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Role of phospholipase A2 and prostaglandin E in growth and differentiation of myeloid leukemic cells
Biochimica et Biophysica Acta, 720 (1982) I 1 I - I 19 Elsevier Biomedical Press
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BBA 11013
R O L E OF P H O S P H O L I P A S E A 2 AND P R O S T A G L A N D I N E IN G R O W T H AND
D I F F E R E N T I A T I O N O F MYELOID LEUKEMIC CELLS RABI SIMANTOV and LEO SACHS
Department of Genetics, Weizmann Institute of Science, Rehovot 76100 (Israel) (Received Julf20th, 1981)
Key words: Phospholipase A 2," Prostaglandin E; Growth regulation; Differentiation; Tumor promoter," (Leukemic cell) P h o s p h o l i p a s e A 2 activity and prostaglandin E synthesis have been studied in different clones of myeloid
leukemic cells, which differ in their competence to be induced to differentiate by the macrophage and granulocyte differentiation-inducing protein or the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA). Clones that could be induced to differentiate by this protein showed a higher basal phospholipase A 2 activity than clones that could not be induced to differentiate by this protein inducer. Cell competence to be induced to differentiate by TPA did not show this correlation, and the clone with the least ability to respond to TPA showed the lowest number of binding sites for [20-3H]phorbol 12,13-dibutyrate. Differentiation induced by the protein was accompanied by a 7-14-fold increase in prostaglandin E synthesis, whereas differentiation induced by TPA did not show this increase. Externally added prostaglandin E 1 did not induce differentiation but inhibited cell proliferation and the degree of inhibition in the different clones was related to the basal phospholipase A 2 activity. The results indicate that increase of prostaglandin E synthesis was not an essential pre-requisite for differentiation, that prostaglandin E seems to be involved in the inhibition of cell proliferation in association with phospholipase A 2 , and that the differentiation-inducing protein and TPA can induce differentiation by different pathways. The amount of basal phospholipase A 2 activity was also related to previously found differences in the ability of the clones to develop desensitization to ~-adrenergic hormones or prostaglandin E 1.
Introduction The key role of phospholipase A 2 in the control of prostaglandin synthesis has been established in several tissues [1-4]. Activation of prostaglandin E synthesis by various external stimuli was associated with activation of phospholipase A 2. Studies with astrocytoma cells have indicated, that the activation of phospholipase A 2 in the local domain of the fl-adrenergic receptor may be involved in desensitization to fl-adrenergic hormones [5]. Experiments with myeloid leukemic cells have also shown an association between the ability of different clones of these leukemic cells to desensitize to fl-adrenergic hormones and to prostaglandin El, 0167-4889/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
and their ability to be induced to differentiate by the macrophage and granulocyte differentiationinducing protein. Clones that could be induced to differentiate by this protein ( M G I + D ÷ clones) desensitized to fl-adrenergic hormones and prostaglandin El, whereas clones that could not be induced to differentiate by this protein (MGI - D clones) could not desensitize to these hormones [6,7]. These clones of leukemic cells also differ in their ability to be induced to differentiate by the tumor promoter TPA, and cell competence to be induced to differentiate by the protein was not necessarily associated with cell competence to beinduced to differentiate by TPA [8]. The present experiments were, therefore, undertaken with these
112
different clones of myeloid leukemic cells to determine: (A) The relationship between basal phospholipase A2, hormone desensitization and cell competence for induction of differentiation by the protein differentiation inducer and the phorbol ester TPA; (B) the role of prostaglandin E synthesis in the control of cell growth and in cell differentiation induced by the protein or TPA, and (C) whether the protein and TPA may induce differentiation by different pathways. Materials and Methods
Cells and Cell culture. The leukemic cells used were clones of mouse myeloid leukemic cells isolated from a spontaneous or X-irradiation-induced myeloid leukemias [8]. Clones 7-M18 and 11 belong to the type of MGI + D + and clones 1 and 6 to the type of M G I - D , based on their sensitivity to be induced to differentiate in culture by the differentiation-inducing form of the macrophage and granulocyte-inducing protein, using the criteria for differentiation including lysozyme synthesis previously described [9,10]. Cells were cultured in Eagle's medium with a 4-fold concentration of amino acids and vitamins (H-21, Grand Island Biological Co.) and 10% inactivated fetal calf serum as described [6]. For the experiments cells were seeded at 2 - 5 . 10 4 cells per Petri dish (Nunc, 60 mm) in 5 ml medium. Total lysozyme was measured using the turbidometric assay with Mierococcus lysodeikticus as described [11] and protein concentrations were determined according to Lowry et al. [12]. The differentiation inducing protein (MGI-2 [10]) was obtained from Krebs ascites cells
[81. Phospholipase A 2 activity and thin layer chromatography. Basal phospholipase A 2 activity was tested according to Hong and Levine [13] with minor modifications. Cells grown to a density of 1 . 5 - 2 . 0 . 1 0 6 cells/ml culture medium were collected, washed once with culture medium and then incubated for 3 h at 5 : 10 6 cells/ml medium containing 2.5 nM 3H-arachidonic acid (New England Nuclear, 78.2 Ci/mmol). The cells were then centrifuged at low speed (500 × g), the medium removed, the cells washed with fresh culture medium and re-suspended in fresh medium at 2 . 6 - 3 . 0 - 10 6 cells/ml. At 0-120 min later, aliquots
of 2.5 ml of the cell suspension were taken, centrifuged (500 × g) and the amount of radioactivity released by the cells was determined. Thin layer chromatograms were developed according to Honma et al. [14] with a mixture of ethyl acetate/2,2,4-trimethyl pentane/acetic a c i d / H 2 0 ( 1 1 : 5 : 2 : 1 0 v/v), dried, cut into strips of 0.5-cm and the radioactivity determined. Binding assay of [20--~H]-phorbol 12,13dibutyrate to leukemic cells. The binding of [20 -3 H] -phorbol 12,13-dibutyrate to membrane preparations from the four clones of myeloid leukemic cells was performed essentially as described by Driedger and Blumberg [15] with minor modifications. Cells (4.108 ) were collected by low-speed centrifugation, washed with 100 vol. phosphatebuffered saline, and incubated for 15 min in icecold 5 mM Tris-HC1 buffer/5 mM MgC12 (pH 7.4 at 25°C). The cells were homogenized in a Thomas glass homogenizer type B (30 ups and downs)~ centrifuged for 10 min at 40000 × g, the supernatant removed, the pellets re-suspended in I0 ml of the same buffer and re-centrifuged. The pellets were then suspended in 4 ml of 50 mM Tris-HCl buffer, pH 7.4 at 25°C, containing 4 m g / m l bovine serum albumin (Sigma). Duplicate aliquots of 180 /tl of the homogenates were incubated for 20 min at 39°C with 12.5 or 25 nM [20-3H]phorbol 12,13dibutyrate (Lifesystems CO., specific activity 6.4 C i / m m o l ) in the presence or absence of 0.5/~g/ml TPA. It was found that the binding reached saturation within 20 min incubation. The binding was terminated by centrifugation for 10 min at 40000 × g, the supernatant was removed and the pellets washed with l ml Tris-HC1 buffer. They were then dried, lysed in 0.5 ml of 2% sodium dodecyl sulfate and counted in Toluene-Triton Scintillation fluid. To obtain the specific binding, the binding in the presence of 0.5 /~g/ml TPA (Lifesystems Co.) was considered as nonspecific and subtracted from that obtained in the absence of TPA. Prostaglandin E determination. Prostaglandins were extracted from the culture medium as follows. 1 ml medium was mixed with 1 ml 0.5 M citric acid and 15 ml ether, the upper phase was separated and evaporated at 37°C. The remaining material was dissolved in 1 ml phosphate-buffered saline, pH 7.4/0,1% bovine serum albumin. 100-~tl
113
aliquots of the samples were incubated with 400/xl anti-prostaglandin E antiserum diluted 1:10000 and 0.14 nM 3H-prostaglandin E 1 (Radiochemical Centre, 46 C i / m m o l ) prepared in the same buffer in a final volume of 0.8 ml. After incubation for 1 6 - 2 0 h at 4°C, 0.2 ml phosphate-buffered saline/0.1% bovine serum albumin/0.1% Dextran T-70 (Pharmacia)/l% charcoal (Fisher) was added, samples incubated at 4°C for 10 min, centrifuged for 15 min at 3000 × g and 0.5 ml of the supernatant was taken for determination of the radioactivity in Toluene-Triton scintillation fluid. A set of tubes containing 0.3-1000 /:M prostaglandin E j was used in each experiment for quantitation of the samples. The antiprostaglandin E antiserum used was a generous gift from Prof. H. Lindner and Dr. F. Cohen and the specificity of the antibodies has been previously described [16].
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60
90
120
TIME OF INCUBATION
(min)
Fig. 1. Basal release of radioactivity from [3H]archidoniclabeled cells. MGI+D + clones 7-M18 ([3) and 11 (A)and M G I - D - clones 6 (O) and 1 (0) were pre-labeled with [3H]arachidonic acid. The cells were then washed and the amount of radioactivity released to the medium was determined at different times. Data are an average of duplicate samples from a representative experiment repeated 4-6 times. The variation between duplicate samples was less than 5% and between experiments less than 15%.
Results
Basal phospholipase A., activity and cell competence for induction of differentiation Two M G I + D + clones, nos. 7-M18 and 11, and two M G I - D - clones, nos. 1 and 6, that differed in their competence to be induced to differentiate by the normal differentiation-inducing protein and TPA [8] were used in the present studies. Cells were incubated for 3 h with [3H]arachidonic acid and the spontaneous release of the radioactive compound to the culture medium was determined as with other cell types [5,13]. At all time points tested, the release of 3H-arachidonic acid by M G I + D ÷ clones 7-M18 and 11 that can be induced to differentiate by the protein was higher than that of the non-differentiable M G I - D clones 1 and 6 (Fig. 1). The rate of radioactivity released by the various clones was in the order 7-M18>11>6>1. The higher release by the M G I ÷ D + cells reflects higher phospholipase A z activity and not differences in incorporation of [3H]arachidonic acid during the labeling period, since the amount of radioactivity recovered from cells of the various clones was similar (690-+ 55 f m o l / 1 0 6 cells). Thin layer chromatography analysis of the radioactivity released from clones 11 and 1 showed that prostaglandins of the D, E and F series were
not detectable and over 90% of the soluble radioactivity that moved into the chromatogram comigrated with authentic arachidonic acid. The results, therefore, indicate that the basal phospholipase A 2 activity in the various clones correlated with cell competence to be induced to differentiate by the protein. The competence of these clones to be induced to differentiate by the protein did not necessarily correlate with their competence to be induced to differentiate by TPA. Thus TPA induced differentiation in M G I + D + clones 7-M18 and M G I D - clone 6, but not in M G I + D + clone 11 or MGI-Dclone 1 [8]. The correlation between basal phospholipase A 2 activity and cell competence to be induced to differentiate therefore did not apply to TPA.
Prostaglandin E synthesis and the induction of differentiation by the protein inducer or TPA A further comparison of induction of differentiation by the protein inducer and TPA was made by comparing the synthesis of prostaglandin E. Since mammalian cells store very little, if any, prostaglandins, the level of prostaglandin E was measured in the culture medium. Untreated cells of the M G I + D + and MGI - D - clones released similar amounts of prostaglandin E to the culture
WITH
THE
DIFFERENTIATION-INDUCING
PROTEIN
0 IO 100
ng/ml
TPA
pg prostaglandin E/ lo6 Cells 25.6* 18 70.9*43 74.5*40
2.7-cO.4 1.5kO.2 1.1*0.2
of the Protein
Clone 7-Ml8
No. of cells (1’10-6)
(-)
Addition
MGI+D+
0.6 kO.14 0.08 kO.03 0.04*0.02
No. of cells (1.10 --y
(+)
370* 66 7237* 863 16750% I240
pg prostaglandin E/lo6 cells
Cells were incubated for 5 days in culture medium with O-100 q/ml TPA with or without medium were determined by radioimmunoassay. Results are means* SD.
EFFECT OF TREATMENT 7.Ml8 AND 1 I
TABLE I
3.4kO.4 2.OkO.3 1.6kO.3
Clone
II
medium.
16.2’- 12 35.0*20 58.0237
pf prostaglandin E/ IOh cells
of the Protein
No. of cells (1.10-6)
(v)
Addition
MGI+D’
containing
TPA ON PROSTAGLANDIN
15% of the protein
AND/OR
i.2r0.2 0.6 rO.1 0.61-0.1
No. of cells (I IOw6)
(+)
Prostaglandin
E LEVELS
117-’ 47 476-’ 88 553’155
pg prostaglandin E/IO6 cells
E levels in the culture
MGI + D + CLONES
115
medium (16-46 pg/106 cells). When the MGI + D + clones were induced to differentiate by the protein, there was a 14.4-fold increase in prostaglandin released per cell in clone 7-M18 and a 7.3fold increase in clone 11 (Table I). The MGI D clones that were not induced to differentiate by the protein also showed no increase in prostaglandin E synthesis (Table II). In contrast to these results with differentiation induced by the protein, differentiation induced by TPA showed only a small increase (3-fold) in prostaglandin E synthesis in clone 7-M18 (Table I) and no increase in clone 6 (Table II). These results indicate that enhancement of prostaglandin E synthesis was associated with induction of differentiation by the protein but not by TPA. Combined treatment with the protein and TPA showed an enhanced stimulation of prostaglandin E synthesis in clone 7-M18 (Table I) in which TPA can induce the production of the differentiation-inducing protein [8]. But there was no stimulation of prostaglandin E synthesis in clones 6 or 1 (Table II) even though this combined treatment enhanced differentiation in clone 6 and induced differentiation in clone 1 [8]. Stimulation of prostaglandin E synthesis has also been reported after treatment of M G I + D + leukemic cells with the steroid dexamethasone [14] and of normal peritoneal macrophages with the protein [17]. In order to determine whether the difference in
response to TPA was associated with the number of TPA binding sites, membrane preparations of the four clones were tested for specific binding of the tumor promoter [20-3H]phorbol 12,13dibutyrate [15]. The results indicate (Table III) that within the MGI + D + and MGI D - groups, the clones that could be induced to differentiate by TPA showed a higher binding than those that could not be induced to differentiate by TPA. This difference in TPA binding sites was, however, not associated with the response to the protein, since MGI + D + clone 11 that did not respond to TPA showed the lowest number of TPA binding sites of all the clones tested (Table III).
Effect of prostaglandin E 1 and jndomethacin on cell proliferation In order to further study the possible role of prostaglandin E, experiments were carried out on the effects of adding prostaglandin E~ or indomethacin, an inhibitor of prostaglandin synthesis. Prostaglandin E~ is known to be unstable in aqueous solutions and was therefore added to the cultures every day. At 2- 10 -8, 10-7 and 10 -6 M, prostaglandin E~ did not induce differentiation in any of the four clones studied. Addition of 1. 10-9-105 M indomethacin together with the protein did not inhibit induction of differentiation in MGI ÷ D ÷ clone 11 (Fig. 2). However addition of prostaglandin E~ every day to cells not treated
TABLE II E F F E C T OF T R E A T M E N T WITH T H E D I F F E R E N T I A T I O N - I N D U C I N G PROTEIN A N D / O R TPA ON PROST A G L A N D I N E LEVELS IN MGI - D - CLONES 1 A N D 6 Cells were cultured for 5 days in culture medium with 0-1000 n g / m l TPA with or without 15% of the protein containing medium. Prostaglandin E levels in the culture medium were determined as pg/106 cells. Results are m e a n s ± S.D. TPA ng/ml
0 10 100 1000
MGI-D-
Clone 1
MGI-D-
Clone 6
Addition of the Protein
Addition of the Protein
(-)
(+)
(-)
(+)
35.4-+11 21.7-+16 34.8-+ 14 36.4-+17
38.2±17 37.5±18 44.1 ± 2 0 40.0±16
46.3±14 49.7-+21 48.8± 16 45.5-+19
49.3-+16 50.7-+18 47.7-+22 47.5±16
TABLE III B I N D I N G OF [20-3H]PHORBOL 12,13-DIBUTYRATE TO D I F F E R E N T CLONES OF L E U K E M I C CELLS Membrane fractions were prepared and tested for binding. Results are means-+ S.D. [20-3 H]Phorbol 12,13-dibutyrate concentrations 12.5 and 25 nM are given as fmol bound/106 cells Clone Type
MGI +D + MGI +D + MGI-DMGI D
Clone No.
7-M18 I1 6 I
[20 -3 H]phorbol 12,13-dibutyrate concentration 12.5 nM
25 nM
3.1-+ 1.1 0.7±0.3 5.3± 1.7 2.9-+1.3
4.0± 1.4 1.3±0.5 6.6± 1.7 4.1±1.7
116
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Fig. 2. Differentiation of M G I ÷ D ÷ clone 11 cells induced by the differentiation-inducing protein in the presence of indomethacin. Cells were cultured in the presence of 10 % of the protein-containing medium and 0 - 1 0 - S M indomethacin for four days and cell attachment (A) and total lysozyme activity (B) were then tested. Data are averages of two experiments that varied less than 8%.
with the protein or TPA, inhibited cell proliferation in a dose-dependent manner and M G I + D ÷ clones 7-M18 and 11 were more sensitive to this inhibition than M G I - D - clones 1 and 6 (Fig. 3). A single application of prostaglandin E 1 at the same concentrations did not inhibit the proliferation of any of these clones of mouse myel0id
leukemic cells, as has also been reported for human myeloid leukemic cells [ 18,19]. That prostaglandin E plays a role in cell proliferation rather than differentiation was further suggested from the following experiments. M G I ~ D + leukemic cells that were induced to differentiate for several days with the protein, washed and then cultured without the protein, no longer proliferate and the cells die [20]. M G I + D ÷ clone 11 cells were therefore incubated for 4 days with 10% of the protein-containing medium, washed and then cultured without the protein in the presence of 0 - 1 0 ~ M indomethacin. This inhibitor of prostaglandin synthesis had no effect on proliferation of cells that had never been treated with the protein, but after treatment with the protein and its removal indomethacin rescued a small fraction of cells that then continued to proliferate (Fig. 4). This effect of indomethacin was dose-dependent and could not be observed if the protein concentration was too high (20%) for the amount of indomethacin (Fig. 4). The inhibition of prostaglandin synthesis thus permitted the proliferation of some cells that could otherwise not proliferate.
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[PGE,] ,M Fig. 3. Effect of prostaglandin E I on proliferation of the different clones of leukemic cells. M G I + D + clones 7-M18 ( O ) and II ( ~ ) and M G I - D clones 1 (O) and 6 ( A ) were cultured for four days with 0 - 1 0 6 M prostaglandin E I added daily at 0.1% vol. of the culture medium. Cells were counted in the presence of trypan blue dye to exclude cells that stained with the dye. Data are averages of three experiments which varied less then 10%.
Fig. 4. Effect of indomathacin on proliferation of MGI ~ D " clone 11 cells previously incubated with the differentiation inducing protein. Cells were cultured for four days in the presence of 10% of the protein-containing medium (MGI). The medium was then replaced by fresh medium without the protein ( - M G I ) and 0 (©); I ' 1 0 - 1 ° M ( ~ ) ; 1"10 - g M (O) or I-10 8 M ( D ) indomethacin. In one series (A), cells were cultured for four days with 20% protein-containing medium and the medium was then replaced by fresh medium without the protein and 1 0 - s M indomethacin. Data are from a representative experiment repeated three times. The variation between experiments varied less than 20%.
IV
a Macrophage
_
+ t _
a
differentiation-inducing
Differentiationinducing protein
+
+ _
TPA
OF THE DIFFERENT
Differentiation induced by:
and granulocyte
I
7-Ml8 II 6
MGI+D+ MGI+D+ MGI-DMGIID-
type
Clone No.
OF PROPERTIES
Clone
SUMMARY
TABLE
protein
High Medium Low LOW
Phospholipase A, activity
CLONES
LEUKEMIC
High Medium Low Low
Differentiationinducing protein a
CELLS
Low Low Low LOW
TPA
Prostaglandin E synthesis after treatment with:
OF MYELOID
High Medium Low Low
Inhibition of proliferation by adding prostaglandin E,
_
+ +
[61
P-adrenergic
Desensitization
+ + _
[71
prostaglandin
to hormones E,
5
118
Discussion The .present results (summarised in Table IV) indicate that basal phospholipase A 2 activity in the clones of myeloid leukemic cells was associated with cell competence to be induced to differentiate by the normal differentiation-inducing protein and the ability to be induced for prostaglandin E synthesis by this protein. In contrast, cell competence for induction of differentiation by TPA was not associated with basal phospholipase A 2 activity and cells could be induced to differentiate by TPA without an induction of prostaglandin E synthesis. This indicates, that although in some myeloid leukemic cells induction of differentiation ~y TPA can take place via induction of the differentiation-inducing protein [8], this protein and TPA may also induce differentiation by different pathways. The clone with the least ability to respond to TPA showed the lowest number of TPA binding sites. The interaction of TPA with specific receptors may thus mimic some as yet unknown endogenous compound(s) which does not appear to be the same as the differentiation-inducing protein. The availability of these different clones of myeloid leukemic cells with different susceptibilities to TPA, should prove useful in identifying and determining the physiological role of such a compound(s). The results with TPA also indicate that induction of prostaglandin E synthesis is not a prerequisite for differentiation. This was further substantiated by the data that addition of prostaglandin E 1 did not induce differentiation and that addition of indomethacin did not inhibit differentiation induced by the protein. However, addition of prostaglandin E I inhibited cell proliferation and the degree of inhibition was associated with the amount of phospholipase A 2 activity. Addition of prostaglandin E1 can also inhibit the proliferation of normal myeloid cells [18,19]. The results, therefore, indicate that the primary effect of prostaglandin E seems to be in controlling cell proliferation. This was also shown by the continued proliferation of some protein-treated M G I + D + leukemic cells after treatment with indomethacin. It can thus be suggested, that prostaglandin E may play a role in the inhibition of cell proliferation that occurs concomitantly with
differentiation both in the myeloid and other cell types. The finding that high basal phospholipase A 2 activity is correlated with cell competence to be induced to differentiate by the physiological protein inducer, but that induction of prostaglandin E is not necessary for differentiation, suggests that other compounds that result from phospholipase A 2 activity may be involved in the induction of differentiation. Products of phospholipase A 2 may be used, for example, to form fatty acids some of which are involved in differentiation (see Ref. [21]). It is of special interest that the lipid A part of bacterial lipopolysaccharide, which is rich in fatty acids including myristic acid [22], induced differentiation of M G I + D ÷ clones [23] but not of M G I - D _ clones of myeloid leukemic cells. There is also the possibility that deacylated phosphoglycerides, which are also products of phospholipase A 2, may be involved. These compounds are major donors of methyl groups and D N A methylation may play a role in cell differentiation [24]. Thromboxanes [1,25] may also be involved. It can therefore be concluded that the activation of pbospholipid metabolism by the protein may initiate multistep pathways with products which can either induce differentiation or inhibit proliferation (prostaglandin E). The leukemic clones studied differ in their ability to develop desensitization to /3-adrenergic hormones [6] or prostaglandin E~ [7]. The cells with the lowest basal phospholipase A 2 activity are also the ones that do not desensitize to these hormones [6,7], and this supports the suggestion that phosphotipase A 2 activity plays a role in the development of desensitization [5]. Since low basal phospholipase A 2 activity was found in cells that could not be induced to differentiate by the protein, it can be suggested that the response of cells to the normal protein inducer of differentiation is associated with their general capability to desensitize to external inducers.
Acknowledgments We are indebted to Mr. A. Goldenberg and Mrs. H. Nadler for skilful technical assistance. R.S. is incumbent of the C.S. Koshland Career Development Chair. This study was supported by
119 a grant from the Israel Academy of Sciences and Humanities--Basic Research Foundation and the National Council for Research and Development and D.K.F.Z., F.R.G.
References 1 Samuelsson, B., Goldyne, M., Granstrom, E., Hamberg, M., Hammastrom, S. and Malmsten, C. (1978) Ann. Rev. Biochem. 47, 997-1029 2 Kunze, H. and Vogt, W. (1971) Ann. N.Y. Acad. Sci. 180, 123-125 3 Flower, R.J. and Blackwell, G.J. (1976) Biochem. Pharmacol. 25, 285-291 4 Levine, L. (1979) Cold Spring Harbor Conferences on Cell Proliferation 6, 623-640 5 Mallorga, P., Tallman, J.F., Henneberry, R.C., Hirata, F., Strittmatter, W.T. and Axelrod, J. (1980) Proc. Natl. Acad. Sci. USA 77, 1341-1345 6 Simantov, R. and Sachs, L. (1978) Proc. Natl. Acad. Sci. USA 75, 1805-1809 7 Simantov, R., Shkolnik, T. and Sachs, L. (1980) Proc. Natl. Acad. Sci. USA 77, 4798-4802 8 Lotem, J. and Sachs, L. (1979) Proc. Natl. Acad. Sci. USA 76, 5158-5162 9 Sachs, L. (1978) Nature 274, 535-539 10 Sachs, L. (1980) Proc. Natl. Acad. Sci. USA 77, 6152-6156
11 Krystosek, A. and Sachs, L. (1976) Cell 9, 675-684 12 Lowry, D.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 13 Hong, S.L. and Levine, L. (1976) Proc. Natl. Acad. Sci. USA 73, 1730-1734 14 Honma, Y., Kasukabe, T., Hozumi, M. and Koshihara, Y. (1980) J. Cell. Physiol. 104, 349-357 15 Driedger, P.E. and Blumberg, P.M. (1980) Proc. Natl. Acad. Sci. USA 77, 567-571 16 Bauminger, S. (1976) J. Immunol. Methods 13, 253-259 17 Kurland, J.I., Pelus, L.M., Ralph, P., Bockman, R.S. and Moore, M.A.S. (1979) Proc. Natl. Acad. Sci. USA 76, 2326 - 2330 18 Aglietta, M., Piacibello, W. and Gavosto, F. (1980) Cancer Res. 40, 2507-251 I 19 Pelus, L.M., Broxmeyer, H.E., Clarkson, B.D. and Moore, M.A.S. (1980) Cancer Res. 40, 2512-2515 20 Fibach, E. and Sachs, L. (1976) J. Cell Physiol. 89, 259-266 21 Dulbecco, R., Bologna, M. and Unger, M. (1980) Proc. Natl. Acad. Sci. USA 77, 1551-1555 22 Reitschel, E.T.L., Gottert, H., Li~deritz, O. and Westphal, O. (1972) Eur. J. Biochem. 28, 166-173 23 Weiss, B. and Sachs, L. (1978) Proc. Natl. Acad. Sci. USA 75, 1374-1378 24 Jones, P.A. and Taylor, S,M. (1980) Cell 20, 85-93 25 Morley, J., Bray, M.A., Jones, R.W., Nugteren, D.H. and Van Dorp, D.A. (1979) Prostaglandins 17, 729-736
111
BBA 11013
R O L E OF P H O S P H O L I P A S E A 2 AND P R O S T A G L A N D I N E IN G R O W T H AND
D I F F E R E N T I A T I O N O F MYELOID LEUKEMIC CELLS RABI SIMANTOV and LEO SACHS
Department of Genetics, Weizmann Institute of Science, Rehovot 76100 (Israel) (Received Julf20th, 1981)
Key words: Phospholipase A 2," Prostaglandin E; Growth regulation; Differentiation; Tumor promoter," (Leukemic cell) P h o s p h o l i p a s e A 2 activity and prostaglandin E synthesis have been studied in different clones of myeloid
leukemic cells, which differ in their competence to be induced to differentiate by the macrophage and granulocyte differentiation-inducing protein or the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA). Clones that could be induced to differentiate by this protein showed a higher basal phospholipase A 2 activity than clones that could not be induced to differentiate by this protein inducer. Cell competence to be induced to differentiate by TPA did not show this correlation, and the clone with the least ability to respond to TPA showed the lowest number of binding sites for [20-3H]phorbol 12,13-dibutyrate. Differentiation induced by the protein was accompanied by a 7-14-fold increase in prostaglandin E synthesis, whereas differentiation induced by TPA did not show this increase. Externally added prostaglandin E 1 did not induce differentiation but inhibited cell proliferation and the degree of inhibition in the different clones was related to the basal phospholipase A 2 activity. The results indicate that increase of prostaglandin E synthesis was not an essential pre-requisite for differentiation, that prostaglandin E seems to be involved in the inhibition of cell proliferation in association with phospholipase A 2 , and that the differentiation-inducing protein and TPA can induce differentiation by different pathways. The amount of basal phospholipase A 2 activity was also related to previously found differences in the ability of the clones to develop desensitization to ~-adrenergic hormones or prostaglandin E 1.
Introduction The key role of phospholipase A 2 in the control of prostaglandin synthesis has been established in several tissues [1-4]. Activation of prostaglandin E synthesis by various external stimuli was associated with activation of phospholipase A 2. Studies with astrocytoma cells have indicated, that the activation of phospholipase A 2 in the local domain of the fl-adrenergic receptor may be involved in desensitization to fl-adrenergic hormones [5]. Experiments with myeloid leukemic cells have also shown an association between the ability of different clones of these leukemic cells to desensitize to fl-adrenergic hormones and to prostaglandin El, 0167-4889/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
and their ability to be induced to differentiate by the macrophage and granulocyte differentiationinducing protein. Clones that could be induced to differentiate by this protein ( M G I + D ÷ clones) desensitized to fl-adrenergic hormones and prostaglandin El, whereas clones that could not be induced to differentiate by this protein (MGI - D clones) could not desensitize to these hormones [6,7]. These clones of leukemic cells also differ in their ability to be induced to differentiate by the tumor promoter TPA, and cell competence to be induced to differentiate by the protein was not necessarily associated with cell competence to beinduced to differentiate by TPA [8]. The present experiments were, therefore, undertaken with these
112
different clones of myeloid leukemic cells to determine: (A) The relationship between basal phospholipase A2, hormone desensitization and cell competence for induction of differentiation by the protein differentiation inducer and the phorbol ester TPA; (B) the role of prostaglandin E synthesis in the control of cell growth and in cell differentiation induced by the protein or TPA, and (C) whether the protein and TPA may induce differentiation by different pathways. Materials and Methods
Cells and Cell culture. The leukemic cells used were clones of mouse myeloid leukemic cells isolated from a spontaneous or X-irradiation-induced myeloid leukemias [8]. Clones 7-M18 and 11 belong to the type of MGI + D + and clones 1 and 6 to the type of M G I - D , based on their sensitivity to be induced to differentiate in culture by the differentiation-inducing form of the macrophage and granulocyte-inducing protein, using the criteria for differentiation including lysozyme synthesis previously described [9,10]. Cells were cultured in Eagle's medium with a 4-fold concentration of amino acids and vitamins (H-21, Grand Island Biological Co.) and 10% inactivated fetal calf serum as described [6]. For the experiments cells were seeded at 2 - 5 . 10 4 cells per Petri dish (Nunc, 60 mm) in 5 ml medium. Total lysozyme was measured using the turbidometric assay with Mierococcus lysodeikticus as described [11] and protein concentrations were determined according to Lowry et al. [12]. The differentiation inducing protein (MGI-2 [10]) was obtained from Krebs ascites cells
[81. Phospholipase A 2 activity and thin layer chromatography. Basal phospholipase A 2 activity was tested according to Hong and Levine [13] with minor modifications. Cells grown to a density of 1 . 5 - 2 . 0 . 1 0 6 cells/ml culture medium were collected, washed once with culture medium and then incubated for 3 h at 5 : 10 6 cells/ml medium containing 2.5 nM 3H-arachidonic acid (New England Nuclear, 78.2 Ci/mmol). The cells were then centrifuged at low speed (500 × g), the medium removed, the cells washed with fresh culture medium and re-suspended in fresh medium at 2 . 6 - 3 . 0 - 10 6 cells/ml. At 0-120 min later, aliquots
of 2.5 ml of the cell suspension were taken, centrifuged (500 × g) and the amount of radioactivity released by the cells was determined. Thin layer chromatograms were developed according to Honma et al. [14] with a mixture of ethyl acetate/2,2,4-trimethyl pentane/acetic a c i d / H 2 0 ( 1 1 : 5 : 2 : 1 0 v/v), dried, cut into strips of 0.5-cm and the radioactivity determined. Binding assay of [20--~H]-phorbol 12,13dibutyrate to leukemic cells. The binding of [20 -3 H] -phorbol 12,13-dibutyrate to membrane preparations from the four clones of myeloid leukemic cells was performed essentially as described by Driedger and Blumberg [15] with minor modifications. Cells (4.108 ) were collected by low-speed centrifugation, washed with 100 vol. phosphatebuffered saline, and incubated for 15 min in icecold 5 mM Tris-HC1 buffer/5 mM MgC12 (pH 7.4 at 25°C). The cells were homogenized in a Thomas glass homogenizer type B (30 ups and downs)~ centrifuged for 10 min at 40000 × g, the supernatant removed, the pellets re-suspended in I0 ml of the same buffer and re-centrifuged. The pellets were then suspended in 4 ml of 50 mM Tris-HCl buffer, pH 7.4 at 25°C, containing 4 m g / m l bovine serum albumin (Sigma). Duplicate aliquots of 180 /tl of the homogenates were incubated for 20 min at 39°C with 12.5 or 25 nM [20-3H]phorbol 12,13dibutyrate (Lifesystems CO., specific activity 6.4 C i / m m o l ) in the presence or absence of 0.5/~g/ml TPA. It was found that the binding reached saturation within 20 min incubation. The binding was terminated by centrifugation for 10 min at 40000 × g, the supernatant was removed and the pellets washed with l ml Tris-HC1 buffer. They were then dried, lysed in 0.5 ml of 2% sodium dodecyl sulfate and counted in Toluene-Triton Scintillation fluid. To obtain the specific binding, the binding in the presence of 0.5 /~g/ml TPA (Lifesystems Co.) was considered as nonspecific and subtracted from that obtained in the absence of TPA. Prostaglandin E determination. Prostaglandins were extracted from the culture medium as follows. 1 ml medium was mixed with 1 ml 0.5 M citric acid and 15 ml ether, the upper phase was separated and evaporated at 37°C. The remaining material was dissolved in 1 ml phosphate-buffered saline, pH 7.4/0,1% bovine serum albumin. 100-~tl
113
aliquots of the samples were incubated with 400/xl anti-prostaglandin E antiserum diluted 1:10000 and 0.14 nM 3H-prostaglandin E 1 (Radiochemical Centre, 46 C i / m m o l ) prepared in the same buffer in a final volume of 0.8 ml. After incubation for 1 6 - 2 0 h at 4°C, 0.2 ml phosphate-buffered saline/0.1% bovine serum albumin/0.1% Dextran T-70 (Pharmacia)/l% charcoal (Fisher) was added, samples incubated at 4°C for 10 min, centrifuged for 15 min at 3000 × g and 0.5 ml of the supernatant was taken for determination of the radioactivity in Toluene-Triton scintillation fluid. A set of tubes containing 0.3-1000 /:M prostaglandin E j was used in each experiment for quantitation of the samples. The antiprostaglandin E antiserum used was a generous gift from Prof. H. Lindner and Dr. F. Cohen and the specificity of the antibodies has been previously described [16].
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60
90
120
TIME OF INCUBATION
(min)
Fig. 1. Basal release of radioactivity from [3H]archidoniclabeled cells. MGI+D + clones 7-M18 ([3) and 11 (A)and M G I - D - clones 6 (O) and 1 (0) were pre-labeled with [3H]arachidonic acid. The cells were then washed and the amount of radioactivity released to the medium was determined at different times. Data are an average of duplicate samples from a representative experiment repeated 4-6 times. The variation between duplicate samples was less than 5% and between experiments less than 15%.
Results
Basal phospholipase A., activity and cell competence for induction of differentiation Two M G I + D + clones, nos. 7-M18 and 11, and two M G I - D - clones, nos. 1 and 6, that differed in their competence to be induced to differentiate by the normal differentiation-inducing protein and TPA [8] were used in the present studies. Cells were incubated for 3 h with [3H]arachidonic acid and the spontaneous release of the radioactive compound to the culture medium was determined as with other cell types [5,13]. At all time points tested, the release of 3H-arachidonic acid by M G I + D ÷ clones 7-M18 and 11 that can be induced to differentiate by the protein was higher than that of the non-differentiable M G I - D clones 1 and 6 (Fig. 1). The rate of radioactivity released by the various clones was in the order 7-M18>11>6>1. The higher release by the M G I ÷ D + cells reflects higher phospholipase A z activity and not differences in incorporation of [3H]arachidonic acid during the labeling period, since the amount of radioactivity recovered from cells of the various clones was similar (690-+ 55 f m o l / 1 0 6 cells). Thin layer chromatography analysis of the radioactivity released from clones 11 and 1 showed that prostaglandins of the D, E and F series were
not detectable and over 90% of the soluble radioactivity that moved into the chromatogram comigrated with authentic arachidonic acid. The results, therefore, indicate that the basal phospholipase A 2 activity in the various clones correlated with cell competence to be induced to differentiate by the protein. The competence of these clones to be induced to differentiate by the protein did not necessarily correlate with their competence to be induced to differentiate by TPA. Thus TPA induced differentiation in M G I + D + clones 7-M18 and M G I D - clone 6, but not in M G I + D + clone 11 or MGI-Dclone 1 [8]. The correlation between basal phospholipase A 2 activity and cell competence to be induced to differentiate therefore did not apply to TPA.
Prostaglandin E synthesis and the induction of differentiation by the protein inducer or TPA A further comparison of induction of differentiation by the protein inducer and TPA was made by comparing the synthesis of prostaglandin E. Since mammalian cells store very little, if any, prostaglandins, the level of prostaglandin E was measured in the culture medium. Untreated cells of the M G I + D + and MGI - D - clones released similar amounts of prostaglandin E to the culture
WITH
THE
DIFFERENTIATION-INDUCING
PROTEIN
0 IO 100
ng/ml
TPA
pg prostaglandin E/ lo6 Cells 25.6* 18 70.9*43 74.5*40
2.7-cO.4 1.5kO.2 1.1*0.2
of the Protein
Clone 7-Ml8
No. of cells (1’10-6)
(-)
Addition
MGI+D+
0.6 kO.14 0.08 kO.03 0.04*0.02
No. of cells (1.10 --y
(+)
370* 66 7237* 863 16750% I240
pg prostaglandin E/lo6 cells
Cells were incubated for 5 days in culture medium with O-100 q/ml TPA with or without medium were determined by radioimmunoassay. Results are means* SD.
EFFECT OF TREATMENT 7.Ml8 AND 1 I
TABLE I
3.4kO.4 2.OkO.3 1.6kO.3
Clone
II
medium.
16.2’- 12 35.0*20 58.0237
pf prostaglandin E/ IOh cells
of the Protein
No. of cells (1.10-6)
(v)
Addition
MGI+D’
containing
TPA ON PROSTAGLANDIN
15% of the protein
AND/OR
i.2r0.2 0.6 rO.1 0.61-0.1
No. of cells (I IOw6)
(+)
Prostaglandin
E LEVELS
117-’ 47 476-’ 88 553’155
pg prostaglandin E/IO6 cells
E levels in the culture
MGI + D + CLONES
115
medium (16-46 pg/106 cells). When the MGI + D + clones were induced to differentiate by the protein, there was a 14.4-fold increase in prostaglandin released per cell in clone 7-M18 and a 7.3fold increase in clone 11 (Table I). The MGI D clones that were not induced to differentiate by the protein also showed no increase in prostaglandin E synthesis (Table II). In contrast to these results with differentiation induced by the protein, differentiation induced by TPA showed only a small increase (3-fold) in prostaglandin E synthesis in clone 7-M18 (Table I) and no increase in clone 6 (Table II). These results indicate that enhancement of prostaglandin E synthesis was associated with induction of differentiation by the protein but not by TPA. Combined treatment with the protein and TPA showed an enhanced stimulation of prostaglandin E synthesis in clone 7-M18 (Table I) in which TPA can induce the production of the differentiation-inducing protein [8]. But there was no stimulation of prostaglandin E synthesis in clones 6 or 1 (Table II) even though this combined treatment enhanced differentiation in clone 6 and induced differentiation in clone 1 [8]. Stimulation of prostaglandin E synthesis has also been reported after treatment of M G I + D + leukemic cells with the steroid dexamethasone [14] and of normal peritoneal macrophages with the protein [17]. In order to determine whether the difference in
response to TPA was associated with the number of TPA binding sites, membrane preparations of the four clones were tested for specific binding of the tumor promoter [20-3H]phorbol 12,13dibutyrate [15]. The results indicate (Table III) that within the MGI + D + and MGI D - groups, the clones that could be induced to differentiate by TPA showed a higher binding than those that could not be induced to differentiate by TPA. This difference in TPA binding sites was, however, not associated with the response to the protein, since MGI + D + clone 11 that did not respond to TPA showed the lowest number of TPA binding sites of all the clones tested (Table III).
Effect of prostaglandin E 1 and jndomethacin on cell proliferation In order to further study the possible role of prostaglandin E, experiments were carried out on the effects of adding prostaglandin E~ or indomethacin, an inhibitor of prostaglandin synthesis. Prostaglandin E~ is known to be unstable in aqueous solutions and was therefore added to the cultures every day. At 2- 10 -8, 10-7 and 10 -6 M, prostaglandin E~ did not induce differentiation in any of the four clones studied. Addition of 1. 10-9-105 M indomethacin together with the protein did not inhibit induction of differentiation in MGI ÷ D ÷ clone 11 (Fig. 2). However addition of prostaglandin E~ every day to cells not treated
TABLE II E F F E C T OF T R E A T M E N T WITH T H E D I F F E R E N T I A T I O N - I N D U C I N G PROTEIN A N D / O R TPA ON PROST A G L A N D I N E LEVELS IN MGI - D - CLONES 1 A N D 6 Cells were cultured for 5 days in culture medium with 0-1000 n g / m l TPA with or without 15% of the protein containing medium. Prostaglandin E levels in the culture medium were determined as pg/106 cells. Results are m e a n s ± S.D. TPA ng/ml
0 10 100 1000
MGI-D-
Clone 1
MGI-D-
Clone 6
Addition of the Protein
Addition of the Protein
(-)
(+)
(-)
(+)
35.4-+11 21.7-+16 34.8-+ 14 36.4-+17
38.2±17 37.5±18 44.1 ± 2 0 40.0±16
46.3±14 49.7-+21 48.8± 16 45.5-+19
49.3-+16 50.7-+18 47.7-+22 47.5±16
TABLE III B I N D I N G OF [20-3H]PHORBOL 12,13-DIBUTYRATE TO D I F F E R E N T CLONES OF L E U K E M I C CELLS Membrane fractions were prepared and tested for binding. Results are means-+ S.D. [20-3 H]Phorbol 12,13-dibutyrate concentrations 12.5 and 25 nM are given as fmol bound/106 cells Clone Type
MGI +D + MGI +D + MGI-DMGI D
Clone No.
7-M18 I1 6 I
[20 -3 H]phorbol 12,13-dibutyrate concentration 12.5 nM
25 nM
3.1-+ 1.1 0.7±0.3 5.3± 1.7 2.9-+1.3
4.0± 1.4 1.3±0.5 6.6± 1.7 4.1±1.7
116
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Fig. 2. Differentiation of M G I ÷ D ÷ clone 11 cells induced by the differentiation-inducing protein in the presence of indomethacin. Cells were cultured in the presence of 10 % of the protein-containing medium and 0 - 1 0 - S M indomethacin for four days and cell attachment (A) and total lysozyme activity (B) were then tested. Data are averages of two experiments that varied less than 8%.
with the protein or TPA, inhibited cell proliferation in a dose-dependent manner and M G I + D ÷ clones 7-M18 and 11 were more sensitive to this inhibition than M G I - D - clones 1 and 6 (Fig. 3). A single application of prostaglandin E 1 at the same concentrations did not inhibit the proliferation of any of these clones of mouse myel0id
leukemic cells, as has also been reported for human myeloid leukemic cells [ 18,19]. That prostaglandin E plays a role in cell proliferation rather than differentiation was further suggested from the following experiments. M G I ~ D + leukemic cells that were induced to differentiate for several days with the protein, washed and then cultured without the protein, no longer proliferate and the cells die [20]. M G I + D ÷ clone 11 cells were therefore incubated for 4 days with 10% of the protein-containing medium, washed and then cultured without the protein in the presence of 0 - 1 0 ~ M indomethacin. This inhibitor of prostaglandin synthesis had no effect on proliferation of cells that had never been treated with the protein, but after treatment with the protein and its removal indomethacin rescued a small fraction of cells that then continued to proliferate (Fig. 4). This effect of indomethacin was dose-dependent and could not be observed if the protein concentration was too high (20%) for the amount of indomethacin (Fig. 4). The inhibition of prostaglandin synthesis thus permitted the proliferation of some cells that could otherwise not proliferate.
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[PGE,] ,M Fig. 3. Effect of prostaglandin E I on proliferation of the different clones of leukemic cells. M G I + D + clones 7-M18 ( O ) and II ( ~ ) and M G I - D clones 1 (O) and 6 ( A ) were cultured for four days with 0 - 1 0 6 M prostaglandin E I added daily at 0.1% vol. of the culture medium. Cells were counted in the presence of trypan blue dye to exclude cells that stained with the dye. Data are averages of three experiments which varied less then 10%.
Fig. 4. Effect of indomathacin on proliferation of MGI ~ D " clone 11 cells previously incubated with the differentiation inducing protein. Cells were cultured for four days in the presence of 10% of the protein-containing medium (MGI). The medium was then replaced by fresh medium without the protein ( - M G I ) and 0 (©); I ' 1 0 - 1 ° M ( ~ ) ; 1"10 - g M (O) or I-10 8 M ( D ) indomethacin. In one series (A), cells were cultured for four days with 20% protein-containing medium and the medium was then replaced by fresh medium without the protein and 1 0 - s M indomethacin. Data are from a representative experiment repeated three times. The variation between experiments varied less than 20%.
IV
a Macrophage
_
+ t _
a
differentiation-inducing
Differentiationinducing protein
+
+ _
TPA
OF THE DIFFERENT
Differentiation induced by:
and granulocyte
I
7-Ml8 II 6
MGI+D+ MGI+D+ MGI-DMGIID-
type
Clone No.
OF PROPERTIES
Clone
SUMMARY
TABLE
protein
High Medium Low LOW
Phospholipase A, activity
CLONES
LEUKEMIC
High Medium Low Low
Differentiationinducing protein a
CELLS
Low Low Low LOW
TPA
Prostaglandin E synthesis after treatment with:
OF MYELOID
High Medium Low Low
Inhibition of proliferation by adding prostaglandin E,
_
+ +
[61
P-adrenergic
Desensitization
+ + _
[71
prostaglandin
to hormones E,
5
118
Discussion The .present results (summarised in Table IV) indicate that basal phospholipase A 2 activity in the clones of myeloid leukemic cells was associated with cell competence to be induced to differentiate by the normal differentiation-inducing protein and the ability to be induced for prostaglandin E synthesis by this protein. In contrast, cell competence for induction of differentiation by TPA was not associated with basal phospholipase A 2 activity and cells could be induced to differentiate by TPA without an induction of prostaglandin E synthesis. This indicates, that although in some myeloid leukemic cells induction of differentiation ~y TPA can take place via induction of the differentiation-inducing protein [8], this protein and TPA may also induce differentiation by different pathways. The clone with the least ability to respond to TPA showed the lowest number of TPA binding sites. The interaction of TPA with specific receptors may thus mimic some as yet unknown endogenous compound(s) which does not appear to be the same as the differentiation-inducing protein. The availability of these different clones of myeloid leukemic cells with different susceptibilities to TPA, should prove useful in identifying and determining the physiological role of such a compound(s). The results with TPA also indicate that induction of prostaglandin E synthesis is not a prerequisite for differentiation. This was further substantiated by the data that addition of prostaglandin E 1 did not induce differentiation and that addition of indomethacin did not inhibit differentiation induced by the protein. However, addition of prostaglandin E I inhibited cell proliferation and the degree of inhibition was associated with the amount of phospholipase A 2 activity. Addition of prostaglandin E1 can also inhibit the proliferation of normal myeloid cells [18,19]. The results, therefore, indicate that the primary effect of prostaglandin E seems to be in controlling cell proliferation. This was also shown by the continued proliferation of some protein-treated M G I + D + leukemic cells after treatment with indomethacin. It can thus be suggested, that prostaglandin E may play a role in the inhibition of cell proliferation that occurs concomitantly with
differentiation both in the myeloid and other cell types. The finding that high basal phospholipase A 2 activity is correlated with cell competence to be induced to differentiate by the physiological protein inducer, but that induction of prostaglandin E is not necessary for differentiation, suggests that other compounds that result from phospholipase A 2 activity may be involved in the induction of differentiation. Products of phospholipase A 2 may be used, for example, to form fatty acids some of which are involved in differentiation (see Ref. [21]). It is of special interest that the lipid A part of bacterial lipopolysaccharide, which is rich in fatty acids including myristic acid [22], induced differentiation of M G I + D ÷ clones [23] but not of M G I - D _ clones of myeloid leukemic cells. There is also the possibility that deacylated phosphoglycerides, which are also products of phospholipase A 2, may be involved. These compounds are major donors of methyl groups and D N A methylation may play a role in cell differentiation [24]. Thromboxanes [1,25] may also be involved. It can therefore be concluded that the activation of pbospholipid metabolism by the protein may initiate multistep pathways with products which can either induce differentiation or inhibit proliferation (prostaglandin E). The leukemic clones studied differ in their ability to develop desensitization to /3-adrenergic hormones [6] or prostaglandin E~ [7]. The cells with the lowest basal phospholipase A 2 activity are also the ones that do not desensitize to these hormones [6,7], and this supports the suggestion that phosphotipase A 2 activity plays a role in the development of desensitization [5]. Since low basal phospholipase A 2 activity was found in cells that could not be induced to differentiate by the protein, it can be suggested that the response of cells to the normal protein inducer of differentiation is associated with their general capability to desensitize to external inducers.
Acknowledgments We are indebted to Mr. A. Goldenberg and Mrs. H. Nadler for skilful technical assistance. R.S. is incumbent of the C.S. Koshland Career Development Chair. This study was supported by
119 a grant from the Israel Academy of Sciences and Humanities--Basic Research Foundation and the National Council for Research and Development and D.K.F.Z., F.R.G.
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11 Krystosek, A. and Sachs, L. (1976) Cell 9, 675-684 12 Lowry, D.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 13 Hong, S.L. and Levine, L. (1976) Proc. Natl. Acad. Sci. USA 73, 1730-1734 14 Honma, Y., Kasukabe, T., Hozumi, M. and Koshihara, Y. (1980) J. Cell. Physiol. 104, 349-357 15 Driedger, P.E. and Blumberg, P.M. (1980) Proc. Natl. Acad. Sci. USA 77, 567-571 16 Bauminger, S. (1976) J. Immunol. Methods 13, 253-259 17 Kurland, J.I., Pelus, L.M., Ralph, P., Bockman, R.S. and Moore, M.A.S. (1979) Proc. Natl. Acad. Sci. USA 76, 2326 - 2330 18 Aglietta, M., Piacibello, W. and Gavosto, F. (1980) Cancer Res. 40, 2507-251 I 19 Pelus, L.M., Broxmeyer, H.E., Clarkson, B.D. and Moore, M.A.S. (1980) Cancer Res. 40, 2512-2515 20 Fibach, E. and Sachs, L. (1976) J. Cell Physiol. 89, 259-266 21 Dulbecco, R., Bologna, M. and Unger, M. (1980) Proc. Natl. Acad. Sci. USA 77, 1551-1555 22 Reitschel, E.T.L., Gottert, H., Li~deritz, O. and Westphal, O. (1972) Eur. J. Biochem. 28, 166-173 23 Weiss, B. and Sachs, L. (1978) Proc. Natl. Acad. Sci. USA 75, 1374-1378 24 Jones, P.A. and Taylor, S,M. (1980) Cell 20, 85-93 25 Morley, J., Bray, M.A., Jones, R.W., Nugteren, D.H. and Van Dorp, D.A. (1979) Prostaglandins 17, 729-736