(4) YOSHIDA S, TANAKA R, TAKAI N, ET AL: Local administration of autologous
(13) YOUNG JD-E, HENGARTNER H, PODACK ER, ET AL: Purification and charac-
lymphokine-activated killer cells and recombinant interleukin 2 to patients with malignant brain tumors. Cancer Res 48:5011-5016, 1988
terization of a cytolytic pore-forming protein from granules of cloned lymphocytes with natural killer cell activity. Cell 44:849-859, 1986 RUSSELL JH, DOBOS CB: Mechanism of immune lysis. II. CTL-induced nuclear disintegration of the target begins within minutes of cell contact. J Immunol 125:1256-1261, 1980. MUELLER-KLIESER W: Multicellular spheroids. A review on cellular aggregates in cancer research. J Cancer Res Clin Oncol 113:101^122, 1987 ACKER H: Microenvironmental conditions in multicellular spheroids grown under liquid-overlay tissue culture conditions. In Recent Results in Cancer Research, vol 95: Spheroids in Cancer Research (Acker H, Carlsson J, Durand R, et al, eds). Berlin: Springer-Verlag, 1984, pp 116-133 LADMAN AJ, MARTINEZ AO: Cell contacts and surface features of three murine tumors grown as multicellular spheroids. Eur J Cell Biol 45: 224-229,1987 CARLSSON J, BRUNK U: The fine structure of three-dimensional colonies of human glioma cells in agarose culture. Acta Pathol Microbiol Scand [A] 85:183-192, 1977
(5) OKAMOTO Y, SHIMIZU K, TAMURA K, ET AL: An adoptive immunotherapy of
(14)
patients with medulloblastoma by lymphokine-activated killer cells (LAK). Acta Neurochir (Vienna) 94:47-52, 1988 (6) MERCHANT RE, MERCHANT LH, COOK SHS, ET AL: Intralesiona! infusion of
lymphokine-activated killer (LAK) cells and recombinant interleukin-2 (rIL-2) for the treatment of patients with malignant brain tumor. Neurosurgery 23:725-732, 1988
(/5) (16)
(7) BARBA D, SARIS SC, HOLDER C, ET AL: Intratumoral LAK cell and.
interleukin-2 therapy of human gliomas. J Neurosurg 70:175-182, 1989 (8) HOOK GR, GREENWOOD MA, BARBA D, ET AL: Morphology of interleukin-
2-stimulated human peripheral blood mononuclear cells killing gliomaderived tumor cells in vitro. J Natl Cancer Inst 80:171-177, 1988
(17)
(9) JAASKELAINEN J, KALLIOMAKI P, PAETAU A, ET AL: Effect of LAK cells
(18)
(19) GLIMELIUS B, NORLING B, NEDERMAN T, ET AL: Extracellular matrices in
multicellular spheroids of human glioma origin: Increased incorporation of proteoglycans and fibronectin as compared to monolayer cultures. APMIS 96:433-444, 1988 (20) NEDERMAN T, CARLSSON J, KUOPPA K: Penetration of substances into
tumour tissue. Model studies using saccharides, thymidine and thymidine5'-triphosphate in cellular spheroids. Cancer Chemother Pharmacol 22: 21-25, 1988
Phorbol Ester-Induced, Cell-Cycle-Specific, Growth Inhibition of Human B-Lymphoma Cell Lines Margaret Beckwith,* Dan L. Longo, Catherine D. O'Cornell, Chantal M. Moratz, Walter J. Urba
The activation, growth, and differentiation of three B-celland one non-B-cell-derived human lymphoma cell lines were examined after treatment with protein kinase C-activating phorbol esters. Treatment with these agents resulted in early activation events similar to those observed in normal B cells. However, in contrast to their growth-promoting effect on normal human B lymphocytes, exposure to these phorbol esters induced profound growth inhibition of the three Bcell-derived lymphoma lines. Maximal inhibition was achieved within 24 hours of culture initiation and could be reversed if the phorbol ester was removed after 12, but not 20, hours in culture. Cell-cycle analysis of phorbol ester-treated lymphoma cells revealed a G,/S block in one line, whereas cells from the other two lines accumulated in G2/M. These data demonstrate that protein kinase C-binding phorbol esters can interrupt the cell cycle in two places in actively dividing human B-lymphoma cells. These findings may prove valuable with regard to potential therapy of human malignant lymphomas. [J Natl Cancer Inst 82:501-509,1990]
Vol. 82, No. 6, March 21, 1990
In murine and human systems, it has been shown {1-3) that resting B lymphocytes can be activated polyclonally by external signals that mimic antigen, including antibodies directed to surface immunoglobulin (Ig). The mechanisms by which surface molecules transmit extracellular signals through the membrane to
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against three-dimensional tumor tissue. In vitro study using multi-cellular human glioma spheroids as targets. J Immunol 142:1036-1045, 1989 (10) CARLSSON J, YUHAS JM: Liquid-overlay culture of cellular spheroids. In Recent Results in Cancer Research, vol 95: Spheroids in Cancer Research (Acker H, Carlsson J, Durand R, et al, eds). Berlin: Springer-Verlag, 1984," pp 1-23 (//) MATTER A: Microcinematographic and electron microscopic analysis of target cell lysis induced by cytotoxic T lymphocytes. Immunology 36: 179-197, 1979 (12) CARPEN O, VIRTANEN I, SAKSELA E: infrastructure of human natural killer cells: Nature of the cytolytic contacts in relation to cellular secretion. J Immunol 128:2691-2697, 1982
Received August 21, 1989; revised December 18, 1989; accepted December 28, 1989. Supported in part by Public Health Service contract N01-CO-74102 from the National Cancer Institute (Program Resources, Inc.), National Institutes of Health, Department of Health and Human Services. M. Beckwith, C. D. O'Connell, C. M. Moratz, W. J. Urba (Clinical Immunology Services, Program Resources, Inc.), D. L. Longo (Biological Response Modifiers Program, Division of Cancer Treatment), National Cancer Institute, Frederick Cancer Research Facility, Frederick, MD. We thank Dr. Bill Kopp, Louise Finch, Ken Green, and Gina Stievenart for their contributions to the flow cytometry analysis; Dr. Suzanne Beckner for the methyl-12-0-tetradecanoylphorbol-13-acetate; and Hamblin Phillips for his assistance with the computer graphics. We also thank Debbie Beachley for her assistance in the preparation of this manuscript. Correspondence to: Margaret Beckwith, Ph.D., Bldg. 567, Rm. 213, Frederick Cancer Research Facility, Frederick, MD 21701-1013.
ARTICLES 5 0 1
We have recently begun to investigate the activation, growth, and differentiation responses of several human lymphoma cell lines to agents previously shown to result in growth of normal human B cells. In this study, we examined the response of three human B-lymphoma cell lines and one lymphoid cell line of uncertain lineage to PKC-binding phorbol esters, such as PMA and PdBu and the Ca 2+ ionophore ionomycin. We demonstrate here that treatment of the B-lymphoma cells with these agents resulted in changes similar to those associated with early activation of normal B lymphocytes such as increased expression of mRNA for the oncogene c-fos and cell-surface expression of class II major histocompatibility (MHC) antigens. Furthermore, although the phorbol esters significantly inhibited the growth of all three B-lymphoma lines without causing direct cytotoxicity, the mechanism appears to differ from line to line. One line exhibited a G,/S block after exposure to phorbol ester, but the other two were stopped in G2/M. Near maximal inhibition occurred within 24 hours after culture initiation and could be reversed if the phorbol ester was removed 12, but not 20 or 24, hours after culture initiation.
Materials and Methods Cell Lines and Culture Conditions The four cell lines, RL, HT, DB, and SR, were grown from the ascites or pleural effusion of four patients with lymphoma. The lines are passed twice weekly in RPMI-1640 (CellGro, Mediatech, Washington, DC), containing 2 mA/ L-glutamine, 1,000 U of penicillin/mL, 100 fig of streptomycin/mL, and 10% fetal bovine serum (GIBCO Laboratories Inc., Grand Island, NY). No exogenous growth factors are added. All four lines have been shown by Southern blot analysis to be Epstein-Barr virus genome negative. Reagents PMA (Sigma Chemical Co., St. Louis, MO) was dissolved in 100% ethanol, and PdBu (Sigma Chemical .Co.) was dissolved in 100% dimethyl sulfoxide (DMSO) (Fisher Scientific, Fair Lawn, NJ); both stocks of 100 ng/mL were stored in the dark at - 2 0 °C. Methyl-12-O-tetradecanoylphorbol-13-acetate (M-TPA), a nonPKC-binding analog of PMA, was stored at 4 °C in 100% ethanol
502
at 10 n-g/mL, and ionomycin (Behring Diagnostics, Calbiochem Biochemicals, La Jolla, CA) was kept in the dark at 4 °C in 100% DMSO at 5 mg/mL. Proliferation Assays Lymphoma cell lines were split 18-24 hours before assays were performed. Cells were resuspended in culture medium to a concentration of 1 x lO^mL, and 100 jxL was plated in 96-well, flat-bottomed, microtiter plates (Costar Inc., Cambridge, MA) already containing 100 |xL of appropriately diluted reagents. Sixty to 72 hours later, 100-p.L aliquots of the supernatants were removed for enzyme-linked immunosorbent assay of Ig secretion, and 1 p,Ci of [3H]thymidine/well [sp act, 6.7 Ci/mmol; (New England Nuclear Research Products, Boston, MA)] was added for the final 8 to 18 hours of culture. In some experiments, cells were exposed to PdBu for various lengths of time, washed thoroughly, and plated in microtiter wells as described above. [3H]Thymidine was added 8 hours before harvesting at 72 hours. Cultures were harvested onto glass fiber filters with a PhD Cell Harvesting system (Cambridge Technology, Inc., Cambridge, MA), and [3H]thymidine uptake was assessed by liquid scintillation on an LKB beta counter (LKB Instruments, Inc., Turku, Finland). Flow Cytometry Analysis Lymphoma cells were incubated for 24, 48, or 72 hours with phorbol ester before being stained for flow cytometry. Cells were aliquoted at 1 x 106 per well of a 96-well, round-bottomed microtiter plate (Costar Inc.), and incubated for 30 minutes in phosphate-buffered saline (PBS) containing 1% bovine serum albumin, 0.1 % sodium azide, and 5% human AB serum to block potential Fc receptor-mediated binding. Plates were spun for 1 minute at 1,500 rpm, and liquid was aspirated from the cells. Then 20 |xL of fluorescein isothiocyanate (FITC)-labeled antibodies was added directly to the cell pellet and incubated for 30 minutes at 4 °C. Cells were then washed once, transferred to 10x 75-mm tubes, and analyzed on an Ortho Cytofluorograf 50. The labeled antibodies were obtained from several sources: FITC-B1 antibody (Coulter Immunology, Hialeah, FL); FITCHLA-DR (Becton Dickinson, Mountain View, CA); and FITCgoat anti-IgM [human mu chain specific; (Cappel Laboratories, Cooper Biomedical, Inc., Malvern, PA)]. Cell-Cycle Analysis Cell-cycle analysis was performed as described by Pallavicini et al. (79). Cells were treated with phorbol ester for 24 hours, washed, and placed in serum-free media containing 10 mA/ of bromodeoxyuridine (Sigma Chemical Co.) for 30 minutes at 37 CC. This thymidine analogue is incorporated into DNA as the cells pass through S phase. The cells were then fixed for 30 minutes with 70% ethanol and permeabilized with detergent (PBS containing 0.5% Tween 20); the DNA was partially denatured by incubation with 2% HC1 for 30 minutes. Then they were stained with an FITC-labeled antibody specific for bromodeoxyuridine incorporated into single-stranded DNA (Becton Dickinson) and with propidium iodide, which binds to all doublestranded DNA present in the cells. Analysis for green and red fluorescence was done on a Coulter Profile. Doublet discrimination was performed by setting a bit map around a population Journal of the National Cancer Institute
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result in specific activation events are not completely understood. There is evidence to suggest that anti-Ig-mediated B-cell activation occurs at least in part via the phospholipase C-mediated hydrolysis of phosphatidyl-inositol 4,5-bisphosphate to second messenger compounds inositol 1,4,5 trisphosphate (4) and diacylglyceride (5). By using phorbol esters such as phorbol myristate (PMA) and phorbol 12,13 dibutyrate (PdBu), which have been shown to bind directly to protein kinase C (PKC) (6,7), and Ca2+-mobilizing ionophores, such as ionomycin, one can modulate the pathways of these two second messengers independently. Thus several groups (8-13) have demonstrated that treatment of resting B cells with phorbol esters resulted in early Go to G, activation events similar to those observed with anti-Ig treatment of murine and human B cells. Furthermore, PMA alone was shown to be mitogenic for normal human B lymphocytes (14,15), and a synergistic response similar to that observed for' murine B lymphocytes (16) was obtained with a combination of PMA and ionomycin (17,18).
Table 1. Cell-surface phenotype of human lymphoma cell lines* Cell lines
Cluster designation
Antigen
Bl Leu 12
B2 Leu 14 HLA-DQ HLA-DR Kappa Lambda
IgM IgG IgD TAC TcR
Normal distribution
RL
CD20 CD19 CD21 CD22 —.
DB
SR
B cells B cells Mature B cells B cells - B cells, monocytes B, monocytes, activated T cells slg/B cells slg/B cells slg/B cells slg/B cells slg/B cells Activated T, B, natural killer cells Mature T cells Leukocytes
— — — — — — CD25
— —
HLe-1
HT
*slg = surface immunoglobulin. TcR = T-cell receptor.
Effect of Phorbol Esters on Expression of Cell Surface Markers
In both the murine (12) and the human (13) systems, increased cell surface expression of class II MHC antigens has been associated with early B-cell activation, and levels of CD20 have Characteristics of Human Lymphoma Lines been shown to increase after phorbol ester treatment (20). The cell lines that have been used in these experiments were Therefore, we examined the four lymphoma cell lines for the derived from four patients with diffuse, large cell lymphomas. effects of phorbol esters on the expression of several surface Tables 1 and 2 outline some of the characteristics of each cell line, markers including class II MHC antigens, CD20 (B1), and Ig. As thus identifying the RL, DB, and HT cells as being of B-cell shown in figure 1, incubation of the RL cells with PMA resulted origin. Each of these lines is different: RL expresses surface IgM in a significant increase in levels of Bl and HLA-DR. In contrast lambda and IgD and secretes low levels of IgM; DB secretes IgG lambda; HT expresses mRNA for both mu and kappa and expresses low levels of kappa on the surface. We are currently analyzing the expression of other heavy-chain isotypes by this HLA-DR Bl SKi RLr~7T cell line. The SR cell line is of undetermined cellular origin because it expresses no markers unique to B, T, natural killer, or monocyte-lineage cells (data not shown). It does express high \ levels of class II MHC antigens, and the 55-kilodalton interleuHT kin-2 receptor TAC, as well as the general leukocyte marker HLe-1. We have used these cell lines as a means to investigate the response of malignant B cells to signals involved in the activation, proliferation, and differentiation of normal human B lymphocytes.
Results
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defined by the peak versus integral propidium iodide signal, thereby eliminating cell doublets from the analysis.
SR
Table 2. Characterization of Ig and TcR genes in human lymphoma cell lines Cell lines OREEN FLUORESCENCE
Ig/TcR gene
RL Secreted Ig Rearranged Ig genes Rearranged TcR beta mRNA TcR beta (1.0 kilobase)
Vol. 82, No. 6, March 21, 1990
IgM
HT
DB IgG
SR Figure 1. Phorbol ester-induced changes in cell-surface expression of HLA-DR Bl, and surface Ig. Lymphoma cells were cultured for 72 hr in the presence of M-TPA (dotted lines) or PMA (solid lines) before being stained directly with FITC-HLA-DR (A, D, G, J), FTTC-B1 (B, E, H, K), or FITC-anti-surface IgM (C, L) or IgG (F, I). Unstained cells are shown as a dashed line in each panel. Data shown for RL and SR are from the same experiment as are those for HT and DB, and each are representative of two or three identical expts.
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Effects of Phorbol Esters on Proliferation of Lymphoma Cell Lines The PKC-binding phorbol esters, such as PMA and PdBu, have been shown to stimulate normal human B lymphocytes to proliferate (14,15). However, when the human B-lymphomacell lines RL, HT, and DB were treated with these phorbol esters, their growth was dramatically inhibited (fig. 2A-C). For each of
0
Effect of PMA in Combination With Ionomycin on Cell Growth Significant proliferation of normal B lymphocytes can be induced with a combination of PKC-binding phorbol esters and Ca 2+ ionophores such as ionomycin (16-18). Although it is clear that PMA alone had significant effects on the B-lymphoma cells, we wished to determine if a synergistic interaction could be observed by using subthreshold PMA concentrations in the presence of ionomycin. In the experiments shown in figure 4, one can see that, if RL cells were treated with 0.1 ng of PMA/mL in conjunction with 0.25, 0.5, or 1.0 |xg of ionomycin/mL, the proliferative response remaining after PMA treatment was com-
8. HT
, A. RL
i-
these lines, inhibition was nearly maximal at 1.0 ng of PMA or PdBu/mL. This agrees with other reports demonstrating activation of PKC at 1.0 ng of PMA/mL (21). Furthermore, M-TPA had no effect on cell growth, which indicated that activation of PKC is involved in the inhibition of growth. Interestingly, inhibition of the SR cell line was not observed, even with phorbol ester concentrations of 100 ng/mL or greater (fig. 2D). This finding suggested that the growth inhibition of the other lines induced by PMA is not due to direct cellular cytotoxicity. We examined this directly, and the results for the RL and SR cell lines are shown in figure 3. By 72 hours in culture with PMA, RL cell viability had decreased slightly from 100% to 80%; cell growth was almost completely inhibited by 24 hours in culture with PMA but not with M-TPA. Similar results were obtained for the HT and DB cell lines (data not shown). Growth of SR cells was unaffected by PMA or M-TPA, with viability remaining between 90% and 95% and cell growth occurring normally at each time point.
.001
.01
0.1
1.0
10
.001
.01
0.1
1.0
10
100
.001
.01
0.1
.001
.01
0.1
1.0
10
100
10
100
a. o
100
0
Figure 2. Phorbol esterinduced growth inhibition of B-lymphoma cell lines. Lymphoma cells were plated at I x 104 cells/well in the presence of various concentrations of M-TPA (•) or PMA ( A ) . Plates were labeled with 1 u.Ci of [3H]thymidine/well 64 hr after initiation of culture and were harvested 8 hr later. Data are expressed as counts per minute and are representative of six to nine expts for each cell line.
PHORBOL ESTER CONCENTRATION ( n g / m l )
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Journal of the National Cancer Institute
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(fig. 1C), surface IgM decreased dramatically; levels of mRNA for the mu Ig heavy chain began to drop in RL cells after 24 hours in culture with PMA and continued to decline until 48 hours (data not shown). Although there was no significant shift in intensity of staining for class IIMHC antigens in PMA-treated HT cells, there was a significant increase in CD20 expression. Surprisingly, PMA was able to induce low levels of expression of HLA-DR class II MHC antigens on DB, which is normally negative for both HLA-DR and -DQ (table 1). Similar to results observed for RL cells, CD20 expression also increased and surface IgG decreased. The decrease in surface IgG was not observed until 72 hours of culture. No effect was observed with M-TPA, the non-PKC-binding analogue of PMA, thus indicating that early activation of PKC may be involved in these responses. In contrast to these three B-cell lines, the level of HLA-DR expression on the SR cell line was not significantly altered in the presence of PMA, nor did PMA induce expression of CD20 or surface IgM. Thus these data demonstrate that PMA induces changes in early gene expression in malignant B cells similar to those observed during activation of normal B cells. However, these changes do not necessarily predict the ultimate biologic consequences of PMA treatment, as will be demonstrated in the following sections.
responses obtained with PdBu were similar to those seen in PMA-treated cultures.
A. RL
so
100-
Reversibility of Inhibition by PdBu
40
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80-
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72
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24
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Cell-cycle analysis was performed by two-color immunofluorescence (79). Green fluorescence indicates uptake of bromodeoxyuridine into S-phase cells, whereas red fluorescence measures propidium iodide uptake by all double-stranded DNA. Figure 7
A. RL
B. HT
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pletely abolished. A synergistic inhibition was also observed with 0.01 ng of PMA/mL (data not shown). The HT cells demonstrated an additive effect of PMA and ionomycin, with a moderate degree of synergy observed only at the highest concentration of ionomycin. No synergy was observed with either the DB cell line or the SR cell line at any dose of PMA or ionomycin tested. All four cell lines were sensitive to concentrations of ionomycin alone greater than 0.25 (jug/mL for SR, and 1.0 (xg/mL for RL, HT, and DB (data not shown). Thus it appears that for the RL and HT cell lines, activation of PKC in conjunction with mobilization of intracellular Ca 2+ stores when a combination of very low concentrations of phorbol ester and ionomycin is used can result in a significant biologic response. In the DB cell line, the second signal seems to be less important, and similar growth inhibition occurs with either agent alone. Furthermore, in RL and HT, use of higher concentrations of either agent makes the second signal less effective. The ability of ionomycin to act with PMA is the greatest in RL, less effective in HT, and not present in DB, and therefore appears to decrease with the later stages of differentiation of the B-cell tumors.
Cell-Cycle Analysis
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Figure 3. Cell numbers and viabilities after culture with phorbol esters. We cultured 0.5 x 106 lymphoma cells in six-well plates in the presence of media ( • ) , M-TPA (•), or PMA (A). At 24,48, and 72 hr afterculture initiation, cells were harvested and counted (closed symbols), and viability was determined by trypan blue dye exclusion (open symbols). Data are representative of three expts for each cell line.
Next we wished to determine if the phorbol ester-induced growth inhibition was reversible. PdBu was used because it is less lipophilic than PMA and is more easily washed out of the cells (7). RL cells were treated with DMSO or PdBu for various lengths of time, washed five times, plated at 1 x lO'Vwell, and incubated for an additional 72 hours. [3H]Thymidine was added during the final 8 hours of culture. It is clear from the data shown in figure 6 that if cells were removed from PdBu at 12 hours or before, growth inhibition was not observed. However, cells cultured with PdBu for 20 hours or longer were irreversibly committed to growth arrest. Taken together, these data suggest that in RL cells, a PKC-mediated event occurring between 12 and 20 hours after initial exposure to phorbol ester results in an irreversible inhibitory signal that prevents the cells from proliferating.
100A.
Kinetics of Growth Inhibition To begin addressing potential mechanisms involved in phorbol ester-induced growth inhibition, we examined the growth kinetics of the four cell lines after exposure to PdBu. We used DMSO as a diluent control for the PdBu, and similar results have been obtained with the PMA diluent ethanol. Cells were incubated for 24,48, or 72 hours in the presence of DMSO, PMA, or PdBu (fig. 5). For RL, HT, and DB, nearly maximal inhibition of [3H]thymidine uptake was observed by 24 hours after culture initiation, whereas SR remained unaffected by PMA throughout 72 hours in culture. These data are consistent with the viable cell numbers shown in figure 3 and demonstrate that a powerful inhibitory signal is generated within the first 24 hours of exposure to the phorbol ester. These results also demonstrate that the Vol. 82, No. 6, March 21, 1990
80604020-
250 500 750 1000 0 250 500 750 1000 IONOMYCIN CONCENTRATION (ng/ml) Figure 4. Increased growth inhibition observed with PMA plus ionomycin. We cultured 1 x 104 lymphoma cells for 72 hr with various concentrations of ionomycin in the presence ( A ) or absence (A) of O.I ng of PMA/mL. [3H]Thymidine was added 8 hr before harvesting. Data are expressed as percent control proliferation relative to a media control and are representative of three expts.
ARTICLES
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A. RL
B. HT
125
Figure 5. Time course of phorbol ester-induced growth inhibition. We plated 1 x 104 lymphoma cells with various concentrations of DMSO (•), PdBu (•), or PMA (A), and 1 ^.Ci/well of [3H]thymidine was added 8 hr before harvesting at 24, 48, and 72 hr. Data are expressed relative to a media control for each time point and are an average of two to three expts. Similar results were obtained with the PMA diluent ethanol as a control instead of DMSO.
D. SR
A
A PUA
• — • PDBU 24
36
48
60
72
12
24
36
48
60
HOURS IN CULTURE
illustrates cell-cycle analysis of RL, HT, and DB cells after 24 hours in culture with PMA. RL cells cultured with ethanol showed approximately 40% of the cells in GQ/G, , 36% in S phase, and 19% in G2/M (fig. 7A). After a 24-hour exposure to PMA, the number of cells in GQ/G, increased to 66%, but in S phase, it decreased to approximately 8% (fig. 7B). The proportion in G2/M remained approximately the same. Surprisingly, the DB (fig. 7C-D) and HT (fig. 7E-F) cell lines showed a different pattern of cell-cycle progression, with most of the PMA-treated cells accumulating in G2/M instead of GQ/G,. These data suggest that in RL, PMA induces a block in the cell cycle before cells enter the S phase, whereas in DB and HT, the block occurs in the G2/M phase
Figure 6. Reversibility of phorbol ester-induced inhibition of RL cells. RL cells were exposed to 10 ng or equivalent volume of PdBu (•) or DMSO/mL (•), respectively, for 4, 12, 20, 24, or 48 hr. Cells were then washed five times, and 1 x 104 were plated in microtiter wells for an additional 72 hr of culture. [3H]Thymidine was added 8 hr before harvesting. Data represent an average of four expts and are expressed relative to a media control for each time point.
506
of the cell cycle, thus preventing mitosis and progression through the cycle.
Discussion Recently, we began to investigate the requirements for activation, proliferation, and differentiation of malignant human B lymphocytes. We measured the biologic responses of a panel of human B-cell lymphomas to agents that have been shown to influence normal B-cell development. In this report, we described the responses of three B-cell-derived lymphoma cell lines (RL, HT, and DB) and one cell line of undetermined lineage (SR) to several PKC-activating phorbol esters. It has recently been demonstrated in human and murine B cells that the sequence of anti-Ig-stimulated activation events occurs, at least in part, through hydrolysis of phosphatidyl inositol to diacylglyceride and 1,4,5-trisphosphate and subsequent activation of PKC (3); therefore, activation can be mimicked through the use of PKC-binding phorbol esters alone or in conjunction with calcium-mobilizing agents (8-13). Thus PMA treatment of murine (70) or human (11) B cells resulted in increased expression of mRNA for the oncogenes c-fos and c-myc, as well as increased mRNA and cell-surface expression of class II MHC antigens (12,13). This paper contains a description of our findings that similar early events occurred following phorbol ester treatment of human B-lymphoma cells. Two of the three B-cell lines showed increased cell-surface expression of class II MHC antigens, and all three manifested large increases in the human B-cell marker CD20, detected by the Bl monoclonal antibody (fig. 1). Furthermore, RNA from PMA-treated RL and SR cells demonstrated early increases in expression of the c-fos oncogene (data not shown), an event that has been associated with early PKC-mediated activation (22,23). Therefore, because all three Journal of the National Cancer Institute
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• — • OMSO
G 1 40.1 S 36.3 G 2 19.2
B
G1 65.8 S 8.0 G2 20.0
G1 26.9 S 24.2 G2 41.2
8
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Red Fluorescence Figure 7. Cell-cycle analysis of phorbol ester-treated lymphoma cells. RL (A, B), HT (C, D), or DB (E, F) cells were incubated overnight with 10 ng of PMA/mL (B, D, F) or with an equivalent amount of ethanol (A, C, E). Cells were then treated with bromodeoxyuridine and stained with an FITC-antibromodeoxyuridine monoclonal antibody (green fluorescence), followed by propidium iodide (red fluorescence), as described in the Materials and Methods section. A representative experiment is shown for each cell line except SR, for which no difference between treated and untreated cells was seen.
B-cell lines are significantly growth inhibited by PMA and normal B cells and SR cells are not, no correlation is apparent between the effect of PMA on early activation events and proliferation. A similar dissociation of anti-Ig-induced increases in c-fos expression (10), Ca 2+ levels (24,25), and inositol phosphate hydrolysis (25) from normal B-cell proliferation has been described in murine (10,24) and human (25) systems. In addition to these early activation events, PMA and ionomycin have been shown to promote synergistically cellular proliferVol. 82, No. 6, March 21, 1990
Second, increasing evidence suggests that normal cells and their malignant counterparts of the same lineage demonstrate opposite responses to activating signals. Investigators demonstrated that the growth of murine T-cell hybridomas was irreversibly blocked at the G,/S interface when they were exposed to specific antigen (25) or to antibodies directed to the TcR (29), both of which are growth-promoting signals for normal T lymphocytes. This was termed "activation-induced growth inhibition" (29). In a B-cell system, several investigators reported the inhibitory effect of anti-Ig antibodies on the murine B-lymphoma cell line WEHI-231 (30-33) and a panel of antigen-specific B-lymphoma cell lines (34). Similarly, we observed anti-IgMmediated growth inhibition of the RL cell line (manuscript in preparation). Scott and co-workers demonstrated that anti-Ig induced a G,/S cell-cycle block in WEHI-231 (31) and that this could be overcome by the early addition of T-cell-derived factors (32). Recently, Page and DeFranco (33) showed that growth arrest of WEHI-231 could also be induced by phorbol esters and ionomycin; however, several other similar B-cell tumors were not susceptible to phorbol ester-mediated growth inhibition (33).
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ation of normal murine (16) and human (17,18) B lymphocytes, as well as chronic lymphocytic leukemia B cells (26). In the human system, several investigators (14,15) have shown that PMA alone can stimulate purified normal B cells to divide. In contrast to PMA-induced proliferation, we demonstrated that although 1 ng of PMA or PdBu/mL resulted in profound inhibition of the growth of the three B-lymphoma cell lines RL, DB, and HT, no such effect was seen in proliferation of the SR line (fig. 2). Furthermore, increased inhibition was observed in the RL and HT cells when a combination of PMA and ionomycin was used, effectively lowering the concentration of PMA required for maximal inhibition and suggesting that a requirement for calcium can be overcome with higher concentrations of phorbol ester (fig. 4). There are several nonexclusive interpretations of these observations. First, it is possible that what is being measured is phorbol ester-induced terminal differentiation of the B-cell lines to plasma cells. Chronic lymphocytic leukemia cells can be induced to differentiate by phorbol esters alone (27) or in combination with Ca 2+ ionophores (26). After exposure to these agents, such cells demonstrate increased Ig secretion, decreased surface Ig, and decreased levels of other antigens such as B1 and B2 (26,27). In contrast, we observed a significant inhibition of IgM secretion from the RL cells and a moderate decrease in IgG secretion from DB (data not shown), accompanied by PMA-induced increases in HLA-DR and Bl (markers not usually found on plasma cells), which remained elevated throughout 72 hours in culture (fig. 1). Therefore, there does not appear to be a link between differentiation and growth arrest in these cells.
Our data are consistent with this interpretation. However, it is also possible that what we observed is a differential susceptibility of continually cycling cells versus resting cells to a critical activation event. Breitmeyer and associates (35) recently reported that growth of phytohemagglutinin-stimulated human T lymphocytes could be inhibited by the addition of anti-TcR antibodies, and Nau et al. (36) found inhibition of proliferation of interleukin2-driven murine T-cell clones by high concentrations of immobilized anti-TcR monoclonal antibody. In addition, in a nonlymphocyte system, Huang et al. (21) demonstrated that the addition of PMA to a-thrombin-stimulated vascular smooth muscle cells ARTICLES
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In summary, we have demonstrated that PKC-binding phorbol esters induce a profound growth inhibition of three human B-lymphoma cell lines. It is clear that differences exist in the pattern of response of normal cells and some malignant cells to the same external stimuli. This system should allow us to investigate precise biochemical events important to cell proliferation , and it may be a useful approach to our understanding of the cellular response of normal lymphocytes and their malignant counterparts. We are currently investigating the effects of natural PKC-activating compounds such as the bryostatins (43,44) as potential therapeutic agents for treatment of human malignant lymphomas. It is also possible that some antitumor effects observed in patients treated with anti-idiotypic antibodies (45) are related to direct inhibition of cell proliferation through signal transduction involving PKC activation.
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of calcium ionophore A23187 and phorbol ester TPA on B-chronic lymphocytic leukemia cells. Blood 70:1536-1542, 1987 (27) GORDON J, MELLSTEDT H, AMAN P, ET AL: Phenotypic modulation of chronic
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Journal of the National Cancer Institute
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resulted in profound growth inhibition at the G,/S interface. These authors suggested that the activation of PKC in resting cells resulted in cell growth, but the activation of PKC during G, led to growth inhibition. In all three B-lymphoma cell lines, maximal phorbol ester-induced inhibition occurred by 24 hours in culture (fig. 5). In the RL cell line, inhibition could be prevented if the activating agent was removed before 20-24 hours in culture (fig. 6). These data, as well as those of Ashwell et al. (28) and Page and DeFranco (33), suggest that a critical event, which is probably PKC mediated, occurs at some point during G, to prevent RL cells from undergoing DNA synthesis. This was confirmed by cell-cycle analysis of PMA-treated RL cells that demonstrated a block in G, that prevented cells from entering S phase (fig. 7). Interestingly, although cell-cycle analysis of DB and HT also revealed a decrease in the number of S-phase cells, there was a significant increase in the number of cells in G2/M. Thus it appears that PMA is able to block cell-cycle progression at two points in the cycle. Evidence has shown that Chinese hamster ovary cells treated with doxorubicin (37), cisplatin (38), or inhibitors of topoisomerase II (39,40) are arrested at the G2/M interface. A reduction in topoisomerase II has been associated with PMA-induced differentiation of HL-60 cells (40) and therefore may be mediated via PKC. Furthermore, it has recently been proposed that phosphorylation of a mammalian protein (p34 CDC2 ), homologous to the yeast cell-cycle control protein cdc2+, may be involved in regulation of exit from (G2/M) and re-entry into (G,) the mitotic cycle (41,42). It is possible that PKC activation by phorbol ester results in phosphorylation of a key regulatory protein that functions at control points in G, and in G2/M in these lymphoma lines, thus halting their progression through the cell cycle. Because RL appears to be less differentiated than does HT or DB, the point at which the regulatory event occurs may be a function of the stage of differentiation of a cell; a comparison of the phosphorylated substrates in these two cell types after PKC activation will be important.
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proliferation of murine T lymphocyte clones by supraoptimal levels of immobilized anti-T cell receptor monoclonal antibody. J Immunol 139:114-122, 1987
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