EXPERIMENTALCELL
RESEARCH
187,4-10
(1990)
Reversible Gl Arrest in the Cell Cycle of Human Lymphoid Cell Lines by Dimethyl Sulfoxide MASAJI SAWAI,* Kozo TAKASE,~ HIROBUMI TERAOKA,* AND KINJI TSUKADA*T’ *Department of Pathological Biochemistry, Medical Research Institute, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101, Japan; and TDepartment of Pediatrics, Faculty of Medicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113, Japan
the course of our studies on cell proliferation and differentiation, we investigated the effect of DMSO on the cell cycle of lymphoid cell lines by flow cytometry. In this paper, we describe that DMSO induces reversible Cl arrest in the cell cycle of lymphoid cell lines without any detectable differentiation. DMSO seems to be a useful agent for elucidating the regulatory mechanisms of cell differentiation and the proliferation of lymphoid cells.
Proliferation of human B- and T-lymphoid cell lines including Raji and Akata cells was found to be arrested at the Gl stage in the cell cycle by dimethyl sulfoxide (DMSO). The Gl arrest by DMSO occurred gradually and was completed within 96 h after addition of 1.5% DMSO concomitantly with a decrease in growth rate. Progression of Gl-phase cells containing a larger amount of RNA into S-phase began 9-12 h after removal of DMSO. At 24 h, the DNA pattern of the cell cycle was similar to that of nontreated log-phase cells. The expression of six differentiation markers on the lymphoid cells was not appreciably changed by treatment with DMSO. On the other hand, the expression of transferrin receptor (one of the growth-related markers) on Gl-phase cells 96 h after addition of DMSO was decreased to one-fourth that on log-phase cells and was completely restored 24 h after removal of DMSO. These results indicate that DMSO, known as an inducer of differentiation in several myeloid cell lines, acts as an agent inducing Gl arrest in the cell cycle of the lymphoid cells. 0 1990 Academic Press, Inc.
MATERIALS
Materials. DMSO (gas chromatography grade) was purchased from Wako Pure Chemicals (Osaka, Japan); propidium iodide (PI), from Sigma (St. Louis, MO) and Calbiochem Behring (La Jolla, CA); acridine orange (AO), from Sigma; ribonuclease, from Worthington Diagnostic Systems (Freehold, NJ); neutral mouse serum immunoglobulin (Ig) and monoclonal antibodies (mAbs) Bl (CD20), B2 (CD21), B3 (CD22), B5, B6 (CD23), and PCAl, from Coulter Immunology (Hialeah, Fla); anti-transferrin receptor mAb (CD71), from Becton Dickinson (Mountain View, CA); human Ig, from Green Cross (Osaka, Japan); fluorescein isothiocyanate (FITC)-conjugated F(ab’), of sheep anti-mouse IgG antibody (H chain- and L chain-specific), from Silenus (Hawthorn, Australia). All other reagents were of the highest purity available. Cell culture. Human B-cell lines (Raji [5], Akata [6], BJAB [7], and NC-37 [8]) and a T-cell line (MOLT-4 [9]) were grown as a suspension culture in RPMI-1640 medium (GIBCO, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (KC Biological, Lenexa, KS), 100 IU/ml penicillin, and 100 pg/ml streptomycin at 37°C in a humidified atmosphere of 5% C02/95% air. Cells were seeded at an initial density of 4 X lo5 cells/ml and maintained at a density below 1.2 X 10s cells/ml (log-phase culture). To obtain DMSO-treated cells, we incubated the cells with 1.5% DMSO (v/v) for 96 h, unless otherwise specified. For reversibility experiments, the DMSO-treated cells were washed twice with phosphatebuffered saline (PBS), seeded at 4 X lo5 cells/ml, and grown without DMSO. Cells cultured for 5 days without dilution were harvested after an additional 2-3 days, and were referred to as stationary-phase cells. Cell viability was determined by the trypan blue exclusion test [lo]. DNA synthesis was determined by [3H]Thymidine incorporation. incorporation of [6-3H]thymidine (Amersham Japan). Briefly, cells were incubated with [6-3H]thymidine (1 &i/ml culture) for 60 min at 37°C. They were then washed with PBS and completely dissolved in 0.1 N NaOH followed by precipitation with 7% trichloroacetic acid/ 1% sodium pyrophosphate. The precipitate dissolved in 0.1 N NaOH was spotted on a 3MM paper, washed extensively with 7% trichloroacetic acid/l% sodium pyrophosphate, and counted in a liquid scintillation counter.
INTRODUCTION Dimethyl sulfoxide (DMSO) is well known as an inducer of differentiation in several myeloid cell lines, such as mouse erythroleukemia (Friend) cells [l] and human promyelocytic leukemia cells (HL-60) [2]. The relationship between cell differentiation and proliferation, however, has not necessarily been fully understood, several lines of evidence indicate that a transient growth arrest is accompanied by myeloid cell differentiation [ 3,4], but it remains obscure whether growth arrest is essential for cell differentiation. In contrast to that on myeloid cells, little attention has been focused on the effect of DMSO on lymphoid cells, because DMSO has been believed to be ineffective in differentiation of lymphoid cells. During ’ To whom reprint requests should be addressed at: Department of Pathological Biochemistry, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101, Japan.
0014-4827/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
4
REVERSIBLE qoes -
Gl ARREST
BY DMSO
a
5905 -
1
b
2103 -
0
&---A 100
0
200
256
Time
(h)
FIG. 2. Effect of DMSO on proliferation of Raji cells. Cells were initially seeded at 4 X lo5 cells/ml and diluted at a concentration of 4 X 10s cells/ml every day in the presence of 0% (0), 0.5% (A), 1% (O), 1.5% (O), 2% (A), or 2.5% (m) DMSO.
Distribution of cellular DNA content was determined PI staining. by hypotonic PI staining [ 111. Briefly, 1 X 10’ cells were suspended in 1 ml of PI solution (50 pg/ml of 0.1% sodium citrate and 0.2% Nonidet P-40) and analyzed with a FACS440, a FACScan (Becton-Dickinson), or an EPICS C (Coulter). Excitation was carried out with the 488-nm line of an argon ion laser operating at a continuous output of 200 mW. Cell cycle analysis by DNA distribution was computed by the polynomial method reported by Dean [ 121.
TABLE Effect
1
of DMSO on Cell Cycle Configuration in Lymphoid Cell Lines
I
vso -
Percentage of cells in the phase of
e
Cell line
Treatment with 1.5% DMSO
WI5 -
0.
0
I IO0
Relative
\
ZOO
Raji
-
Akata
+ +a -
BJAB
+ -
NC-37
+ -
MOLT-4
+ -
256
DNA content
FIG. 1. Cell cycle configuration of DMSO-treated and nontreated Raji cells. Log-phase cells (a) were obtained as described under Materials and Methods and then maintained at a density of below 1.2 X lo6 cells/ml in the presence of 1.5% DMSO for 24 (b), 48 (c), 60 (d), and 96 h (e). Cell cycle analysis was carried out by a PI staining method.
+ a Data from Fig. le.
Gl
S
G2/M
34.5 89.0 88.5 26.1 83.2 42.5 79.0 59.8 73.5 49.7 92.9
50.6 7.1 5.4 60.9 12.3 42.9 14.0 27.3 11.9 41.3 6.2
14.9 3.9 6.1 13.0 4.5 14.6 7.0 12.9 14.6 8.9 0.9
6
SAWAI
Log-phase Raji cells
Gl-arrested 670
a
Raji cells
ET AL.
Log-phase Akata cells
Gl-arrested
Akata cells
a
283
228 L
0k 281
b
b
114
0
382
C 169
0 l-/L
0h 223
285
d
d
133
0k
0 -i’_
528
871 e
e
=LlL 0
=i--A0 393
197
214
0
0
:;[A , , ljgh , , jgi. , , rjgh , , 0
100
200
Fluorescence
266
intensity
0
100
(channel
200 266
number)
0
100
200
256
Fluorescence intensity
0
100
(channel
200
253
number)
FIG. 3. Expression of differentiation markers on log-phase and DMSO-treated B-lymphoid cells. Log-phase cells and the cells exposed to 1.5% DMSO for 96 h were obtained as described under Materials and Methods. Raji (A) and Akata cells (B) were treated with mouse serum IgG (a), Bl (b), B2 (c), B3 (d), B5 (e), B6 (f) or PCAl (g) and then stained with FITC-conjugated F(ab’), of sheep anti-mouse IgG antibody. Surface immurwphenotyping. Cells were washed once with PBS and suspended in RPMI-1640 medium containing 10% FBS to a density of 2 X lo6 cells/ml. A 0.1.ml aliquot of the cell suspension was placed in a plastic tube and then treated with human Ig at a final concentration of 15 mg/ml at 4°C for 15 min to reduce the nonspecific binding of antibody via Fc receptors [13]. The cells were incubated
with an appropriate amount of mAb at 4’C for 30 min and then washed with PBS at 4°C. The cells were further stained with FITC-conjugated F(ab’)* of sheep anti-mouse IgG (H and L chain-specific) antibody. Fluorescence-stained cells were analyzed with a FACS440 (BectonDickinson). The background fluorescence was determined by incubating cells with neutral mouse serum IgG instead of the specific mAb.
REVERSIBLE
RaJi cells
Gl ARREST
Akata cells
BY DMSO
7
mixed with 0.5 ml of PI solution (30 rg/ml PBS) and kept at 0°C for 60 min. PI/F’ITC two-color flow cytometry was carried out with a FACS440 equipped with a 488-nm argon ion laser and an optic system with a 560-nm dichroic mirror, a 530-nm band-pass filter, and a 6.20. nm long-pass filter. The expression of TFR on the cells was evaluated on the FITC fluorescence intensity distribution derived from integration of the columns at the position of Gl phase in PI fluorescence. Mean fluorescence intensity corresponding to the surface TFR expression was estimated through a curve fitting method with simplex optimization [15].
I::
RESULTS
L:-100
Relative
100
200
DNA content
FIG. 4. DNA configuration of Raji and Akata cells released from Gl arrest. DNA content in Raji (left panels) and in Akata cells (right panels) was determined by a PI staining method as described under Materials and Methods. Log-phase cells (a) were treated with 1.5% DMSO for 96 h (b). The DMSO-treated cells were cultured in the absence of DMSO for 12 (c), 21 (d), and 24 h (e).
Simultaneous measurement of DNA and RNA. Simultaneous quantitation of cellular DNA and RNA was carried out as reported by Darzynkiewicz et al. [14]. Briefly, we mixed 0.2 ml of cell suspension with 0.4 ml of chilled 0.1% (v/v) Triton X-100, 0.08 N HCI, and 0.15 M NaCl. One minute later, we added 1.2 ml of 14 pg/ml recrystallized AO, 1 mM EDTA-Na, and 0.15 M NaCl to the mixture and then analyzed the solution with an EPICS C. Cellular DNA content and RNA content were measured with a 590-nm dichroic mirror, a 525-nm bandpass filter, and a 610-nm long-pass filter. We displayed the distribution pattern in dot plotting. Dual staining anulysis of cell surface TFR and DNA content. Raji cells were stained with anti-TFR mAb as described above. The stained cells were washed once with chilled PBS and fixed in 3 ml of 70% ethanol at 0°C for 6 h. The fixed cells were washed once with PBS and incubated in 0.5 ml PBS containing 0.15 mg ribonuclease at room temperature for 30 min in the dark, and then the cell suspension was
Effect of DMSO on Cell Cycle and Proliferation The cell cycle distribution of asynchronous cultures of Raji cells treated with or without 1.5% (v/v) DMSO was determined by flow cytometry (Fig. 1). The cell density was maintained below lo6 cells/ml by nearly daily dilution. In a log-phase growth of Raji cells, the percent Gl-phase and the percent S-phase were 34.5 and 50.6, respectively. After incubation for 24 h with DMSO, a notable decrease in cells in S-phase, especially at early S-phase, was observed (Fig. lb). Then the population of cells in S-phase decreased gradually, and at 96 h it was reduced to 5% concomitantly with a 54% increase in Glphase (Fig. le). The growth rate of Raji cells treated with 1.5% DMSO decreased gradually (Fig. 2, solid circles) with increase in cell population at Gl-phase (see Fig. 1). In contrast to DMSO-treated cells, the cell cycle configuration of stationary-phase cells (Gl-phase = 4862%; S-phase = 44-26%) was not drastically different from that of the control log-phase cells, but [3H]thymidine incorporation in stationary-phase cells was negligible compared with that in log-phase cells. When Raji cells were cultured in the presence of DMSO at an initial density of 4 X lo5 cells/ml without dilution for 96 h, the cell cycle configuration was quite similar to that in Fig. le (see Table 1). The data suggest that DMSO-induced Gl accumulation occurs independently of the cell density in culture. On the basis of dose-dependent experiments (0.5-2.5% DMSO), 1.5% DMSO was fully effective for Gl arrest of Raji cells with a good cell viability of more than 95% (Fig. 2). Although an efficient growth arrest was also observed in the presence of 2 or 2.5% DMSO, cell viability was about 90% in the case of daily dilution of culture (Fig. 2) and worse without dilution. Table 1 shows cell cycle configurations of four B-cell lines and one T-cell line (MOLT-4) that were treated with 1.5% DMSO without dilution. Cell viability was always more than 95%. In these cell lines treated with DMSO, the percent Gl-phase increased reciprocally with the decrease in percent S-phase, indicating that Gl accumulation induced by DMSO is commonly observed in lymphoid cells. Differentiation Stage of Lymphoid Cells Exposed to DMSO To elucidate any transitional changes in differentiation stages of two B-cell lines Raji and Akata by DMSO
8
SAWAI
a
ET AL.
d
b
::...;:j ,*
e 4
1
h
f
“. .’ I------
;:
,,..., .:..j:. ',
____ ._ .r::-: :.: -I
.' ... :
:.. . .. .._::_. . . . . . . . .i.::...i.: ., . .,:::, . “’
.:
J
L Relative
1
RNA content
FIG. 5. Patterns of DNA and RNA contents in Raji cells released from Gl arrest. Log-phase cells (a) were treated with 1.5% DMSO for 96 h (b). The DMSO-treated cells were cultured in the absence of DMSO for 6 (c), 9 (d), 12 (e), 16 (f), 20 (g), 24 (h), 28 (i), 32 (j), 36 (k) and 40 h (1). Two-dimensional distribution patterns of DNA and RNA contents were determined by flow cytometry following A0 staining.
treatment, we examined the immunophenotyping with mAbs against B-lymphoid cell-associated antigens related to various stages of maturation (Bl, B2, B3, B5, B6, and PCAl). No appreciable change in the expression of these surface markers on Raji and Akata cells was observed (Fig. 3). Raji cells were positive for Bl, B2, B3, and B5 and negative for B6 and PCAl (Fig. 3A), and Akata cells were positive for Bl, B3, and B5 and negative for B2, B6, and PCAl (Fig. 3B). Release from Gl Arrest We examined reversibility of the DMSO-induced Gl arrest by determining alteration in cellular DNA contents after removal of DMSO (Fig. 4). Raji and Akata cells synchronized at Gl-phase by DMSO treatment were transferred to DMSO-free medium. No progression into S-phase was observed within 9 h after removal of
DMSO. The cell cycle pattern at 12 h demonstrated the progression of Gl-phase cells into S-phase, and the pattern at 24 h appeared similar to that of control log-phase cells (Figs. 4a and 4e). Interestingly, the percent G2/Mphase as well as the percent S-phase of Raji and Akata cells reached a maximum at 21 h (Fig. 4d). These observations indicate that DMSO induces reversible Gl arrest. Next, we carried out simultaneous quantitations of cellular DNA and RNA to analyze precisely this reversible Gl arrest in Raji cells by DMSO treatment (Fig. 5). In addition to the low RNA content group at Gl-phase (GlA) predominantly observed in log-phase cells, higher RNA content cells at Gl-phase (GlB) were accumulated by treatment with DMSO for 96 h (Fig. 5b). The GlB cells observed after DMSO treatment started to progress into S-phase 12 h after release from DMSO (Fig. 5e). GlB cells gradually decreased with increase in S- and
REVERSIBLE
Cl
ARREST
9
BY DMSO
and cellular DNA and the histogram patterns of TFR expression in Gl-phase. The expression of TFR on DMSO-treated cells in Gl-phase was reduced to onefourth that on log-phase cells in our calibration and was restored 24 h after release from DMSO treatment. Similarly, the 4 to i reduction of TFR expression on Akata and MOLT-4 cells was observed 96 h after 1.5% DMSO treatment (data not shown). DISCUSSION b
,/ 2c DNA content
4\? (PI)
--
\ 50
25 TFR expre~~lo"
(channel
number!
FIG. 6. Contour plotting pattern of cell surface TFR expression and cellular DNA content in Raji cells. Log-phase cells (a) were obtained as described under Materials and Methods. The cells exposed to 1.5% DMSO for 96 h (b) were cultured in the absence of DMSO for 24 h (c). The vertical and horizontal dimensions represent the cellsurface expression of TFR in a logarithmic scale and PI fluorescence corresponding to the DNA content in a linear scale, respectively (A). The one-dimensional histogram patterns of TFR expression (log FITC-fluorescence) at Gl phase are shown in (B). The fluorescence intensities of TFR expression at Gl-phase are 36.3 f 2.9 for a, 27.3 f 2.5 for b, and 39.6 + 2.9 for c (mean + SD in channel number).
G2/M-phase (Figs. 5e-5g), but even at 24 h the DNARNA pattern was not restored to the control log-phase (Fig. 5h). The distribution pattern similar to the logphase cells was observed 36 and 40 h after release from DMSO (Figs. 5k and 51). This implies that cell cycle progression of Raji cells does not necessarily correlate with RNA content. Dud Staining Analysis of Cell Surface Transferrin Receptor (TFR) and DNA Content Finally, we investigated the relationship between cell cycle phase and expression of one of the growth-related markers, TFR, on Raji cells. Figure 6 shows contour plotting patterns of simultaneous analyses of the TFR
In this paper, we have revealed that DMSO acts as an efficient agent inducing Gl arrest in several B- and Tlymphoid cell lines. By a simple treatment of these cell lines with 1.5% DMSO for 96 h, we obtained more than 74% Gl-phase with good cell viability. In general, Gl/ GO arrest in adherent cells would be easily induced by contact inhibition or serum starvation. In contrast, neither serum starvation in Raji and Akata cells (unpublished results) nor a complete growth arrest at the stationary phase in these cells (this work) resulted in Gl arrest. Blomhoff et al. [16, 171 have explored Gl-accumulating effects of forskolin and 12-O-tetradecanoylphorbol-13-acetate on B-lymphoid cell lines. The population of Gl-phase cells after treatment with forskolin was increased 22% for Raji cells and 20% for Reh cells, and the cell viability was unknown. Therefore, this efficient Gl arrest by DMSO could be very useful for investigation of lymphocytic proliferation and differentiation. We suggest that DMSO induces GlB arrest in Raji cells because a large number of GlB cells were found in Gl-phase after treatment with DMSO (Fig. 5b). Since the GlB cells started to progress into S-phase 9-12 h after removal of DMSO (see Figs. 5d and 5e). Transiently, large populations of S- and G2/M-phase were observed at 21 h (Fig. 4d), and the DNA pattern at 24 h was similar to that in control log-phase cells. In this reversible process, expression of TFR, one of growth-related markers, is well correlated with Gl arrest (Fig. 6): it was reduced to one-fourth in Gl-phase (GlA + GlB) by DMSO treatment and restored 24 h after release from DMSO. TFR was previously regarded as a candidate for cell cycle progression signal at the transition from the early Gl-phase (GlA) to the late Gl-phase (GlB) [18]. Cells in GlB as well as in GlA at DMSO-induced Gl arrest have only one-fourth the TFR expression of cells at GlA observed mainly in log-phase culture, implying that TFR is a proliferation signal expressed at a higher level in actively growing cells but not a cell cycle progression signal. The DNA distribution pattern 24 h after removal of DMSO returned to that of control log-phase, whereas the DNA-RNA pattern was not restored at 24 h but returned nearly to the control pattern at 36-40 h (see Fig.
10
SAWAI
5). In particular, an appreciable population of cells at GlB-phase remained 24 h after release from DMSO. Why is the pattern of the subpopulation of Gl-phase cells restored 12 h later than that of DNA configuration? The influence of DMSO on the cellular RNA content in lymphoid cells is likely to continue longer than that on the apparent DNA configuration pattern. DMSO is widely used as a cryoprotectant and as a simple, lipophilic solvent. In addition, a variety of effects of DMSO on biological function and structure have been reported. DMSO acts as an inducer for differentiation of several myeloid cell lines [l, 21, an activator for several enzymes [ 19-221, and a modulator for cytoskeletal organization [23]. At present, it seems impossible to explain reasonably the mechanism of Gl arrest in lymphoid cell lines as well as that of DMSO-induced differentiation in myeloid cell lines. As for growth arrest during the course of differentiation of myeloid cells by DMSO, contradictory observations have been reported [3, 4, 24, 251. If a transient, stage-specific arrest in Gl-phase is essential for differentiation of several myeloid cell lines, a common mechanism is likely to be present at the earlier stage during incubation of lymphoid and myeloid cells with DMSO. As in the case of DMSO, trichostatin A, a fungistatic antibiotic, has been reported to elicit both differentiation of murine erythroleukemia cells [ 261 and reversible arrest at Gl- and G2-phase in proliferation of 3Yl cells [27]. We expect to find new agents that easily induce differentiation of B- and T-lymphoid cell lines arrested at Gl-phase by DMSO. We are now investigating the mechanism of DMSO-induced Gl arrest in lymphoid cells as well as the relationship between differentiation and proliferation of lymphoid cells. We are grateful to Professor Kohtaro Yamamoto, Medical Research Institute, Tokyo Medical and Dental University, for supplying lymphoid cell lines. We also thank him for his encouragement and helpful discussions during this work. This work was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare of Japan (62-7) to K. Tsukada and from the Ministry of Education, Science and Culture of Japan (01010032) to H. Teraoka.
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Collins, (1978) Terada, (1977) Rubin,
S. J., Ruscetti, F. W., Gallagher, R. E., and Gallo, R. C. Proc. Natl. Acad. Sci. USA 75,2458-2462. M., Fried, J., Nudel, U., Rifkind, R. A., andMarks, P. A. Proc. Natl. Acad. Sci. USA 74,248-252. C. S. (1978) J. Cell. Physiol. 94,57-68. R. J. V. (1964) Lancet
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Durr, F. E., Monroe, J. H., Schmitter, R. Traul, K. A., and Hirshaut, Y. (1970) Znt. J. Cancer 6,436-449.
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Dean, P. N. (1980) Cell Tissue Kinet. 13,299-303. Takase, K., Okawa, H., Minato, K., and Yata, J. (1988) Leuk.
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Darzynkiewicz, Z., Traganos, F., Sharpless, T., and Melamed, M. R. (1976) Proc. Natl. Acad. Sci. USA 73,2881-2884. Takase, K., Iwaki, K., Gunji, T., and Yata, J. (1989) J. Zmmunol. Methods 118,129-138. Lemo, J., Holte, H., deLange Davies, C., Ruud, E., Laukas, M., Smeland, E. B., Godal, T., and Blomhoff, H. K. (1987) Exp. Cell Res. 172,84-91. Blomhoff, H. K., Blomhoff, R., Stokke, T., deLange Davies, C., Brevik, K., Smeland, E. B., Funderud, S., and Godal, T. (1988) J. Cell. Physiol. 137,583-587. Neckers, L. M., and Cossman, J. (1983) Proc. Natl. Acad. Sci. USA 80,3494-3498. Ishida, H., Mori, M., and Tatibana, M. (1977) Arch. Biochem. Biophys. 182.258-265. Okada, G., Teraoka, H., and Tsukada, K. (1981) Biochemistry 20.934-940. Srivastava, A. K. (1985) Biochem. Biaphys. Res. Commun. 126, 104221047. Lee, M. Y. W. T., and Toomey, N. L. (1986) Nucleic Acids Res.
14,1719-1726. 23.
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26. 27.
RESEARCH
187,4-10
(1990)
Reversible Gl Arrest in the Cell Cycle of Human Lymphoid Cell Lines by Dimethyl Sulfoxide MASAJI SAWAI,* Kozo TAKASE,~ HIROBUMI TERAOKA,* AND KINJI TSUKADA*T’ *Department of Pathological Biochemistry, Medical Research Institute, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101, Japan; and TDepartment of Pediatrics, Faculty of Medicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113, Japan
the course of our studies on cell proliferation and differentiation, we investigated the effect of DMSO on the cell cycle of lymphoid cell lines by flow cytometry. In this paper, we describe that DMSO induces reversible Cl arrest in the cell cycle of lymphoid cell lines without any detectable differentiation. DMSO seems to be a useful agent for elucidating the regulatory mechanisms of cell differentiation and the proliferation of lymphoid cells.
Proliferation of human B- and T-lymphoid cell lines including Raji and Akata cells was found to be arrested at the Gl stage in the cell cycle by dimethyl sulfoxide (DMSO). The Gl arrest by DMSO occurred gradually and was completed within 96 h after addition of 1.5% DMSO concomitantly with a decrease in growth rate. Progression of Gl-phase cells containing a larger amount of RNA into S-phase began 9-12 h after removal of DMSO. At 24 h, the DNA pattern of the cell cycle was similar to that of nontreated log-phase cells. The expression of six differentiation markers on the lymphoid cells was not appreciably changed by treatment with DMSO. On the other hand, the expression of transferrin receptor (one of the growth-related markers) on Gl-phase cells 96 h after addition of DMSO was decreased to one-fourth that on log-phase cells and was completely restored 24 h after removal of DMSO. These results indicate that DMSO, known as an inducer of differentiation in several myeloid cell lines, acts as an agent inducing Gl arrest in the cell cycle of the lymphoid cells. 0 1990 Academic Press, Inc.
MATERIALS
Materials. DMSO (gas chromatography grade) was purchased from Wako Pure Chemicals (Osaka, Japan); propidium iodide (PI), from Sigma (St. Louis, MO) and Calbiochem Behring (La Jolla, CA); acridine orange (AO), from Sigma; ribonuclease, from Worthington Diagnostic Systems (Freehold, NJ); neutral mouse serum immunoglobulin (Ig) and monoclonal antibodies (mAbs) Bl (CD20), B2 (CD21), B3 (CD22), B5, B6 (CD23), and PCAl, from Coulter Immunology (Hialeah, Fla); anti-transferrin receptor mAb (CD71), from Becton Dickinson (Mountain View, CA); human Ig, from Green Cross (Osaka, Japan); fluorescein isothiocyanate (FITC)-conjugated F(ab’), of sheep anti-mouse IgG antibody (H chain- and L chain-specific), from Silenus (Hawthorn, Australia). All other reagents were of the highest purity available. Cell culture. Human B-cell lines (Raji [5], Akata [6], BJAB [7], and NC-37 [8]) and a T-cell line (MOLT-4 [9]) were grown as a suspension culture in RPMI-1640 medium (GIBCO, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (KC Biological, Lenexa, KS), 100 IU/ml penicillin, and 100 pg/ml streptomycin at 37°C in a humidified atmosphere of 5% C02/95% air. Cells were seeded at an initial density of 4 X lo5 cells/ml and maintained at a density below 1.2 X 10s cells/ml (log-phase culture). To obtain DMSO-treated cells, we incubated the cells with 1.5% DMSO (v/v) for 96 h, unless otherwise specified. For reversibility experiments, the DMSO-treated cells were washed twice with phosphatebuffered saline (PBS), seeded at 4 X lo5 cells/ml, and grown without DMSO. Cells cultured for 5 days without dilution were harvested after an additional 2-3 days, and were referred to as stationary-phase cells. Cell viability was determined by the trypan blue exclusion test [lo]. DNA synthesis was determined by [3H]Thymidine incorporation. incorporation of [6-3H]thymidine (Amersham Japan). Briefly, cells were incubated with [6-3H]thymidine (1 &i/ml culture) for 60 min at 37°C. They were then washed with PBS and completely dissolved in 0.1 N NaOH followed by precipitation with 7% trichloroacetic acid/ 1% sodium pyrophosphate. The precipitate dissolved in 0.1 N NaOH was spotted on a 3MM paper, washed extensively with 7% trichloroacetic acid/l% sodium pyrophosphate, and counted in a liquid scintillation counter.
INTRODUCTION Dimethyl sulfoxide (DMSO) is well known as an inducer of differentiation in several myeloid cell lines, such as mouse erythroleukemia (Friend) cells [l] and human promyelocytic leukemia cells (HL-60) [2]. The relationship between cell differentiation and proliferation, however, has not necessarily been fully understood, several lines of evidence indicate that a transient growth arrest is accompanied by myeloid cell differentiation [ 3,4], but it remains obscure whether growth arrest is essential for cell differentiation. In contrast to that on myeloid cells, little attention has been focused on the effect of DMSO on lymphoid cells, because DMSO has been believed to be ineffective in differentiation of lymphoid cells. During ’ To whom reprint requests should be addressed at: Department of Pathological Biochemistry, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101, Japan.
0014-4827/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
4
REVERSIBLE qoes -
Gl ARREST
BY DMSO
a
5905 -
1
b
2103 -
0
&---A 100
0
200
256
Time
(h)
FIG. 2. Effect of DMSO on proliferation of Raji cells. Cells were initially seeded at 4 X lo5 cells/ml and diluted at a concentration of 4 X 10s cells/ml every day in the presence of 0% (0), 0.5% (A), 1% (O), 1.5% (O), 2% (A), or 2.5% (m) DMSO.
Distribution of cellular DNA content was determined PI staining. by hypotonic PI staining [ 111. Briefly, 1 X 10’ cells were suspended in 1 ml of PI solution (50 pg/ml of 0.1% sodium citrate and 0.2% Nonidet P-40) and analyzed with a FACS440, a FACScan (Becton-Dickinson), or an EPICS C (Coulter). Excitation was carried out with the 488-nm line of an argon ion laser operating at a continuous output of 200 mW. Cell cycle analysis by DNA distribution was computed by the polynomial method reported by Dean [ 121.
TABLE Effect
1
of DMSO on Cell Cycle Configuration in Lymphoid Cell Lines
I
vso -
Percentage of cells in the phase of
e
Cell line
Treatment with 1.5% DMSO
WI5 -
0.
0
I IO0
Relative
\
ZOO
Raji
-
Akata
+ +a -
BJAB
+ -
NC-37
+ -
MOLT-4
+ -
256
DNA content
FIG. 1. Cell cycle configuration of DMSO-treated and nontreated Raji cells. Log-phase cells (a) were obtained as described under Materials and Methods and then maintained at a density of below 1.2 X lo6 cells/ml in the presence of 1.5% DMSO for 24 (b), 48 (c), 60 (d), and 96 h (e). Cell cycle analysis was carried out by a PI staining method.
+ a Data from Fig. le.
Gl
S
G2/M
34.5 89.0 88.5 26.1 83.2 42.5 79.0 59.8 73.5 49.7 92.9
50.6 7.1 5.4 60.9 12.3 42.9 14.0 27.3 11.9 41.3 6.2
14.9 3.9 6.1 13.0 4.5 14.6 7.0 12.9 14.6 8.9 0.9
6
SAWAI
Log-phase Raji cells
Gl-arrested 670
a
Raji cells
ET AL.
Log-phase Akata cells
Gl-arrested
Akata cells
a
283
228 L
0k 281
b
b
114
0
382
C 169
0 l-/L
0h 223
285
d
d
133
0k
0 -i’_
528
871 e
e
=LlL 0
=i--A0 393
197
214
0
0
:;[A , , ljgh , , jgi. , , rjgh , , 0
100
200
Fluorescence
266
intensity
0
100
(channel
200 266
number)
0
100
200
256
Fluorescence intensity
0
100
(channel
200
253
number)
FIG. 3. Expression of differentiation markers on log-phase and DMSO-treated B-lymphoid cells. Log-phase cells and the cells exposed to 1.5% DMSO for 96 h were obtained as described under Materials and Methods. Raji (A) and Akata cells (B) were treated with mouse serum IgG (a), Bl (b), B2 (c), B3 (d), B5 (e), B6 (f) or PCAl (g) and then stained with FITC-conjugated F(ab’), of sheep anti-mouse IgG antibody. Surface immurwphenotyping. Cells were washed once with PBS and suspended in RPMI-1640 medium containing 10% FBS to a density of 2 X lo6 cells/ml. A 0.1.ml aliquot of the cell suspension was placed in a plastic tube and then treated with human Ig at a final concentration of 15 mg/ml at 4°C for 15 min to reduce the nonspecific binding of antibody via Fc receptors [13]. The cells were incubated
with an appropriate amount of mAb at 4’C for 30 min and then washed with PBS at 4°C. The cells were further stained with FITC-conjugated F(ab’)* of sheep anti-mouse IgG (H and L chain-specific) antibody. Fluorescence-stained cells were analyzed with a FACS440 (BectonDickinson). The background fluorescence was determined by incubating cells with neutral mouse serum IgG instead of the specific mAb.
REVERSIBLE
RaJi cells
Gl ARREST
Akata cells
BY DMSO
7
mixed with 0.5 ml of PI solution (30 rg/ml PBS) and kept at 0°C for 60 min. PI/F’ITC two-color flow cytometry was carried out with a FACS440 equipped with a 488-nm argon ion laser and an optic system with a 560-nm dichroic mirror, a 530-nm band-pass filter, and a 6.20. nm long-pass filter. The expression of TFR on the cells was evaluated on the FITC fluorescence intensity distribution derived from integration of the columns at the position of Gl phase in PI fluorescence. Mean fluorescence intensity corresponding to the surface TFR expression was estimated through a curve fitting method with simplex optimization [15].
I::
RESULTS
L:-100
Relative
100
200
DNA content
FIG. 4. DNA configuration of Raji and Akata cells released from Gl arrest. DNA content in Raji (left panels) and in Akata cells (right panels) was determined by a PI staining method as described under Materials and Methods. Log-phase cells (a) were treated with 1.5% DMSO for 96 h (b). The DMSO-treated cells were cultured in the absence of DMSO for 12 (c), 21 (d), and 24 h (e).
Simultaneous measurement of DNA and RNA. Simultaneous quantitation of cellular DNA and RNA was carried out as reported by Darzynkiewicz et al. [14]. Briefly, we mixed 0.2 ml of cell suspension with 0.4 ml of chilled 0.1% (v/v) Triton X-100, 0.08 N HCI, and 0.15 M NaCl. One minute later, we added 1.2 ml of 14 pg/ml recrystallized AO, 1 mM EDTA-Na, and 0.15 M NaCl to the mixture and then analyzed the solution with an EPICS C. Cellular DNA content and RNA content were measured with a 590-nm dichroic mirror, a 525-nm bandpass filter, and a 610-nm long-pass filter. We displayed the distribution pattern in dot plotting. Dual staining anulysis of cell surface TFR and DNA content. Raji cells were stained with anti-TFR mAb as described above. The stained cells were washed once with chilled PBS and fixed in 3 ml of 70% ethanol at 0°C for 6 h. The fixed cells were washed once with PBS and incubated in 0.5 ml PBS containing 0.15 mg ribonuclease at room temperature for 30 min in the dark, and then the cell suspension was
Effect of DMSO on Cell Cycle and Proliferation The cell cycle distribution of asynchronous cultures of Raji cells treated with or without 1.5% (v/v) DMSO was determined by flow cytometry (Fig. 1). The cell density was maintained below lo6 cells/ml by nearly daily dilution. In a log-phase growth of Raji cells, the percent Gl-phase and the percent S-phase were 34.5 and 50.6, respectively. After incubation for 24 h with DMSO, a notable decrease in cells in S-phase, especially at early S-phase, was observed (Fig. lb). Then the population of cells in S-phase decreased gradually, and at 96 h it was reduced to 5% concomitantly with a 54% increase in Glphase (Fig. le). The growth rate of Raji cells treated with 1.5% DMSO decreased gradually (Fig. 2, solid circles) with increase in cell population at Gl-phase (see Fig. 1). In contrast to DMSO-treated cells, the cell cycle configuration of stationary-phase cells (Gl-phase = 4862%; S-phase = 44-26%) was not drastically different from that of the control log-phase cells, but [3H]thymidine incorporation in stationary-phase cells was negligible compared with that in log-phase cells. When Raji cells were cultured in the presence of DMSO at an initial density of 4 X lo5 cells/ml without dilution for 96 h, the cell cycle configuration was quite similar to that in Fig. le (see Table 1). The data suggest that DMSO-induced Gl accumulation occurs independently of the cell density in culture. On the basis of dose-dependent experiments (0.5-2.5% DMSO), 1.5% DMSO was fully effective for Gl arrest of Raji cells with a good cell viability of more than 95% (Fig. 2). Although an efficient growth arrest was also observed in the presence of 2 or 2.5% DMSO, cell viability was about 90% in the case of daily dilution of culture (Fig. 2) and worse without dilution. Table 1 shows cell cycle configurations of four B-cell lines and one T-cell line (MOLT-4) that were treated with 1.5% DMSO without dilution. Cell viability was always more than 95%. In these cell lines treated with DMSO, the percent Gl-phase increased reciprocally with the decrease in percent S-phase, indicating that Gl accumulation induced by DMSO is commonly observed in lymphoid cells. Differentiation Stage of Lymphoid Cells Exposed to DMSO To elucidate any transitional changes in differentiation stages of two B-cell lines Raji and Akata by DMSO
8
SAWAI
a
ET AL.
d
b
::...;:j ,*
e 4
1
h
f
“. .’ I------
;:
,,..., .:..j:. ',
____ ._ .r::-: :.: -I
.' ... :
:.. . .. .._::_. . . . . . . . .i.::...i.: ., . .,:::, . “’
.:
J
L Relative
1
RNA content
FIG. 5. Patterns of DNA and RNA contents in Raji cells released from Gl arrest. Log-phase cells (a) were treated with 1.5% DMSO for 96 h (b). The DMSO-treated cells were cultured in the absence of DMSO for 6 (c), 9 (d), 12 (e), 16 (f), 20 (g), 24 (h), 28 (i), 32 (j), 36 (k) and 40 h (1). Two-dimensional distribution patterns of DNA and RNA contents were determined by flow cytometry following A0 staining.
treatment, we examined the immunophenotyping with mAbs against B-lymphoid cell-associated antigens related to various stages of maturation (Bl, B2, B3, B5, B6, and PCAl). No appreciable change in the expression of these surface markers on Raji and Akata cells was observed (Fig. 3). Raji cells were positive for Bl, B2, B3, and B5 and negative for B6 and PCAl (Fig. 3A), and Akata cells were positive for Bl, B3, and B5 and negative for B2, B6, and PCAl (Fig. 3B). Release from Gl Arrest We examined reversibility of the DMSO-induced Gl arrest by determining alteration in cellular DNA contents after removal of DMSO (Fig. 4). Raji and Akata cells synchronized at Gl-phase by DMSO treatment were transferred to DMSO-free medium. No progression into S-phase was observed within 9 h after removal of
DMSO. The cell cycle pattern at 12 h demonstrated the progression of Gl-phase cells into S-phase, and the pattern at 24 h appeared similar to that of control log-phase cells (Figs. 4a and 4e). Interestingly, the percent G2/Mphase as well as the percent S-phase of Raji and Akata cells reached a maximum at 21 h (Fig. 4d). These observations indicate that DMSO induces reversible Gl arrest. Next, we carried out simultaneous quantitations of cellular DNA and RNA to analyze precisely this reversible Gl arrest in Raji cells by DMSO treatment (Fig. 5). In addition to the low RNA content group at Gl-phase (GlA) predominantly observed in log-phase cells, higher RNA content cells at Gl-phase (GlB) were accumulated by treatment with DMSO for 96 h (Fig. 5b). The GlB cells observed after DMSO treatment started to progress into S-phase 12 h after release from DMSO (Fig. 5e). GlB cells gradually decreased with increase in S- and
REVERSIBLE
Cl
ARREST
9
BY DMSO
and cellular DNA and the histogram patterns of TFR expression in Gl-phase. The expression of TFR on DMSO-treated cells in Gl-phase was reduced to onefourth that on log-phase cells in our calibration and was restored 24 h after release from DMSO treatment. Similarly, the 4 to i reduction of TFR expression on Akata and MOLT-4 cells was observed 96 h after 1.5% DMSO treatment (data not shown). DISCUSSION b
,/ 2c DNA content
4\? (PI)
--
\ 50
25 TFR expre~~lo"
(channel
number!
FIG. 6. Contour plotting pattern of cell surface TFR expression and cellular DNA content in Raji cells. Log-phase cells (a) were obtained as described under Materials and Methods. The cells exposed to 1.5% DMSO for 96 h (b) were cultured in the absence of DMSO for 24 h (c). The vertical and horizontal dimensions represent the cellsurface expression of TFR in a logarithmic scale and PI fluorescence corresponding to the DNA content in a linear scale, respectively (A). The one-dimensional histogram patterns of TFR expression (log FITC-fluorescence) at Gl phase are shown in (B). The fluorescence intensities of TFR expression at Gl-phase are 36.3 f 2.9 for a, 27.3 f 2.5 for b, and 39.6 + 2.9 for c (mean + SD in channel number).
G2/M-phase (Figs. 5e-5g), but even at 24 h the DNARNA pattern was not restored to the control log-phase (Fig. 5h). The distribution pattern similar to the logphase cells was observed 36 and 40 h after release from DMSO (Figs. 5k and 51). This implies that cell cycle progression of Raji cells does not necessarily correlate with RNA content. Dud Staining Analysis of Cell Surface Transferrin Receptor (TFR) and DNA Content Finally, we investigated the relationship between cell cycle phase and expression of one of the growth-related markers, TFR, on Raji cells. Figure 6 shows contour plotting patterns of simultaneous analyses of the TFR
In this paper, we have revealed that DMSO acts as an efficient agent inducing Gl arrest in several B- and Tlymphoid cell lines. By a simple treatment of these cell lines with 1.5% DMSO for 96 h, we obtained more than 74% Gl-phase with good cell viability. In general, Gl/ GO arrest in adherent cells would be easily induced by contact inhibition or serum starvation. In contrast, neither serum starvation in Raji and Akata cells (unpublished results) nor a complete growth arrest at the stationary phase in these cells (this work) resulted in Gl arrest. Blomhoff et al. [16, 171 have explored Gl-accumulating effects of forskolin and 12-O-tetradecanoylphorbol-13-acetate on B-lymphoid cell lines. The population of Gl-phase cells after treatment with forskolin was increased 22% for Raji cells and 20% for Reh cells, and the cell viability was unknown. Therefore, this efficient Gl arrest by DMSO could be very useful for investigation of lymphocytic proliferation and differentiation. We suggest that DMSO induces GlB arrest in Raji cells because a large number of GlB cells were found in Gl-phase after treatment with DMSO (Fig. 5b). Since the GlB cells started to progress into S-phase 9-12 h after removal of DMSO (see Figs. 5d and 5e). Transiently, large populations of S- and G2/M-phase were observed at 21 h (Fig. 4d), and the DNA pattern at 24 h was similar to that in control log-phase cells. In this reversible process, expression of TFR, one of growth-related markers, is well correlated with Gl arrest (Fig. 6): it was reduced to one-fourth in Gl-phase (GlA + GlB) by DMSO treatment and restored 24 h after release from DMSO. TFR was previously regarded as a candidate for cell cycle progression signal at the transition from the early Gl-phase (GlA) to the late Gl-phase (GlB) [18]. Cells in GlB as well as in GlA at DMSO-induced Gl arrest have only one-fourth the TFR expression of cells at GlA observed mainly in log-phase culture, implying that TFR is a proliferation signal expressed at a higher level in actively growing cells but not a cell cycle progression signal. The DNA distribution pattern 24 h after removal of DMSO returned to that of control log-phase, whereas the DNA-RNA pattern was not restored at 24 h but returned nearly to the control pattern at 36-40 h (see Fig.
10
SAWAI
5). In particular, an appreciable population of cells at GlB-phase remained 24 h after release from DMSO. Why is the pattern of the subpopulation of Gl-phase cells restored 12 h later than that of DNA configuration? The influence of DMSO on the cellular RNA content in lymphoid cells is likely to continue longer than that on the apparent DNA configuration pattern. DMSO is widely used as a cryoprotectant and as a simple, lipophilic solvent. In addition, a variety of effects of DMSO on biological function and structure have been reported. DMSO acts as an inducer for differentiation of several myeloid cell lines [l, 21, an activator for several enzymes [ 19-221, and a modulator for cytoskeletal organization [23]. At present, it seems impossible to explain reasonably the mechanism of Gl arrest in lymphoid cell lines as well as that of DMSO-induced differentiation in myeloid cell lines. As for growth arrest during the course of differentiation of myeloid cells by DMSO, contradictory observations have been reported [3, 4, 24, 251. If a transient, stage-specific arrest in Gl-phase is essential for differentiation of several myeloid cell lines, a common mechanism is likely to be present at the earlier stage during incubation of lymphoid and myeloid cells with DMSO. As in the case of DMSO, trichostatin A, a fungistatic antibiotic, has been reported to elicit both differentiation of murine erythroleukemia cells [ 261 and reversible arrest at Gl- and G2-phase in proliferation of 3Yl cells [27]. We expect to find new agents that easily induce differentiation of B- and T-lymphoid cell lines arrested at Gl-phase by DMSO. We are now investigating the mechanism of DMSO-induced Gl arrest in lymphoid cells as well as the relationship between differentiation and proliferation of lymphoid cells. We are grateful to Professor Kohtaro Yamamoto, Medical Research Institute, Tokyo Medical and Dental University, for supplying lymphoid cell lines. We also thank him for his encouragement and helpful discussions during this work. This work was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare of Japan (62-7) to K. Tsukada and from the Ministry of Education, Science and Culture of Japan (01010032) to H. Teraoka.
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