphenylamine reaction was complete) was equal to the sum of the absorbances of dAMP and the aliquot of the cell lysate tested separately. Second, when different concentrations of cell lysates were measured using the diphenylamine reaction, the absorbance values extrapolated to zero as did the dAMP standard. Acknowledgments We are grateful to Ms. Danuta Hornig for excellent technical assistance. These studies were supported by grants from the NIH. C.L.S. is a recipient of a Hirsch1 Career Scientist Award. E.A.F. was supported by an NIH postdoctoral fellowship from the National Cancer Intitute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 16 U.S.C. Section 1734 solely to indicate this fact. Received

June

22,1977;

revised

September

6, 1977

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