Printed in Sweden Copyright © 1977by AcademicPress, Inc. All rights of reproduction in anyform reserved ISSN 0014-4827
Experimental Cell Research 108 (1977) 7-14
A COMPARATIVE STUDY OF PROSTAGLANDIN AND ACTIVATION OF ADENYLATE
E 1
BINDING
CYCLASE IN NORMAL,
MALIGNANT AND HYBRID MAMMALIAN CELLS S. R. AYAD and G. R. J. BURNS D e p a r t m e n t o f Biochemistry, University o f M a n c h e s t e r , M anc he s t e r , M13 9PL, U K
SUMMARY Partially purified plasma membranes have been prepared from a number of normal, malignant and hybrid cell lines which exhibit a wide variation in basal and prostaglandin El-stimulated adenylate cyclase activity. The two lymphoma cell lines, EB-2 and P388F-36, are both characterized by low basal and hormonally stimulated levels of cAMP in intact cells. In broken cell preparations, P388F-36 exhibits a striking increase in hormonal response whilst the poor responsiveness of EB-2 cells is maintained. Hormone binding studies of P388F-36 correlate well with hormone activation. No specific binding of prostaglandin E1 can be observed in plasma membrane preparations of EB-2 indicating that low levels of the receptor may be the cause of the poor hormone responsiveness. The data thus suggests that distinct biochemical alterations have resulted in abnormally low levels of cAMP which are observed in the malignant cell lines. Comparison of hormone binding and activation in PCMI, a hybrid cell line produced by fusion of P388F-36 and CH23 cell lines suggests that the receptors of both parental lines are expressed in the hybrid but that the large variation in the affinities of the two types of receptor for the hormone results in the functional expression of only the high affinity form.
In recent years, evidence has accumulated which indicates that cAMP may play an important role in the control of cellular growth and division, although this relationship is not firmly established [1, 2]. Moreover, malignant cells which are characterised by the loss of such control, often exhibit abnormally low levels of cAMP [3, 4, 5]. It is therefore of considerable interest to determine which specific alterations associated with the malignant state, result in these altered levels of cAMP. It is well established that malignant cells exhibit profound alterations in the plasma membrane [6, 7] which include changes in
membrane fluidity [8] and reductions or loss of certain protein components [9, 10]. The adenylate cyclase of malignant cells often exhibits reduced basal levels and/or reduced sensitivity to hormonal stimulation [11, 12] but it is not clear whether these alterations are the result of general changes in the nature of the plasma membrane, resulting in decreased enzyme activity and inability of the hormone-receptor complex to interact with the enzyme, or whether the low activities are the result of the reduction or loss of the concentration of the enzyme or receptor. In order to investigate the latter possiExp Cell Res 108 (1977)
8
AyadandBurns P388F-36, a mouse lymphatic leukaemia cell line, CH23, a Chinese hamster fibroblast, and PCM1, a somatic cell hybrid produced by fusion of P388F-36 and CH23, were grown in Eagles Minimal Essential Medium supplemented with 10% newborn calf serum at 37°C in an atmosphere of 5 % CO2 and 95 % air. The origins and hormonal responsiveness of these cell lines have been described previously [13, 14, 15]. For the present experiments, cells were seeded at 1-2×106 cells in 1 litre glass Roux bottles. At confluency in the case of AP9 CH23 and PCMI or at limiting cell densities in the case of EB2 and P388F36, cells were harvested and homogenates prepared.
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Preparation of partially purified plasma membranes
8
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1-5
Fig. 1. Abscissa: log prostaglandin E1 (/zM); ordinate: spec. act. of adenyl cyclase (x 10-2) in pmoles cAMP/ 10 min/mg protein. Dose-response curves of adenyl cyclase with prostaglandin El. O - - G , EB-2; []--[], CH23; A - - A , P388F-36; I I - - i , AP-9; O - - O , PCM1; arrow, basal level.
Monolayer cells were scraped off the glass with a rubber policeman and the cells were collected by centrifugation at 4°C (150 g, 5 min). The cells were washed twice in buffer by resuspension and centrifugation and finally resuspended in a few ml of the buffer (50 mM Tris HC1, pH 7.9, containing 0.25 M sucrose, 5 mM MgSO4, 1 mM dithioerythritol and 1 mM ethyleneglycol-bis (/3-amino-ethyl ether) N,N'-tetra-acetic acid). Suspension cells were washed and resuspended in the same manner, ceils were then homogenised in a Dounce all-glass hand homogeniser or using a rotating 'Teflon' pestle. The conditions for homogenisation as judged by optimum adenylate cyclase activity varied somewhat among the cell lines and were as follows: AP9, CH23 and PCMI : 5 strokes hand homogenisation; EB-2:15 strokes hand homogenisation; P388F36 : 10 strokes rotating Teflon pestle (Speed 4, 'Tri-R' Stirrer).
bility, the binding of radioactively labelled prostaglandin E1 has been studied. In the present paper, we report the results of such studies in a number of normal, malignant and hybrid mammalian cells.
1-2
.1.0
.0.8
MATERIALS AND METHODS ~0"6
Cell culture The cell lines used in this study were as follows: AP9, Human embryonic lung fibroblasts isolated by ICI Pharmaceuticals, Alderley Edge, Cheshire, UK. These cells are a primary line which grow as a monolayer. EB-2, Burkitt lymphoma cells, obtained from Flow Laboratories Ltd., Irvine, KA12 8NB, Ayrshire, Scotland, are a malignant cell line which grow in suspension to high density. Both these cell lines were grown in Eagle's Minimal Essential Medium supplemented with 10 % foetal calf serum at 37°C in an atmosphere of 5 % CO~ and 95 % air.
Exp Cell Res 108 (1977)
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0.12
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Fig. 2. Abscissa: [3H]PGE1 (/zM); ordinate: (right) bound [3H]PGE1 (pmoles/mg protein); (left) bound [3H]PGE~ (cpna/mg protein x 10-4). Binding of [ZH]PGE1 to AP-9 membrane. O - - O , Total binding; A - - A , non-specific binding; I I - - n , specific binding.
Prostaglandin E1 binding and adenylate cyclase activation 1"0
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Fig. 3. Abscissa: [3H]PGE1 (/~M); ordinate: (right) bound [3H]PGE1 (pmoles/mg protein); (left) bound [3H]PGE1 (cpm/mg protein x 10-3). 0 - - 0 , Total binding; A---J,, non-specific binding; I - - I I , specific binding. Binding of [3H]PGEa to P388F-36 plasma membranes.
Partially purified plasma membranes were prepared from the homogenates using a discontinuous sucrose gradient centrifugation method [16]. The bands of particulate material which separated during centrifugation were assayed for adenylate cyclase activity and those bands containing the bulk of the enzyme were pooled and diluted with buffer (10 mM Tris-HC1, pH 7.4, containing 0.25 M sucrose and 1 mM dithioerythritol). The membranes were then collected by centrifugation (40000 g, 20 min) and resuspended in the same buffer at a protein concentration of 2-5 mg/ ml. The suspensions were sealed in ampoules and stored under liquid nitrogen until required.
Assay of adenylate cyclase Adenylate cyclase reactions were initiated by the addition of 20-50 /xg of protein to a final volume of 200/xl containing 1.8 mM ATP 50 mM Tris-HC1, pH 7.4 at 30°C, bovine serum albumin 0.1 mg/ml, 0.1 mM EGTA, 1 mM 1-methyl-3-isobutylxanthine, 5 mM MgSO4 and 1 mM dithioerythritol. In some experiments, prostaglandin Ea (a generous gift from ICI Pharmaceuticals Ltd), was included in the assay. Incubations were carried out for 10 or 15 min at 30°C and the reactions were terminated by the addition of 200 /~1 of 0.2 M HCI. After heating at 90°C for 60 rain to hydrolyse the remaining ATP, 80/zl of 2.5 M Tris-HC1, pH 7.4, were added and the cAMP content was determined by a protein binding assay [17]. Samples were also prepared in which membranes were added to incubation mixture after addition of the 0.2 M HCI and then immediately heated at 90°C. This mixture was incubated with standard cAMP samples which were used to construct a standard curve, and serves to correct for any endogenous cAMP present in the membrane preparations. Under these conditions, enzyme activity was a linear function of protein concentration and time of incubation.
9
Assay of [3H]prostaglandin E 1 binding The method followed was essentially that of Powell et al. [18]. Prostaglandin El-[5,6-3H] ([3H]PGE0 was obtained from the Radiochemical Centre, Amersham, Bucks, U K (59 Ci/mmol, 166 mCi/mg), and diluted with distilled water to provide a range of concentrations. Five/xl aliquots of the solutions were added to 1.5 ml plastic Eppendorf tubes, which contained either 5/zl of 25 % ethanol or 5/zl of unlabelled prostaglandin E~ (3 mM) in 25 % ethanol. The final concentration of labelled hormone was varied from 0-0.3 ~M. The frozen plasma membrane preparations were thawed and centrifuged at 40000 g for 20 min and then resuspended in 10 mM Tris-HCl buffer, pH 7.5, containing 0.15 M NaC1. One hundred twenty-five /xl of the suspension were added to the reaction mixtures and incubated at 30°C. After 1 h, 100/zl aliquots were removed and placed on top of a column (8x0.6 cm) of Sephadex G-50 Fine (Pharmacia, Uppsala, Sweden). The column was eluted with buffer at room temperature and the plasma membranes were collected in scintillation vials. Radioactivity was determined by counting in a liquid scintillation spectrometer (Packard Model 3385) after the addition of 100 ml of scintillator (2,5-diphenyl oxazole, 4 g/l; and 1,4-bis-(4methyl-5-phenyloxazolyl)-benzene, 0.2 g/l, dissolved in a mixture of toluene and Triton X-100, 2 : 1 by volume). Independent experiments verified that bound and unbound prostaglandin E~ were completely separated using this procedure. Specific binding was measured by subtraction of the counts remaining bound in the presence of excess unlabelled hormone from the counts observed in the absence of unlabelled hormone. Triplicate values agreed within 10 % of the mean and were averaged to a single representative figure.
Protein determination All protein determinations were performed using the method of Lowry et al. [ 19] with bovine serum albumin as standard. 25
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Fig. 4. Abscissa: [3H]PGE1 (/xM); ordinate: (right) bound [sH]PGE1 (pmoles/mg protein); (left) bound [3H]PGE1 (cpm/mg protein x 10-3). O - - O , Total binding; A---A, non-specific binding; II----I, specific binding. Binding of [3H]PGE~ to EB-2 plasma membrane.
Exp CellRes 108 (1977)
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Ayad and Burns
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, O.OO
, 0.12
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Fig. 5. Abscissa: [aH]PGE1 (~M); ordinate: (right) bound [3H]PGE1 (pmoles/mg protein); (left) bound [3H]PGE1 (cpm/mg protein x 10-8). e - - e , Total bindrag; A---&, non-specific binding; ~ - B , specific binding. Binding of [3H]PGE1 to CH23 plasma membranes.
RESULTS
Prostaglandin E1 activation of adenylate cyclase The dose-response curves of the plasma membrane preparations are shown in fig. 1. It may be noted that the apparent dissociation constants (KD for mouse lymphoma P388F-36 and the hybrid PCM1 are very similar (approx. 3x10 -8 M) although the maximum activity of the adenylate cyclase differs appreciably in the two cell lines. The apparent Kd of AP9 plasma membranes is approx. 30x 10-s M, or about one order of magnitude greater than that observed in P388F-36 and PCM1. Nevertheless, a marked hormone response is observed. In the case of CH23 and EB2, a very poor response is observed, particularly in the latter case. Moreover, no value for the apparent Ka can be obtained since saturation is not achieved even at PGE1 concentration up to 30/xM. Basal activity of adenylate cyclase also shows considerable variation, but is notably low in CH23 and EB2 preparations. In intact cells, the responsiveness of P388F-36 to PGE, is negligible [14] and contrasts markedly with broken cell Exp Cell Res 108 (1977)
preparations or partially purified plasma membranes. It seems possible that this observation may be due to the very high cyclic nucleotide phosphodiesterase activity found in this cell line, and conversely, the very high accumulation of cAMP in the hybrid, PCM1, may be partially due to the low activity of this enzyme [14]. In contrast to the P388F-36 cell line, EB2 cells retain their poor responsiveness to PGE1 in broken cell preparations. The similarity of the apparent dissociation constants for activation of adenylate cyclase by PGE1 in the parental cell line P388F-36 and the hybrid PCM1, in contrast to the low affinity of CH23 parent, suggest that P388F-36, and not the CH23 receptors are functionally expressed in the hybrid cell line.
[3H]Prostaglandin E, binding Figs 2-6 show the results of [3H]PGE1 binding studies. In the case of P388F-36 and 40
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01,8 0.24
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Fig. 6. Abscissa: [3H]PGE1 (~M); ordinate: (right) bound [sH]PGE, (pmoles/mg protein); (left) bound [~H]PGE~ (cpm/mg protein × 10-8). O - - e , Total binding; &--&, non-specific binding; B--B, specific binding. Binding of [sH]PGE, to PCM-1 plasma membrane.
Prostaglandin El binding and adenylate cyclase activation
11
analysis. The data appears, however, to be consistent with a significant rate of dissociation of ligand from low affinity receptors during separation of bound and free PGE1. The data thus suggests that the PCM~ hybrid cell has inherited receptors from both parental cell lines. DISCUSSION
_,.',
_;.,
_ 0'.~
0
Fig. 7. Abscissa: log [SH]PGE1 (log/xM); ordinate: log
specific binding (log cpm/mg protein). Logarithmic relationship between observed specific binding and [3H]PGE1 concentration in CH23 and PCM-1 plasma membranes. I1--11, PCM1; D--D, CH23.
AP9 plasma membranes (figs 2 and 3) the specific binding of the hormone closely parallels hormonal activation of adenylate cyclase and Scatchard plots indicate Ka values of 0.04 and 0.2 /xM respectively. No specific binding of hormone could be detected in the Burkitt lymphoma EB-2 plasma membranes (fig. 4) which is consistent with the very low hormonal response observed (fig. 1). These observations suggest that the low response of this cell line may, unlike that of the mouse lymphoma P388F-36, be the consequence of a low concentration of functional receptors in the plasma membrane. The low basal activity of this cell line might also point to a low concentration of adenylate cyclase. The variation of hormone binding with hormone concentration in PCM1 and CH23 plasma membranes produced unusual binding curves (figs 5 and 6), and the data was not amenable to interpretation by Scatchard
Several observations may be made concerning the origin of the low levels of cAMP observed in the malignant cell lines. PGE1 non-responsive EB-2 Burkitt lymphoma cells are characterised by much reduced concentrations of PGE1 receptors and possibly of adenylate cyclase. In this respect it is pertinent to note that Saez et al. [20] demonstrated a qualitative correlation between the levels of PGE1 binding and the hormone responsiveness of adenylate cyclase in a number of adrenocortical turnouts. P388F-36 mouse lymphoma cells, like EB-2 may be characterised by very low intracellular levels of cAMP and by a low hormone responsiveness. However, in P388F-36, this observation is largely re-
20.
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012
014
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1'.0
Fig. 8. Abscissa: k-1 (min-1); ordinate: ratio of true
binding to observed binding. Variation of the observed binding of [3H]PGE1 with
the ratio constant, k_~, and time. ©--©, 5 min; O--O, 3 min; [~--D, 2 min; A----A, 1 min; B----B,0.5 min. Exp Cell Res 108 (1977)
12
Ayad and Burns
stricted to intact cells [14]. Very high levels of cyclic nucleotide phosphodiesterase have been demonstrated in these cells, and this is probably the dominant factor in the production of the low levels of cAMP. The present report indicates that in plasma membrane preparations of these ceils, the levels of hormone receptors are not abnormally low, and this finding is in agreement with the hormonal responsiveness. It thus appears that although both these cell lines exhibit the low levels of cAMP often associated with the malignant state, the nature of the events leading to this abnormality in the two cell lines is quite distinct. The activation of PCM1 hybrid cell adenylate cyclase by PGE1 is markedly similar to that observed in the P388F-36 parent, and contrasts sharply with that of the CH23 parent• q'hese observations suggested that the P388F-36 receptor is expressed in the hybrid. In both PCM1 and CH23, however, anomalous but similar binding curves are observed suggesting that only the CH23 receptor is expressed in the hybrid. These anomalous binding curves can be rationalised if two assumption--g-are made: (1) the hormone concentration studied is low compared to the dissociation constant Kd; (2) the rate of dissociation of hormone from the hormone receptor complex is significant. Consider the simplest binding process:
k! H+R ~ H R k-1
where H, R and HR denote the concentrations of free hormone, of uncomplexed receptors and of hormone-receptor complex (i.e. bound ligand) respectively. Since the ligand is totally included by Sephadex G50 Exp Cell Res 108 (1977)
and the plasma membranes (receptors) are totally excluded, the concentration of free ligand available to receptors during the separation is very low, and to a first approximation can be neglected. Thus:
dHR dt =k-lXHR Integrating this expression,
logeHRt=loge HRo-k_l Xt
(1)
where HRt is the observed concentration of bound ligand after elution through the column, and HRo is the concentration of bound ligand before elution. If we denote the total receptor concentration a s R t , then
Rt xH HRo=H~
(2)
where Ka is the dissociation constant= k-l/k1 Substituting (2) into (i) loge HRt =lOge Rt xH -k-1 x t
(3)
•K a + H
ifH~Ka, then loge HRt = 1Oge~a + 1OgeH-k_1X t
(4)
which predicts a logarithmic relationship between the observed ligand binding and the concentration of free hormone. Fig. 7 shows a plot of log (observed binding) vs log (hormone concentration). The observed linearity of these plots for PCMI and CH23 indicates that such a relationship exists, and suggests that the anomalous binding curves arise in the manner outlined above. Ifk_lxt is negligible, H R t = HRo, if not then the total receptor concentration observed will be underestimated even where H is not very much less than
Prostaglandin E~ binding and adenylate cyclase activation Ka, although normal saturation curves will be observed in such cases. Brunton et al. [21] have obtained a value for k_~ of 0.15 rain -1 in a murine L cell. These authors separate free and bound ligand by centrifugation, the process taking several minutes to completion. At saturating concentrations of hormone, eq. (3) may be written as:
1
Rt
_k_~xt
Ogl° H g t -
2.306
(3)
Assuming k_1=0.15 min -~ and t~5 min (data ofBrunton et al.)
HRt- Rt
2.1
Thus a significant underestimation of total receptor concentration is observed. Since a logarithmic relationship exists between the ratio of true and observed binding, and k_l×t, relatively small differences in k_~xt can produce correspondingly greater errors in the estimation of the total receptor concentration. In the present studies, the time taken to elute the plasma membrane fraction was 3 min. The ratio of true to observed binding as k_l varies from~0 to 1 is shown in fig. 8, for several separation times. Although reports of kinetic studies of prostaglandin Ea binding are rare, Brunton et al. [21] estimated k_~ as 0.15 min -~ as noted above, and given..this figure, values for k_a between 0.01 and f seem reasonable. Since a value for k_~ of 1 would result in only 5 % of the true binding being observed, it is apparent that great care must be taken in the interpretation of binding data, relying on methods of separation of free and bound ligand which require considerable time to achieve. Millipore filtration techniques were employed to reduce the time required for separation. However, these filters were found
13
to retain high radioactivity even in the absence of membranes which made the evaluation of specific binding difficult. Despite these difficulties, hormonal activation studies demonstrate the presence of a high affinity receptor in PCMa hybrid cell plasma membranes similar to the P388F-36 parent. Hormone binding studies indicate the presence of low affinity receptors similar to the CH23 parent. These data strongly suggest that both parental receptors are expressed in the PCM~ hybrid, but that only the high affinity receptor is able to activate adenylate cyclase, presumably because the concentration of the latter is limiting. The authors wish to express their thanks to Professor G. R. Barker for providing the facilities for this work and Miss Margaret Barber for secretarial assistance. Financial support from the Cancer Research Campaign and the MRC for one of us (G. R. J. B.) is gratefully acknowledged.
REFERENCES 1. Anderson, W B & Pastan, I, Adv cyclic nucleic res 5 (1975) 681. 2. Chlapowski, F T, Kelly, L A & Butcher, R W, Adv cyclic nucleic res 6 (1975) 245. 3. PastaS, I & Johnson, G S, Adv cancer res 19 (1974) 303. 4. Pastan, I, Johnson, G S & Anderson, W B, Ann rev biochem 44 (1975) 491. 5. Ryan, W L & Heidrick, M L, Adv cyclic nucleic res 4 (1974) 81. 6. Hynes, R O, Biochim biophys acta 458 (1976) 73. 7. Nicholson, G L, Biochim biophys acta 458 (1976) 1. 8. Micklem, K J, Abra, R M, Knutton, S, Graham, J M & Pasternak, C A, Biochem j 154 (1976) 561. 9. Shizuta, Y, Davies, P J A, Olden, K & Pastan, I, Nature 261 (1976) 414. 10. Yamada, K M, Yamada, S S & Pastan, I, Proc natl acad sci US 73 (1976) 1222. 11. Polgar, P, Vera, J C, Kelley, P R & Rutenberg, A M, Biochim biophys acta 297 (1973) 378. 12. Prasad, K, Gilmer, J, Sahu, S & Becker, G, Cancer res 35 (1975) 77. 13. Ayad, S R & Delinassios, J G, Biochem genet 12 (1974) 147. 14. Ayad, S R & Foster, S J, Cell 3 (1974) 135. 15. Ayad, S R &White, A, Exp cell res 107 (1976) 201. 16. Glossmann, H & Gips, H, Arch pharmacol 289 (1975) 77.
Exp CellRes 108 (1977)
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17. Brown, B L, Albano, J D M, Etkins, R P & Sgherzi, A M, Biochem j 121 (1971) 561. 18. Powell, W S, Hammerstrom, S & Samuelsson, B, E u r j biochem 41 (1974) 103. 19. Lowry, O H, Rosebrough, N J, Farr, A L & Randall, R J, J biol chem 193 (1951) 265. 20. Saez, J M, Dazord, A & Gallet, D, J clin invest 56 (1975) 536.
Exp Cell Res 108 (1977)
21. Brunton, L L, Wiklund, R A, Van Arsdale, P M & Gilman, A G, J biol chem 251 (1976) 3037. Received March 9, 1977 Accepted March 11, 1977
Experimental Cell Research 108 (1977) 7-14
A COMPARATIVE STUDY OF PROSTAGLANDIN AND ACTIVATION OF ADENYLATE
E 1
BINDING
CYCLASE IN NORMAL,
MALIGNANT AND HYBRID MAMMALIAN CELLS S. R. AYAD and G. R. J. BURNS D e p a r t m e n t o f Biochemistry, University o f M a n c h e s t e r , M anc he s t e r , M13 9PL, U K
SUMMARY Partially purified plasma membranes have been prepared from a number of normal, malignant and hybrid cell lines which exhibit a wide variation in basal and prostaglandin El-stimulated adenylate cyclase activity. The two lymphoma cell lines, EB-2 and P388F-36, are both characterized by low basal and hormonally stimulated levels of cAMP in intact cells. In broken cell preparations, P388F-36 exhibits a striking increase in hormonal response whilst the poor responsiveness of EB-2 cells is maintained. Hormone binding studies of P388F-36 correlate well with hormone activation. No specific binding of prostaglandin E1 can be observed in plasma membrane preparations of EB-2 indicating that low levels of the receptor may be the cause of the poor hormone responsiveness. The data thus suggests that distinct biochemical alterations have resulted in abnormally low levels of cAMP which are observed in the malignant cell lines. Comparison of hormone binding and activation in PCMI, a hybrid cell line produced by fusion of P388F-36 and CH23 cell lines suggests that the receptors of both parental lines are expressed in the hybrid but that the large variation in the affinities of the two types of receptor for the hormone results in the functional expression of only the high affinity form.
In recent years, evidence has accumulated which indicates that cAMP may play an important role in the control of cellular growth and division, although this relationship is not firmly established [1, 2]. Moreover, malignant cells which are characterised by the loss of such control, often exhibit abnormally low levels of cAMP [3, 4, 5]. It is therefore of considerable interest to determine which specific alterations associated with the malignant state, result in these altered levels of cAMP. It is well established that malignant cells exhibit profound alterations in the plasma membrane [6, 7] which include changes in
membrane fluidity [8] and reductions or loss of certain protein components [9, 10]. The adenylate cyclase of malignant cells often exhibits reduced basal levels and/or reduced sensitivity to hormonal stimulation [11, 12] but it is not clear whether these alterations are the result of general changes in the nature of the plasma membrane, resulting in decreased enzyme activity and inability of the hormone-receptor complex to interact with the enzyme, or whether the low activities are the result of the reduction or loss of the concentration of the enzyme or receptor. In order to investigate the latter possiExp Cell Res 108 (1977)
8
AyadandBurns P388F-36, a mouse lymphatic leukaemia cell line, CH23, a Chinese hamster fibroblast, and PCM1, a somatic cell hybrid produced by fusion of P388F-36 and CH23, were grown in Eagles Minimal Essential Medium supplemented with 10% newborn calf serum at 37°C in an atmosphere of 5 % CO2 and 95 % air. The origins and hormonal responsiveness of these cell lines have been described previously [13, 14, 15]. For the present experiments, cells were seeded at 1-2×106 cells in 1 litre glass Roux bottles. At confluency in the case of AP9 CH23 and PCMI or at limiting cell densities in the case of EB2 and P388F36, cells were harvested and homogenates prepared.
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Fig. 1. Abscissa: log prostaglandin E1 (/zM); ordinate: spec. act. of adenyl cyclase (x 10-2) in pmoles cAMP/ 10 min/mg protein. Dose-response curves of adenyl cyclase with prostaglandin El. O - - G , EB-2; []--[], CH23; A - - A , P388F-36; I I - - i , AP-9; O - - O , PCM1; arrow, basal level.
Monolayer cells were scraped off the glass with a rubber policeman and the cells were collected by centrifugation at 4°C (150 g, 5 min). The cells were washed twice in buffer by resuspension and centrifugation and finally resuspended in a few ml of the buffer (50 mM Tris HC1, pH 7.9, containing 0.25 M sucrose, 5 mM MgSO4, 1 mM dithioerythritol and 1 mM ethyleneglycol-bis (/3-amino-ethyl ether) N,N'-tetra-acetic acid). Suspension cells were washed and resuspended in the same manner, ceils were then homogenised in a Dounce all-glass hand homogeniser or using a rotating 'Teflon' pestle. The conditions for homogenisation as judged by optimum adenylate cyclase activity varied somewhat among the cell lines and were as follows: AP9, CH23 and PCMI : 5 strokes hand homogenisation; EB-2:15 strokes hand homogenisation; P388F36 : 10 strokes rotating Teflon pestle (Speed 4, 'Tri-R' Stirrer).
bility, the binding of radioactively labelled prostaglandin E1 has been studied. In the present paper, we report the results of such studies in a number of normal, malignant and hybrid mammalian cells.
1-2
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.0.8
MATERIALS AND METHODS ~0"6
Cell culture The cell lines used in this study were as follows: AP9, Human embryonic lung fibroblasts isolated by ICI Pharmaceuticals, Alderley Edge, Cheshire, UK. These cells are a primary line which grow as a monolayer. EB-2, Burkitt lymphoma cells, obtained from Flow Laboratories Ltd., Irvine, KA12 8NB, Ayrshire, Scotland, are a malignant cell line which grow in suspension to high density. Both these cell lines were grown in Eagle's Minimal Essential Medium supplemented with 10 % foetal calf serum at 37°C in an atmosphere of 5 % CO~ and 95 % air.
Exp Cell Res 108 (1977)
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Fig. 2. Abscissa: [3H]PGE1 (/zM); ordinate: (right) bound [3H]PGE1 (pmoles/mg protein); (left) bound [3H]PGE~ (cpna/mg protein x 10-4). Binding of [ZH]PGE1 to AP-9 membrane. O - - O , Total binding; A - - A , non-specific binding; I I - - n , specific binding.
Prostaglandin E1 binding and adenylate cyclase activation 1"0
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Fig. 3. Abscissa: [3H]PGE1 (/~M); ordinate: (right) bound [3H]PGE1 (pmoles/mg protein); (left) bound [3H]PGE1 (cpm/mg protein x 10-3). 0 - - 0 , Total binding; A---J,, non-specific binding; I - - I I , specific binding. Binding of [3H]PGEa to P388F-36 plasma membranes.
Partially purified plasma membranes were prepared from the homogenates using a discontinuous sucrose gradient centrifugation method [16]. The bands of particulate material which separated during centrifugation were assayed for adenylate cyclase activity and those bands containing the bulk of the enzyme were pooled and diluted with buffer (10 mM Tris-HC1, pH 7.4, containing 0.25 M sucrose and 1 mM dithioerythritol). The membranes were then collected by centrifugation (40000 g, 20 min) and resuspended in the same buffer at a protein concentration of 2-5 mg/ ml. The suspensions were sealed in ampoules and stored under liquid nitrogen until required.
Assay of adenylate cyclase Adenylate cyclase reactions were initiated by the addition of 20-50 /xg of protein to a final volume of 200/xl containing 1.8 mM ATP 50 mM Tris-HC1, pH 7.4 at 30°C, bovine serum albumin 0.1 mg/ml, 0.1 mM EGTA, 1 mM 1-methyl-3-isobutylxanthine, 5 mM MgSO4 and 1 mM dithioerythritol. In some experiments, prostaglandin Ea (a generous gift from ICI Pharmaceuticals Ltd), was included in the assay. Incubations were carried out for 10 or 15 min at 30°C and the reactions were terminated by the addition of 200 /~1 of 0.2 M HCI. After heating at 90°C for 60 rain to hydrolyse the remaining ATP, 80/zl of 2.5 M Tris-HC1, pH 7.4, were added and the cAMP content was determined by a protein binding assay [17]. Samples were also prepared in which membranes were added to incubation mixture after addition of the 0.2 M HCI and then immediately heated at 90°C. This mixture was incubated with standard cAMP samples which were used to construct a standard curve, and serves to correct for any endogenous cAMP present in the membrane preparations. Under these conditions, enzyme activity was a linear function of protein concentration and time of incubation.
9
Assay of [3H]prostaglandin E 1 binding The method followed was essentially that of Powell et al. [18]. Prostaglandin El-[5,6-3H] ([3H]PGE0 was obtained from the Radiochemical Centre, Amersham, Bucks, U K (59 Ci/mmol, 166 mCi/mg), and diluted with distilled water to provide a range of concentrations. Five/xl aliquots of the solutions were added to 1.5 ml plastic Eppendorf tubes, which contained either 5/zl of 25 % ethanol or 5/zl of unlabelled prostaglandin E~ (3 mM) in 25 % ethanol. The final concentration of labelled hormone was varied from 0-0.3 ~M. The frozen plasma membrane preparations were thawed and centrifuged at 40000 g for 20 min and then resuspended in 10 mM Tris-HCl buffer, pH 7.5, containing 0.15 M NaC1. One hundred twenty-five /xl of the suspension were added to the reaction mixtures and incubated at 30°C. After 1 h, 100/zl aliquots were removed and placed on top of a column (8x0.6 cm) of Sephadex G-50 Fine (Pharmacia, Uppsala, Sweden). The column was eluted with buffer at room temperature and the plasma membranes were collected in scintillation vials. Radioactivity was determined by counting in a liquid scintillation spectrometer (Packard Model 3385) after the addition of 100 ml of scintillator (2,5-diphenyl oxazole, 4 g/l; and 1,4-bis-(4methyl-5-phenyloxazolyl)-benzene, 0.2 g/l, dissolved in a mixture of toluene and Triton X-100, 2 : 1 by volume). Independent experiments verified that bound and unbound prostaglandin E~ were completely separated using this procedure. Specific binding was measured by subtraction of the counts remaining bound in the presence of excess unlabelled hormone from the counts observed in the absence of unlabelled hormone. Triplicate values agreed within 10 % of the mean and were averaged to a single representative figure.
Protein determination All protein determinations were performed using the method of Lowry et al. [ 19] with bovine serum albumin as standard. 25
20
0.4
15
1
0-2
5
-
,
0.00
0
0.12
0,18
0.24
0~30
Fig. 4. Abscissa: [3H]PGE1 (/xM); ordinate: (right) bound [sH]PGE1 (pmoles/mg protein); (left) bound [3H]PGE1 (cpm/mg protein x 10-3). O - - O , Total binding; A---A, non-specific binding; II----I, specific binding. Binding of [3H]PGE~ to EB-2 plasma membrane.
Exp CellRes 108 (1977)
10
Ayad and Burns
5
0"10
4
0,08
3 '
0-08
2 •
0"04
1
0"02
, O.OO
, 0.12
0 0.10
0.24
0.30
Fig. 5. Abscissa: [aH]PGE1 (~M); ordinate: (right) bound [3H]PGE1 (pmoles/mg protein); (left) bound [3H]PGE1 (cpm/mg protein x 10-8). e - - e , Total bindrag; A---&, non-specific binding; ~ - B , specific binding. Binding of [3H]PGE1 to CH23 plasma membranes.
RESULTS
Prostaglandin E1 activation of adenylate cyclase The dose-response curves of the plasma membrane preparations are shown in fig. 1. It may be noted that the apparent dissociation constants (KD for mouse lymphoma P388F-36 and the hybrid PCM1 are very similar (approx. 3x10 -8 M) although the maximum activity of the adenylate cyclase differs appreciably in the two cell lines. The apparent Kd of AP9 plasma membranes is approx. 30x 10-s M, or about one order of magnitude greater than that observed in P388F-36 and PCM1. Nevertheless, a marked hormone response is observed. In the case of CH23 and EB2, a very poor response is observed, particularly in the latter case. Moreover, no value for the apparent Ka can be obtained since saturation is not achieved even at PGE1 concentration up to 30/xM. Basal activity of adenylate cyclase also shows considerable variation, but is notably low in CH23 and EB2 preparations. In intact cells, the responsiveness of P388F-36 to PGE, is negligible [14] and contrasts markedly with broken cell Exp Cell Res 108 (1977)
preparations or partially purified plasma membranes. It seems possible that this observation may be due to the very high cyclic nucleotide phosphodiesterase activity found in this cell line, and conversely, the very high accumulation of cAMP in the hybrid, PCM1, may be partially due to the low activity of this enzyme [14]. In contrast to the P388F-36 cell line, EB2 cells retain their poor responsiveness to PGE1 in broken cell preparations. The similarity of the apparent dissociation constants for activation of adenylate cyclase by PGE1 in the parental cell line P388F-36 and the hybrid PCM1, in contrast to the low affinity of CH23 parent, suggest that P388F-36, and not the CH23 receptors are functionally expressed in the hybrid cell line.
[3H]Prostaglandin E, binding Figs 2-6 show the results of [3H]PGE1 binding studies. In the case of P388F-36 and 40
0'8
35
0"6
30
25
0,4
20
15
)'2
10
5
0.00
0.,,
01,8 0.24
0130
Fig. 6. Abscissa: [3H]PGE1 (~M); ordinate: (right) bound [sH]PGE, (pmoles/mg protein); (left) bound [~H]PGE~ (cpm/mg protein × 10-8). O - - e , Total binding; &--&, non-specific binding; B--B, specific binding. Binding of [sH]PGE, to PCM-1 plasma membrane.
Prostaglandin El binding and adenylate cyclase activation
11
analysis. The data appears, however, to be consistent with a significant rate of dissociation of ligand from low affinity receptors during separation of bound and free PGE1. The data thus suggests that the PCM~ hybrid cell has inherited receptors from both parental cell lines. DISCUSSION
_,.',
_;.,
_ 0'.~
0
Fig. 7. Abscissa: log [SH]PGE1 (log/xM); ordinate: log
specific binding (log cpm/mg protein). Logarithmic relationship between observed specific binding and [3H]PGE1 concentration in CH23 and PCM-1 plasma membranes. I1--11, PCM1; D--D, CH23.
AP9 plasma membranes (figs 2 and 3) the specific binding of the hormone closely parallels hormonal activation of adenylate cyclase and Scatchard plots indicate Ka values of 0.04 and 0.2 /xM respectively. No specific binding of hormone could be detected in the Burkitt lymphoma EB-2 plasma membranes (fig. 4) which is consistent with the very low hormonal response observed (fig. 1). These observations suggest that the low response of this cell line may, unlike that of the mouse lymphoma P388F-36, be the consequence of a low concentration of functional receptors in the plasma membrane. The low basal activity of this cell line might also point to a low concentration of adenylate cyclase. The variation of hormone binding with hormone concentration in PCM1 and CH23 plasma membranes produced unusual binding curves (figs 5 and 6), and the data was not amenable to interpretation by Scatchard
Several observations may be made concerning the origin of the low levels of cAMP observed in the malignant cell lines. PGE1 non-responsive EB-2 Burkitt lymphoma cells are characterised by much reduced concentrations of PGE1 receptors and possibly of adenylate cyclase. In this respect it is pertinent to note that Saez et al. [20] demonstrated a qualitative correlation between the levels of PGE1 binding and the hormone responsiveness of adenylate cyclase in a number of adrenocortical turnouts. P388F-36 mouse lymphoma cells, like EB-2 may be characterised by very low intracellular levels of cAMP and by a low hormone responsiveness. However, in P388F-36, this observation is largely re-
20.
16" 12' 8 4
012
014
0,6
0'.8
1'.0
Fig. 8. Abscissa: k-1 (min-1); ordinate: ratio of true
binding to observed binding. Variation of the observed binding of [3H]PGE1 with
the ratio constant, k_~, and time. ©--©, 5 min; O--O, 3 min; [~--D, 2 min; A----A, 1 min; B----B,0.5 min. Exp Cell Res 108 (1977)
12
Ayad and Burns
stricted to intact cells [14]. Very high levels of cyclic nucleotide phosphodiesterase have been demonstrated in these cells, and this is probably the dominant factor in the production of the low levels of cAMP. The present report indicates that in plasma membrane preparations of these ceils, the levels of hormone receptors are not abnormally low, and this finding is in agreement with the hormonal responsiveness. It thus appears that although both these cell lines exhibit the low levels of cAMP often associated with the malignant state, the nature of the events leading to this abnormality in the two cell lines is quite distinct. The activation of PCM1 hybrid cell adenylate cyclase by PGE1 is markedly similar to that observed in the P388F-36 parent, and contrasts sharply with that of the CH23 parent• q'hese observations suggested that the P388F-36 receptor is expressed in the hybrid. In both PCM1 and CH23, however, anomalous but similar binding curves are observed suggesting that only the CH23 receptor is expressed in the hybrid. These anomalous binding curves can be rationalised if two assumption--g-are made: (1) the hormone concentration studied is low compared to the dissociation constant Kd; (2) the rate of dissociation of hormone from the hormone receptor complex is significant. Consider the simplest binding process:
k! H+R ~ H R k-1
where H, R and HR denote the concentrations of free hormone, of uncomplexed receptors and of hormone-receptor complex (i.e. bound ligand) respectively. Since the ligand is totally included by Sephadex G50 Exp Cell Res 108 (1977)
and the plasma membranes (receptors) are totally excluded, the concentration of free ligand available to receptors during the separation is very low, and to a first approximation can be neglected. Thus:
dHR dt =k-lXHR Integrating this expression,
logeHRt=loge HRo-k_l Xt
(1)
where HRt is the observed concentration of bound ligand after elution through the column, and HRo is the concentration of bound ligand before elution. If we denote the total receptor concentration a s R t , then
Rt xH HRo=H~
(2)
where Ka is the dissociation constant= k-l/k1 Substituting (2) into (i) loge HRt =lOge Rt xH -k-1 x t
(3)
•K a + H
ifH~Ka, then loge HRt = 1Oge~a + 1OgeH-k_1X t
(4)
which predicts a logarithmic relationship between the observed ligand binding and the concentration of free hormone. Fig. 7 shows a plot of log (observed binding) vs log (hormone concentration). The observed linearity of these plots for PCMI and CH23 indicates that such a relationship exists, and suggests that the anomalous binding curves arise in the manner outlined above. Ifk_lxt is negligible, H R t = HRo, if not then the total receptor concentration observed will be underestimated even where H is not very much less than
Prostaglandin E~ binding and adenylate cyclase activation Ka, although normal saturation curves will be observed in such cases. Brunton et al. [21] have obtained a value for k_~ of 0.15 rain -1 in a murine L cell. These authors separate free and bound ligand by centrifugation, the process taking several minutes to completion. At saturating concentrations of hormone, eq. (3) may be written as:
1
Rt
_k_~xt
Ogl° H g t -
2.306
(3)
Assuming k_1=0.15 min -~ and t~5 min (data ofBrunton et al.)
HRt- Rt
2.1
Thus a significant underestimation of total receptor concentration is observed. Since a logarithmic relationship exists between the ratio of true and observed binding, and k_l×t, relatively small differences in k_~xt can produce correspondingly greater errors in the estimation of the total receptor concentration. In the present studies, the time taken to elute the plasma membrane fraction was 3 min. The ratio of true to observed binding as k_l varies from~0 to 1 is shown in fig. 8, for several separation times. Although reports of kinetic studies of prostaglandin Ea binding are rare, Brunton et al. [21] estimated k_~ as 0.15 min -~ as noted above, and given..this figure, values for k_a between 0.01 and f seem reasonable. Since a value for k_~ of 1 would result in only 5 % of the true binding being observed, it is apparent that great care must be taken in the interpretation of binding data, relying on methods of separation of free and bound ligand which require considerable time to achieve. Millipore filtration techniques were employed to reduce the time required for separation. However, these filters were found
13
to retain high radioactivity even in the absence of membranes which made the evaluation of specific binding difficult. Despite these difficulties, hormonal activation studies demonstrate the presence of a high affinity receptor in PCMa hybrid cell plasma membranes similar to the P388F-36 parent. Hormone binding studies indicate the presence of low affinity receptors similar to the CH23 parent. These data strongly suggest that both parental receptors are expressed in the PCM~ hybrid, but that only the high affinity receptor is able to activate adenylate cyclase, presumably because the concentration of the latter is limiting. The authors wish to express their thanks to Professor G. R. Barker for providing the facilities for this work and Miss Margaret Barber for secretarial assistance. Financial support from the Cancer Research Campaign and the MRC for one of us (G. R. J. B.) is gratefully acknowledged.
REFERENCES 1. Anderson, W B & Pastan, I, Adv cyclic nucleic res 5 (1975) 681. 2. Chlapowski, F T, Kelly, L A & Butcher, R W, Adv cyclic nucleic res 6 (1975) 245. 3. PastaS, I & Johnson, G S, Adv cancer res 19 (1974) 303. 4. Pastan, I, Johnson, G S & Anderson, W B, Ann rev biochem 44 (1975) 491. 5. Ryan, W L & Heidrick, M L, Adv cyclic nucleic res 4 (1974) 81. 6. Hynes, R O, Biochim biophys acta 458 (1976) 73. 7. Nicholson, G L, Biochim biophys acta 458 (1976) 1. 8. Micklem, K J, Abra, R M, Knutton, S, Graham, J M & Pasternak, C A, Biochem j 154 (1976) 561. 9. Shizuta, Y, Davies, P J A, Olden, K & Pastan, I, Nature 261 (1976) 414. 10. Yamada, K M, Yamada, S S & Pastan, I, Proc natl acad sci US 73 (1976) 1222. 11. Polgar, P, Vera, J C, Kelley, P R & Rutenberg, A M, Biochim biophys acta 297 (1973) 378. 12. Prasad, K, Gilmer, J, Sahu, S & Becker, G, Cancer res 35 (1975) 77. 13. Ayad, S R & Delinassios, J G, Biochem genet 12 (1974) 147. 14. Ayad, S R & Foster, S J, Cell 3 (1974) 135. 15. Ayad, S R &White, A, Exp cell res 107 (1976) 201. 16. Glossmann, H & Gips, H, Arch pharmacol 289 (1975) 77.
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17. Brown, B L, Albano, J D M, Etkins, R P & Sgherzi, A M, Biochem j 121 (1971) 561. 18. Powell, W S, Hammerstrom, S & Samuelsson, B, E u r j biochem 41 (1974) 103. 19. Lowry, O H, Rosebrough, N J, Farr, A L & Randall, R J, J biol chem 193 (1951) 265. 20. Saez, J M, Dazord, A & Gallet, D, J clin invest 56 (1975) 536.
Exp Cell Res 108 (1977)
21. Brunton, L L, Wiklund, R A, Van Arsdale, P M & Gilman, A G, J biol chem 251 (1976) 3037. Received March 9, 1977 Accepted March 11, 1977
