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Phorbol ester-induced effects on cell cycle progression and terminal deoxynucleotidyltransferase (TdT) activity in KM-3 pre-B cell line
Immunology Letters, 35 (1993) 265-270
0165 2478 / 93 / $ 6.00 ~© 1993 ElsevierSciencePublishers B.V. All rights reserved IMLET 01926
Phorbol ester-induced effects on cell cycle progression and terminal deoxynucleotidyltransferase (TdT) activity in KM-3 pre-B cell line O r i a n a T r u b i a n i a, R o b e r t o D i P r i m i o b, L o r i s Z a m a i c, D o m e n i c o B o s c o a, F.J. B o l l u m d a n d M a r c o Vitale e alstituto di Citomorfologia del C.N.R and bIstituto di Morfologia Umana Normale, Facoltgt di Medicina, Universitd di Chieti, Chieti, Italy; Clstituto di Anatomia Umana Normale, Facolt(~ di Medicina, Universitgl di Bologna, Bologna, Italy; dDepartment of Biochemistry, U.S.U.H.S., Bethesda, MD, USA, and eDipartimento di Scienze Biomediehe e Biotecnologie, Sezione Anatomia Umana, Universitd di Brescia, Brescia, Italy
(Received 4 January 1993; accepted 26 January 1993)
1.
Summary
Phorbol myristic acetate (PMA) is a tumorpromoting agent that has been shown to induce differentiation of human leukemia cells and of normal lymphoid cells. We have investigated the ability of PMA to induce inhibition of cell growth of the human KM-3 pre-B leukemic cell line by multiparametric analysis. Our results show that PMA treatment induces cell differentiation with the disappearance of terminal deoxynucleotidyltransferase and a decrease of cell growth, as evaluated by [3H]thymidine uptake. Flow cytometric analysis of BrdU incorporation shows that PMA is able to induce a modification of the cell cycle with a sharp decrease of the percentage of S-phase cells, which is more evident after 24 h of treatment. Comparison between the cell growth kinetics and TdT synthesis and activity shows that differentiated cells are still able to proliferate to a certain extent and that the TdT disappearance and the initial decrease of cell proliferation are two independent effects of PMA. Key words." KM-3; Terminal transferase; PMA; Differentia-
tion; Cell cycle; Flow cytometry Correspondence to: Dr. Oriana Trubiani, lstituto di Citomorfologia del CNR, Facolt~i di Medicina, Universitfi di Chieti, Via dei Vestini 12, 66100 Chieti, Italy.
2.
Introduction
Terminal deoxynucleotidyltransferase (TdT) is a unique D N A polymerase able to generate D N A sequences in the absence of template direction [1]. The enzyme is detected in immature cells of the hemopoietic system undergoing immunoglobulin and T-cell receptor gene rearrangement, suggesting that TdT may play a role in the somatic generation of diversity [2~,]. This consideration is supported by several observations showing that B cells derive from TdT-positive precursors which acquire cytoplasmic #-heavy chains at the pre-Bcell stage [5-7]. These data are derived mainly from analyses of the phenotype of bone marrow B-lymphocyte precursors and of immunoglobulin gene expression [8-10]. 12-O-Tetradecan0Yl phorbol myristate 13-acetate (PMA) is a tumor-promoting agent that induces immature hemopoietic cells to differentiate [10-13]. It has been demonstrated that PMA binds to protein kinase C, whose activation is responsible for most of the observed effects [14,15]. In this respect, PMA treatment of B or T leukemic lymphoblastoid cell lines with an immature cell phenotype has been able to induce differentiation to plasma cells or to mature T cells, respectively [10,16-19]. So far, most studies on the effects of PMA have only considered the initial and final stages of differen265
tiation of the immature cell lines. In order to define the kinetics of the effects of PMA on pre-Bcell proliferation in time course experiments, we examined the perturbation of the KM-3 cell cycle by flow cytometric analysis of bromodeoxyuridine (BrdU) incorporation. Since it has been reported that PMA treatment of different TdT-positive pre-T or pre-B cells induces cellular differentiation and TdT disappearance, we also analyzed TdT localization and enzyme activity in the same cell line. 3.
Materials and Methods
3.1. Cell cultures The KM-3 human lymphoblastoid cell line [20] was maintained in continuous suspension culture in RPMI 1640 medium supplemented with 10% FBS, 4 mM L-glutamine, 100 mM Na-pyruvate, and 25 mM Hepes. Cells were grown at 2.5 x 105/ml, with more than 98% viability as determined by the trypan blue exclusion test. During the log growth phase, the cells were treated with 16 nM PMA for times ranging between 24 and 72 h. Cell growth was evaluated both as total cell number and by [3H]thymidine uptake 24, 48 and 72 h after culture with PMA.
Tris-HC1 buffer pH 7.6 was used for 15 rain at room temperature.
3.3. Enzyme assay TdT activity was determined as described previously [23]. Briefly, cells (2 x 10v) washed with RPMI 1640 medium were lysed in 250 /al of 0.3 M KPi pH 7.4 by freeze-thawing. The lysate was cleared by centrifugation at 15 000 rpm for 30 rain at 4°C and aliquots were assayed using 0.5 mM d(pA)50 as initiator, 5 mM [3H]dGTP (50-150 cpm/pmol) as substrate, 8.0 mM MgC12, 1.0 mM /%mercaptoethanol, 0.1 mg/ml BSA, and 0.2 M K-cacodylate buffer pH 7.5. The reaction products were scored as acid-insoluble radioactivity on glass fiber filters. One unit of enzyme activity was defined as 1 nM d G M P incorporated in 60 min at 36°C.
3.4. FH]Thymidine uptake D N A synthesis in KM-3 cells was measured by adding 2/~Ci of [3H]thymidine (6.7 Ci/mmol) for the final 6 h of each treatment period. Cells were collected using a cell harvester, and [3H]thymidine uptake was measured in a liquid scintillation counter. All data are expressed as mean counts per minute of triplicate determinations.
3.2. Immunocytochemistrv 3.5. BrdU labelling The immunocytochemical localization of TdT was carried out as described previously [21]. Briefly, cells cultured for the different times in the presence of PMA and control cells were fixed for 45 min at room temperature with 1% paraformaldehyde in 0.1 M Na-cacodylate buffer at pH 7.6 immediately after cytocentrifugation. Slides were rinsed three times in fresh cacodylate buffer containing 2% sucrose and 0.1 M glycine and treated for 5 min with normal goat serum and then with 0.2% H202 in cacodylate buffer for 5 rain. Samples were incubated with a rabbit polyclonal anti-TdT antibody [22] for 30 min at room temperature, rinsed twice, and incubated with a goat anti-rabbit IgG peroxidase conjugate antibody. To detect sites of antibody reaction, a solution of 0.05% diaminobenzidine tetrahydrochloride (DAB) containing 0.015% H202 in 0.05 M 266
To evaluate the effect of PMA treatment on the cell cycle, BrdU incorporation was studied by flow cytometry as described [24]. Briefly cells were incubated with 100 ¢tM BrdU for 30 rain at 37°C in a 5% CO2 atmosphere and washed twice in PBS, followed by 70% ethanol fixation at 4°C for 30 min and rinsing in PBS. DNA was denatured with 4 N HC1 at room temperature for 30 min. The cells were then incubated in Na2B407 pH 8.5 for 5 min at room temperature, washed, and treated with Triton X-100 for 3 min. Finally, the cells were incubated with FITC-conjugated anti-BrdU monoclonal antibody for 30 min at 4°C and then counterstained with 5 /~g/ml propidium iodide (PI) before flow cytometric analysis.
3.6.
Flow cytometry and data analysis
Cell cycle analysis was performed by a FACStar PLus flow cytometer (Becton Dickinson, USA) equipped with an argon ion laser tuned at 488 nm, 200 mW output, for excitation of FITC and PI. Data were analyzed as the percentage of cells in different phases of the cell cycle (i.e., Go/ G] S G2/M) and as the mean channel of FITC fluorescence of BrdU-positive cells. Both anti-BrdU fluorescence and PI fluorescence were measured on a linear scale.
3.7.
48 hrs
72 hrs
Time control cells PMA treated ceLls
Fig. 2. [3H]Thymidineuptake in treated and untreated PMA cells. The cpm is defined as the mean of triplicate wells from four different experiments _+ SD.
Sources of materials
RPMI-1640, L-glutamine, PBS, and Na-pyruvate were from Gibco Laboratories (Grand Island, NY). Rabbit polyclonal antibody against 32-kDa calf thymus TdT protein was produced as described [22]. FITC-conjugated anti-BrdU monoclonal antibody was from Becton Dickinson. Peroxidase-conjugated goat anti-rabbit IgG and normal goat serum were from Litton Bionetics (Kensington, MD). All radiolabelled materials were from Amity PG (Geneva, Switzerland). PMA, BrdU, PI, and all other reagent grade materials were from Sigma (St. Louis, MO). 4.
24 h~s
Results and Discussion
The effect of 16 nM P M A treatment at different time intervals on the kinetics of KM-3 growth is shown in Fig. 1. Cell viability was al-
ways more than 98% as evaluated by trypan blue exclusion. The PMA-induced reduction of cell growth is already detectable after 24 h treatment, and this reduction increases with time. This effect is due to a decrease in the number of cells entering the S phase of the cycle following P M A treatment. Thus, both [3H]thymidine uptake (Fig. 2) and the percentage of BrdU-positive cells evaluated by flow cytometry (Fig. 3) show a decrease of the number of S-phase cells during the time interval considered. This effect is particularly marked 24 h after P M A treatment when a decrease in the percentage of S-phase cells occurs. These cells are mainly found in the early S (Es) region of the dot plot (Fig. 4). Although the inhibitory effect of PMA is still present after 72 h, the percentage of S-phase cells
300
E
~, 20o
[
x Io 100
be
0 Ihrs
241hrs
48 Ihrs
72 Ihrs
Time
Fig. l. Cell growth at different times determined as described under Materials and Methods. Continuous line, untreated Cells; dashed line, PMA-treated cells. Means obtained from six different experiments _+ SD.
50
i 0 hrs
i 24 hr$
L 48 hrs
I 72 hrs
Time
Fig. 3. Percentage of S-phase cells after PMA treatment. The decrease is most significant at 24 h of treatment. The data presented are the mean of four different experiments +_ SD. 267
CD
g-
g. A
.
.
~'.-4
¢
B
-
o
,',.
g2 Aei)
ELI
Ei LS '
"2/M
Es ~
I
200 400 600 FL2-H',FL2-He igh t --- >
800
Ls
¢'(X) ~CDT I
~)~
gg
.
I
,..-i .._I t*.
GO/GI '
0
/
0
tOOO
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'
'
i
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.
.
200
.
.
.
.
i
400
'
600
FL2-HxFL2-Height --- >
,
'
,
I
. . . .
800
1000
Fig. 4. BrdU/P| analysis of the cell cycle by flow cytometry. (A) control, (B) after 24 h of PMA treatment. Es, early S; Ms, middle S; Ls, late S.
in the treated samples is closer to that of the untreated controls (Fig. 3). Moreover, the increase of cells in the early S compartment of the cycle is no longer evident after 48 and 72 h of PMA treatment. TdT activity under the same experimental conditions decreases from 98.5 U/ml (control cells) to 15.5 U/ml after 24 h and to 7.5 U/ml after 72 h of PMA treatment (Fig. 5). Immunocytochemical analysis shows that following PMA treatment, TdT levels decrease, too. This effect is related to the time of PMA treatment since > 9 8 % of the cells are TdT-positive in controls,
while 60% of the cells are positive at 24 h and < 30% at 72 h (Fig. 6). Although the enzyme activity in the untreated samples slightly decreased as a function of time, paralleling the decrease in the proliferation rate, the percentage of TdT-positive cells does not decrease, indicating a reduction in the amount of enzyme per cell. Taken together, these data show that PMA induces the differentiation of KM-3 pre-B cells along the B lineage with phenotypic changes in the immunological marker profile [12]. A simultaneous decrease of cell prolif120
--
o"
100
1oo
o
:"
80
Q.
60
E
50
i-a~
40
20 o hrs
24 hrs
48 hrs
72
hrs
0 0 hrs
24 hrs
Time
48 hrs
control cells PMA treated cells
Fig. 5. Units of TdT activity contained in untreated and PMAtreated cells. Means obtained from six parallel cultures _+ SD.
268
T i m e
72 hrs
~Control
cells
m
cells
Trotted
Fig. 6. lmmunocytochemistry analysis of PMA-treated cells. The decrease of TdT-positive cells is detectable starting with the time of treatment. Means obtained from six different experiments 4- SD.
eration is particularly evident after 24 h treatment. A recruitment of proliferating cells occurs for longer treatments (up to 72 h), although the percentage of differentiating cells does not decrease. This suggests that besides the recovery of residual nondifferentiating cells, a proliferation of more mature elements is likely to take place. TdT activity and TdT-positive cells decrease as the differentiation to mature B cells proceeds, and this is due to down-modulation of enzyme synthesis, which can hardly be found in the cell nucleus after 72 h of treatment. The kinetics of TdT disappearance is slower than the decrease of S-phase cells, indicating that the two effects are independent aspects of the differentiation process induced by PMA.
Acknowledgements This work was supported by C N R grants P.F. ACRO, BTBS, and Ministerio del Lavoro-Fondi Infortuni.
References [1] Bollum, F.J. (1974) in: The Enzyme (P.D. Boyer, Ed.) Vol 10, pp. 145-176, Academic Press, New York. [2] Baltimore, D. (1974) Nature 248, 409. [3] Bollum F.J. and Chang, L.M.S. (1986) in: Advances in Cancer Research, Vol. 47, pp. 47-71, Academic Press, New York. [4] Desiderio, S.V., Yacopulos, G.D., Paskind, M., Thomas, E., Boss, M.A., Landau, N., Alt, F.V. and Baltimore, D.
(1984) Nature 311, 752. [5] Cooper, M.D. (1987) N. Engl. J. Med. 317, 1452. [6] Hamaoka, T. and Ono, S. (1986) Ann. Rev. Immunol. 4, 167. [7] Uckun, F.M. (1990) Blood 76, 1908. [8] Kincade, P.W., Lee, G., Pietrangeli, C.E., Hayashi, S. and Gimble, J. (1990) Annu. Rev. Immunol. 7, 111. [9] Loken, M.R., Shah, V.O., Dattilio, K.L. and Civin, C.I. (1987) Blood 70, 1316. [10] Worman, B., Anderson, J., Arthur, D., Silberman, T. and Le Bien, T.W. (1989) J. immunol. 142, 110. [I 1] Miller, A.R and Gralow, J. (1984) J. lmmunol. 133, 3408. [12] Sacchi, N., Van Le Bien, T., Trost, S., Breviario, D. and Bollum, F.J. (1984) Cell. Immunol. 84, 65. [13] Yoshida, N., Radbruch, A. and Rajewsky, K. (1987) EMBO J. 6, 2735. [14] Nishizuka, Y. (1984) Nature 308, 693. [15] Nishizuka, Y. (1989) Cancer 63, 1892. [16] Delia, D., Greaves, M.F., Newman, R.A., Sutherland, D.R., Minowada, J., Kung, P. and Goldstein, G. (1982) Int. J. Cancer 29, 23. [17] Pegoraro, L., Abrahm, J., Cooper, R.A., Levis, A., Lange, B., Meo, P. and Rovera, G. (1980) Blood 55, 859. [18] Tokumine, Y., Waki, K., Nishimori, Y., Kuratsune, H., lnoue, R., Nakamura, Y., Ueda, E., Machii, T. and Kitani, T. (1989) Leukemia 3, 516. [19] Visser, L. and Poppema, S. (1990) Br. J. Haem. 75, 359. [20] Schneider, U., Schwenk, H.U. and Bornkamm, G. (1976) Int. J. Cancer 19, 621. [21] Di Primio, R. and Bollum, F.J. (1987) Hematol. Pathol. 3, 173. [22] Bollum, F.J. (1975) Proc. Natl. Acad. Sci. USA. 72, 4119. [23] Chang, L.M.S. (1971) Biochem. Biophys. Res. Commun. 44, 124. [24] Vitale, M., Neri, L.M., Manzoli, L., Galanzi, A., Rana, R., Antonucci, A. and Papa, S. (1989) Histochemistry 93, 9.
269
0165 2478 / 93 / $ 6.00 ~© 1993 ElsevierSciencePublishers B.V. All rights reserved IMLET 01926
Phorbol ester-induced effects on cell cycle progression and terminal deoxynucleotidyltransferase (TdT) activity in KM-3 pre-B cell line O r i a n a T r u b i a n i a, R o b e r t o D i P r i m i o b, L o r i s Z a m a i c, D o m e n i c o B o s c o a, F.J. B o l l u m d a n d M a r c o Vitale e alstituto di Citomorfologia del C.N.R and bIstituto di Morfologia Umana Normale, Facoltgt di Medicina, Universitd di Chieti, Chieti, Italy; Clstituto di Anatomia Umana Normale, Facolt(~ di Medicina, Universitgl di Bologna, Bologna, Italy; dDepartment of Biochemistry, U.S.U.H.S., Bethesda, MD, USA, and eDipartimento di Scienze Biomediehe e Biotecnologie, Sezione Anatomia Umana, Universitd di Brescia, Brescia, Italy
(Received 4 January 1993; accepted 26 January 1993)
1.
Summary
Phorbol myristic acetate (PMA) is a tumorpromoting agent that has been shown to induce differentiation of human leukemia cells and of normal lymphoid cells. We have investigated the ability of PMA to induce inhibition of cell growth of the human KM-3 pre-B leukemic cell line by multiparametric analysis. Our results show that PMA treatment induces cell differentiation with the disappearance of terminal deoxynucleotidyltransferase and a decrease of cell growth, as evaluated by [3H]thymidine uptake. Flow cytometric analysis of BrdU incorporation shows that PMA is able to induce a modification of the cell cycle with a sharp decrease of the percentage of S-phase cells, which is more evident after 24 h of treatment. Comparison between the cell growth kinetics and TdT synthesis and activity shows that differentiated cells are still able to proliferate to a certain extent and that the TdT disappearance and the initial decrease of cell proliferation are two independent effects of PMA. Key words." KM-3; Terminal transferase; PMA; Differentia-
tion; Cell cycle; Flow cytometry Correspondence to: Dr. Oriana Trubiani, lstituto di Citomorfologia del CNR, Facolt~i di Medicina, Universitfi di Chieti, Via dei Vestini 12, 66100 Chieti, Italy.
2.
Introduction
Terminal deoxynucleotidyltransferase (TdT) is a unique D N A polymerase able to generate D N A sequences in the absence of template direction [1]. The enzyme is detected in immature cells of the hemopoietic system undergoing immunoglobulin and T-cell receptor gene rearrangement, suggesting that TdT may play a role in the somatic generation of diversity [2~,]. This consideration is supported by several observations showing that B cells derive from TdT-positive precursors which acquire cytoplasmic #-heavy chains at the pre-Bcell stage [5-7]. These data are derived mainly from analyses of the phenotype of bone marrow B-lymphocyte precursors and of immunoglobulin gene expression [8-10]. 12-O-Tetradecan0Yl phorbol myristate 13-acetate (PMA) is a tumor-promoting agent that induces immature hemopoietic cells to differentiate [10-13]. It has been demonstrated that PMA binds to protein kinase C, whose activation is responsible for most of the observed effects [14,15]. In this respect, PMA treatment of B or T leukemic lymphoblastoid cell lines with an immature cell phenotype has been able to induce differentiation to plasma cells or to mature T cells, respectively [10,16-19]. So far, most studies on the effects of PMA have only considered the initial and final stages of differen265
tiation of the immature cell lines. In order to define the kinetics of the effects of PMA on pre-Bcell proliferation in time course experiments, we examined the perturbation of the KM-3 cell cycle by flow cytometric analysis of bromodeoxyuridine (BrdU) incorporation. Since it has been reported that PMA treatment of different TdT-positive pre-T or pre-B cells induces cellular differentiation and TdT disappearance, we also analyzed TdT localization and enzyme activity in the same cell line. 3.
Materials and Methods
3.1. Cell cultures The KM-3 human lymphoblastoid cell line [20] was maintained in continuous suspension culture in RPMI 1640 medium supplemented with 10% FBS, 4 mM L-glutamine, 100 mM Na-pyruvate, and 25 mM Hepes. Cells were grown at 2.5 x 105/ml, with more than 98% viability as determined by the trypan blue exclusion test. During the log growth phase, the cells were treated with 16 nM PMA for times ranging between 24 and 72 h. Cell growth was evaluated both as total cell number and by [3H]thymidine uptake 24, 48 and 72 h after culture with PMA.
Tris-HC1 buffer pH 7.6 was used for 15 rain at room temperature.
3.3. Enzyme assay TdT activity was determined as described previously [23]. Briefly, cells (2 x 10v) washed with RPMI 1640 medium were lysed in 250 /al of 0.3 M KPi pH 7.4 by freeze-thawing. The lysate was cleared by centrifugation at 15 000 rpm for 30 rain at 4°C and aliquots were assayed using 0.5 mM d(pA)50 as initiator, 5 mM [3H]dGTP (50-150 cpm/pmol) as substrate, 8.0 mM MgC12, 1.0 mM /%mercaptoethanol, 0.1 mg/ml BSA, and 0.2 M K-cacodylate buffer pH 7.5. The reaction products were scored as acid-insoluble radioactivity on glass fiber filters. One unit of enzyme activity was defined as 1 nM d G M P incorporated in 60 min at 36°C.
3.4. FH]Thymidine uptake D N A synthesis in KM-3 cells was measured by adding 2/~Ci of [3H]thymidine (6.7 Ci/mmol) for the final 6 h of each treatment period. Cells were collected using a cell harvester, and [3H]thymidine uptake was measured in a liquid scintillation counter. All data are expressed as mean counts per minute of triplicate determinations.
3.2. Immunocytochemistrv 3.5. BrdU labelling The immunocytochemical localization of TdT was carried out as described previously [21]. Briefly, cells cultured for the different times in the presence of PMA and control cells were fixed for 45 min at room temperature with 1% paraformaldehyde in 0.1 M Na-cacodylate buffer at pH 7.6 immediately after cytocentrifugation. Slides were rinsed three times in fresh cacodylate buffer containing 2% sucrose and 0.1 M glycine and treated for 5 min with normal goat serum and then with 0.2% H202 in cacodylate buffer for 5 rain. Samples were incubated with a rabbit polyclonal anti-TdT antibody [22] for 30 min at room temperature, rinsed twice, and incubated with a goat anti-rabbit IgG peroxidase conjugate antibody. To detect sites of antibody reaction, a solution of 0.05% diaminobenzidine tetrahydrochloride (DAB) containing 0.015% H202 in 0.05 M 266
To evaluate the effect of PMA treatment on the cell cycle, BrdU incorporation was studied by flow cytometry as described [24]. Briefly cells were incubated with 100 ¢tM BrdU for 30 rain at 37°C in a 5% CO2 atmosphere and washed twice in PBS, followed by 70% ethanol fixation at 4°C for 30 min and rinsing in PBS. DNA was denatured with 4 N HC1 at room temperature for 30 min. The cells were then incubated in Na2B407 pH 8.5 for 5 min at room temperature, washed, and treated with Triton X-100 for 3 min. Finally, the cells were incubated with FITC-conjugated anti-BrdU monoclonal antibody for 30 min at 4°C and then counterstained with 5 /~g/ml propidium iodide (PI) before flow cytometric analysis.
3.6.
Flow cytometry and data analysis
Cell cycle analysis was performed by a FACStar PLus flow cytometer (Becton Dickinson, USA) equipped with an argon ion laser tuned at 488 nm, 200 mW output, for excitation of FITC and PI. Data were analyzed as the percentage of cells in different phases of the cell cycle (i.e., Go/ G] S G2/M) and as the mean channel of FITC fluorescence of BrdU-positive cells. Both anti-BrdU fluorescence and PI fluorescence were measured on a linear scale.
3.7.
48 hrs
72 hrs
Time control cells PMA treated ceLls
Fig. 2. [3H]Thymidineuptake in treated and untreated PMA cells. The cpm is defined as the mean of triplicate wells from four different experiments _+ SD.
Sources of materials
RPMI-1640, L-glutamine, PBS, and Na-pyruvate were from Gibco Laboratories (Grand Island, NY). Rabbit polyclonal antibody against 32-kDa calf thymus TdT protein was produced as described [22]. FITC-conjugated anti-BrdU monoclonal antibody was from Becton Dickinson. Peroxidase-conjugated goat anti-rabbit IgG and normal goat serum were from Litton Bionetics (Kensington, MD). All radiolabelled materials were from Amity PG (Geneva, Switzerland). PMA, BrdU, PI, and all other reagent grade materials were from Sigma (St. Louis, MO). 4.
24 h~s
Results and Discussion
The effect of 16 nM P M A treatment at different time intervals on the kinetics of KM-3 growth is shown in Fig. 1. Cell viability was al-
ways more than 98% as evaluated by trypan blue exclusion. The PMA-induced reduction of cell growth is already detectable after 24 h treatment, and this reduction increases with time. This effect is due to a decrease in the number of cells entering the S phase of the cycle following P M A treatment. Thus, both [3H]thymidine uptake (Fig. 2) and the percentage of BrdU-positive cells evaluated by flow cytometry (Fig. 3) show a decrease of the number of S-phase cells during the time interval considered. This effect is particularly marked 24 h after P M A treatment when a decrease in the percentage of S-phase cells occurs. These cells are mainly found in the early S (Es) region of the dot plot (Fig. 4). Although the inhibitory effect of PMA is still present after 72 h, the percentage of S-phase cells
300
E
~, 20o
[
x Io 100
be
0 Ihrs
241hrs
48 Ihrs
72 Ihrs
Time
Fig. l. Cell growth at different times determined as described under Materials and Methods. Continuous line, untreated Cells; dashed line, PMA-treated cells. Means obtained from six different experiments _+ SD.
50
i 0 hrs
i 24 hr$
L 48 hrs
I 72 hrs
Time
Fig. 3. Percentage of S-phase cells after PMA treatment. The decrease is most significant at 24 h of treatment. The data presented are the mean of four different experiments +_ SD. 267
CD
g-
g. A
.
.
~'.-4
¢
B
-
o
,',.
g2 Aei)
ELI
Ei LS '
"2/M
Es ~
I
200 400 600 FL2-H',FL2-He igh t --- >
800
Ls
¢'(X) ~CDT I
~)~
gg
.
I
,..-i .._I t*.
GO/GI '
0
/
0
tOOO
'
'
'
i
G2/M .
.
.
200
.
.
.
.
i
400
'
600
FL2-HxFL2-Height --- >
,
'
,
I
. . . .
800
1000
Fig. 4. BrdU/P| analysis of the cell cycle by flow cytometry. (A) control, (B) after 24 h of PMA treatment. Es, early S; Ms, middle S; Ls, late S.
in the treated samples is closer to that of the untreated controls (Fig. 3). Moreover, the increase of cells in the early S compartment of the cycle is no longer evident after 48 and 72 h of PMA treatment. TdT activity under the same experimental conditions decreases from 98.5 U/ml (control cells) to 15.5 U/ml after 24 h and to 7.5 U/ml after 72 h of PMA treatment (Fig. 5). Immunocytochemical analysis shows that following PMA treatment, TdT levels decrease, too. This effect is related to the time of PMA treatment since > 9 8 % of the cells are TdT-positive in controls,
while 60% of the cells are positive at 24 h and < 30% at 72 h (Fig. 6). Although the enzyme activity in the untreated samples slightly decreased as a function of time, paralleling the decrease in the proliferation rate, the percentage of TdT-positive cells does not decrease, indicating a reduction in the amount of enzyme per cell. Taken together, these data show that PMA induces the differentiation of KM-3 pre-B cells along the B lineage with phenotypic changes in the immunological marker profile [12]. A simultaneous decrease of cell prolif120
--
o"
100
1oo
o
:"
80
Q.
60
E
50
i-a~
40
20 o hrs
24 hrs
48 hrs
72
hrs
0 0 hrs
24 hrs
Time
48 hrs
control cells PMA treated cells
Fig. 5. Units of TdT activity contained in untreated and PMAtreated cells. Means obtained from six parallel cultures _+ SD.
268
T i m e
72 hrs
~Control
cells
m
cells
Trotted
Fig. 6. lmmunocytochemistry analysis of PMA-treated cells. The decrease of TdT-positive cells is detectable starting with the time of treatment. Means obtained from six different experiments 4- SD.
eration is particularly evident after 24 h treatment. A recruitment of proliferating cells occurs for longer treatments (up to 72 h), although the percentage of differentiating cells does not decrease. This suggests that besides the recovery of residual nondifferentiating cells, a proliferation of more mature elements is likely to take place. TdT activity and TdT-positive cells decrease as the differentiation to mature B cells proceeds, and this is due to down-modulation of enzyme synthesis, which can hardly be found in the cell nucleus after 72 h of treatment. The kinetics of TdT disappearance is slower than the decrease of S-phase cells, indicating that the two effects are independent aspects of the differentiation process induced by PMA.
Acknowledgements This work was supported by C N R grants P.F. ACRO, BTBS, and Ministerio del Lavoro-Fondi Infortuni.
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