Effects of Phosphatidylethanolamine and Phosphatidylcholine in Membrane Phospholipid on Binding of Phorbol Ester in Rat Mammary Carcinoma Cells1

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[CANCER RESEARCH 48, 1528-1532, March 15, 1988J Effects of Phosphatidylethanolamine and Phosphatidylcholine in Membrane Phospholipid on Binding of Phorbol Ester in Rat Mammary Carcinoma Cells1

Tamiko Kano-Sueoka2 and David M. King

Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado S0309

ABSTRACT sphingomyelin are however not altered (5, 6). Etn-responsive cells are not able to synthesize, without an exogenous supply Mammalian cells in culture can be classified as either ethanolamine of Etn, a sufficient amount of PE to maintain growth (6). (Etn)-responsive or Etn-nonresponsive with regard to their growth. Epi Growth and phospholipid compositions of fibroblasts, neuro- thelial cells and some of their transformed derivatives are the Etn- cells, and certain neoplastic cells of epithelial origin, on the responsive type. When these cells are grown without Etn, the content of other hand, are not influenced by Etn in culture medium (2, 5). membrane phospholipid becomes significantly altered. Namely, the con tent of phosphatidylethanolamine is reduced and that of phosphatidyl- When Etn-responsive cells are grown without Etn, as the choline is increased. In addition, the growth rate of these cells is reduced. content of membrane PE is reduced, the growth slows down. Therefore, it is likely that the phosphatidylethanolamine deficiency or The reason as to why PE deficiency leads to the cessation of phosphatidylcholine excess is unsuitable for some membrane-associated cell proliferation could be that the PE synthesis is somehow functions resulting in the cessation of growth. In order to test the above tied to cell growth or the PE deficiency creates unfavorable hypothesis, we examined the binding of a tumor-promoting phorbol ester, conditions for the membrane-associated function, resulting in |'H|phorbol 12,13-dibutyrate (PDB), to an Etn-responsive rat mammary the cessation of growth. In the present study we compared the carcinoma cell line 64-24 grown with (Etn-plus) or without Etn (Etn- binding properties of a tumor-promoting phorbol ester, PDB minus). The time course of binding was very similar between Etn-plus to 64-24 cells grown in the presence or absence of Etn, in order and -minus cells, except that the level of saturation was higher in Etn- to examine the above assumptions. High affinity phorbol ester plus cells, whereas the time course of chase of the bound PDB was significantly different between the two types of cells. Both types of cells receptors have been found in membrane fractions of mamma have one class of binding sites for PDB. The dissociation constant (Ã„J) lian cells (7). Evidence so far accumulated suggests that the for [3H]PDB in Etn-plus cells was 34.0 UM and the number of binding receptor protein is Ca2+-activated, phospholipid-dependent pro sites at saturation was 2.7 x 1012/mg protein or 3.6 x 105/cell. The tein kinase C (8, 9). Further, phorbol esters can fully activate corresponding values in Kin-minus cells were 61.4 UMand 3.2 x 1012/mg this enzyme in the presence of Ca2+ and phospholipids (10). protein or 5.4 x 105/cell, respectively. Although the difference in A*,, Different phospholipids have different effects on the binding of values of the two types of cells was only 2-fold, this difference was phorbol esters to kinase C and also on the enzyme activity (8, statistically significant. On the other hand, the number of binding sites/ 11). Human promyelocytic leukemia cells, whose major polar mg protein in these cells was very similar. Since the amount of protein/ cell was 1.4-fold higher in Etn-minus cells as compared to that of Etn- head groups of phospholipid were choline analogues, have been plus cells, the number of binding sites/cell was larger in Etn-minus cells. shown to have a considerably higher level of PDB binding (12). The present study has shown that (a) 64-24 cells grown with PDB affected the rate of proliferation of 64-24 cells differently, depending or without Etn possess one species of PDB-binding sites; (b) on whether they were grown in the presence or absence of Etn. These results suggest that the phosphatidylethanolamine and/or phosphatidyl however, the binding properties of PDB to these cells were choline content of the membrane phospholipid affects cellular functions distinctly different, although the difference was not so dramatic. mediated by phorbol esters. These results suggest that membrane-associated functions can be influenced by the PE and/or PC content of the membrane phospholipids. INTRODUCTION

Etn,3 which is a structural component of the second most MATERIALS AND METHODS abundant phospholipid in the mammalian cell membrane, PE, is required by a variety of normal and neoplastic mammalian Materials. Horse serum and DME were purchased from Irvine epithelial cells and also by plasmacytoma and hybridoma cells Scientific (Santa Ana, CA). Fetal calf serum was from Flow Laborato ries (Rockville, MD). Etn and PDB were obtained from Sigma Chemical to grow optimally in a defined culture medium (1-4). Compo Co. (St. Louis, MO), and [3H]PDB (specific activity, 12.2 Ci/mmol) sition of membrane phospholipids in these Etn-responsive cells and aqueous scintillation counting fluid, Biofluor, were purchased from is altered when the cells are cultured without Etn (5). The New England Nuclear (Boston, MA). Lux plastic tissue culture dishes content of PE in cells grown without Etn (Etn-minus cells) is were from Miles Scientific (Naperville, IL). about one-third the amount found in those cultured with Etn Cell Culture. The 64-24 cells, clonally isolated from a rat mammary (Etn-plus cells) or in comparable cells in vivo, and the content carcinoma and typically Etn responsive (2, 13) were maintained in of PC in Etn-minus cells increases inversely proportional to DME with 5% horse serum and 2.5% fetal calf serum as described that of PE. The contents of PS, phosphatidylinositol, and previously (13). For the binding experiment, the cells were cultured in DME with 2% fetal calf serum in the presence or absence of 10 MMEtn Received 2/6/87; revised 9/28/87, 12/4/87; accepted 12/9/87. for at least 10 generations. One day before the binding experiment, the The costs of publication of this article were defrayed in part by the payment cells were plated in a serum-free medium (14) or DME containing 2% of page charges. This article must therefore be hereby marked advertisement in fetal calf serum with or without 10 JIMEtn at about 7 x 10s cells and accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by National Science Foundation grant PCS 104480 1 x IO6 cells/35- x 10-mm well, respectively. Etn-nonresponsive rat and NIH Grant CA30545. mammary carcinoma line, 22-1 (13), and Chinese hamster lung fibro- 2To whom requests for reprints should be addressed. 3The abbreviations used are: Etn, ethanolamine; PE, phosphatidylethanola blast line, V-79 (obtained from Dr. David Prescott, University of mine; PDB, phorbol 12,13-dibutyrate; PC, phosphatidylcholine; PS, phosphati- Colorado, Boulder, CO), were also cultured in the same manner as 64- dylserine; DME, Dulbecco's modified Eagle's medium. 24 cells. 1528

PDB-binding Assay. The binding assays were carried out by using intact monolayer cultures in plastic culture dishes according to a nioÃ)Â¡ticaiion of the method of Solanki and Slaga (15). The cells were washed twice with DME at 4Â°Cand incubated with 1 ml DME con taining 15 mM4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid and appropriate amounts of [3H]PDB at 4Â°Cwith gentle rotary shaking at 60 rpm. To estimate the amount of nonspecific binding, 10 Â¿ig/mlof nonradioactive PDB were added in addition to the radioactive PDB. PDB was dissolved in dimethyl sulfoxide; the final concentration of dimethyl sulfoxide in the incubation medium was 1.5%. Duplicate samples were analyzed for each experimental point for both total and nonspecific bindings. The cells were harvested by washing three times in ice-cold phosphate-buffered saline and lysed with 1 ml 0.2% sodium dodecyl sulfate. The radioactivity in each sample was determined by using 0.7 ml sample and 12 ml Biofluor scintillation fluid. Protein was Fig. 1. Time course of binding of PDB to 64-24 cells. 64-24 cells were grown determined by the method of Schacterle and Pollack (16). Analysis of in 60- x 10-mm culture dishes in DME with 2% fetal calf serum in the presence the competition of [3H]PDB binding against varied concentrations of or absence of 10 /,\i Etn. After washing the cells 2 times with DME, the cells were incubated with 1 ml DME containing IS HIM4-(2-hydroxyethyl)-l-pipera- nonradioactive PDB was carried out in order to obtain the K, values. zineethanesulfonic acid and 0.05 nCi [3H]PDB (specific activity, 12.2 Ci/mmol) The KÃ•valueswere calculated from the concentrations of nonradioactive PDB which gave 50% inhibition of [3H]PDB binding according to the for varying lengths of time at 4 ( with gentle shaking. To estimate nonspecific binding, 10 ,1:111!of nonradioactive PDB were added in addition to the radioac method of Yarus (17). tive PDB. PDB was dissolved in dimethyl sulfoxide; the final concentration of Cell Growth Analysis. Cells were grown for 3 days in DME with 2% dimethyl sulfoxide in the medium was 1.5'.. Duplicate samples were analyzed fetal calf serum in the presence or absence of 10 /Â¿MEtnand then were for each experimental point. For the chasing experiment, after 75 min of labeling plated at 1 or 5 x IO4 cells/60- x 10-mm plate in a fresh medium of with radioactive PDB, the cells were rinsed two times, the medium was replaced with medium lacking radioactive PDB, and incubated further. The cells were the same composition or in a serum-free medium (14) with varied harvested at appropriate intervals by washing three times with cold phosphate- amounts of phorbol ester. After 4 days of growth, cells were harvested buffered saline and lysed with 1 ml 0.2% sodium dodecyl sulfate. The radioactivity and the protein amount in each sample were then determined, and the amount of by trypsin treatment and counted in a Coulter Counter. Triplicate radioactive PDB specifically bound was plotted. A, time course of binding; B, samples were counted for each phorbol ester concentration, and the time course of chasing: â€¢,Etn-plus cells; O, Etn-minus cells. experiments were repeated at least once. Table I Chase of specifically bound PDB RESULTS Experiments were carried out as described in Fig. 1. Data from Etn-plus cells were derived from duplicate samples of two separate experiments and those of Kinetics of Binding of PDB. The 64-24 cells were grown in Etn-minus cells were from duplicate samples of three separate experiments. The difference in percentage of radioactivity remaining after 6-min chase between the the presence or absence of 10 /Â¿MEtnfor 10 to 15 generations two types of cells was highly significant (/' < 0.005) and that of 12-min chase was before the binding assays were performed. After more than 10 also significant (0.05 < P < 0.1). generations of growth without Etn, the rate of growth of these boundEtn+6-min % of PDB cells (Etn-minus cells) was very slow and the PE and PC chase101.2 2-minchase87.8 contents of the membrane phospholipid were about 12 and Â±2.0Â° Â±2.975.5 62%, respectively, in contrast to 29 and 44% in the cells grown 85.9 Â±1.71 Â±4.2 with Etn (Etn-plus cells), as previously described (5, 6). The â€¢MeanÂ±SE. time course of binding of the labeled PDB at 37Â°Cindicated that the specific binding (the difference between the binding in rizes the results of the chase experiments. As indicated, during the presence and absence of 10 ng/m\ nonradioactive PDB) to the first 6 min of the chase the specifically bound PDB was both Etn-plus and -minus cells reached a maximum within 3 hardly released from the Etn-plus cells, whereas Etn-minus min after the addition of radioactive PDB, and then the bound cells released about 15% of the bound PDB. The difference radioactivity decreased rapidly. By 30 min, the radioactivity between the two became less significant as the duration of the bound was less than one-half the amount found in 3-min chase became longer. The release of nonspecifically bound PDB labeling (data not shown). Therefore, it was difficult to obtain (20% of total PDB bound at 0-min chase), on the other hand, reproducible results from the binding studies carried out at commenced immediately after the chase was initiated in both 37Â°C.When the binding was carried out at 4"C, the amount of types of cells. This differential loss of nonspecifically and spe PDB specifically bound increased with time and the loss of the cifically bound PDB at the onset of chasing in Etn-plus cells, bound radioactivity was not observed for at least 80 to 100 min and experimental errors in obtaining total and nonspecific in both Etn-plus and -minus cells. Fig. 1 shows a representative counts bound/mg protein resulted in artificially higher levels of result of the time course of specific binding and chasing of the specifically bound PDB after 6-min chase as compared to the bound PDB at 4Â°Cin Etn-plus and -min cells. Kinetics of 0-min samples in some cases (see Table 1). The characteristic binding in the two types of cells were similar except that the chasing pattern of Etn-minus cells was observed even when the amount of specifically bound PDB at a plateau level seemed to pulse and chase were carried out in the presence of 10 ^M Etn be higher with Etn-plus cells (about 25%) (Fig. IA). When the or PE, if the cells were grown in the absence of Etn prior to the binding of PDB was allowed to reach a plateau and the medium experiment. There is hardly any incorporation of Etn or PE was changed to the one without radioactive PDB, the bound into the cell membrane at 4Â°Cfor the duration of the experi radioactivity was gradually released from the cells. In contrast ment. Therefore, neither free Etn nor PE affects the binding to the kinetics of binding, those of the release were significantly characteristics. The above results indicate that Etn-plus cells different between the two types of cells: namely, in Etn-minus seem to bind PDB tighter than Etn-minus cells, and neither cells the release of radioactive PDB always started immediately phospholipids nor their precursors in the medium seem to after the replacement of the medium, whereas in Etn-plus cells influence the binding characteristics of PDB. the loss of the radioactivity from the cells was significantly Dose Saturation Binding of PDB. The binding of PDB to Etn- slower than that in Etn-minus cells (Fig. IB). Table 1 summa- plus and -minus cells was further studied by the Scatchard 1529

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1988 American Association for Cancer Research. PHOSPHOLIPIDS AND PHORBOL ESTER BINDING analysis (18). Varied amounts of radioactive PDB were bound to the cells in mid- to late-log phase in saturation conditions. Nonspecific bindings were determined in the presence of excess nonradioactive PDB (10 ng/ml) and were measured separately for each concentration of radioactive PDB. The level of non specific bindings, which increased linearly with increasing con centrations of radioactive PDB, was not affected by Etn, but was correlated with the cell density: namely, the lighter the cell density, the lower the nonspecific binding per unit amount of 12345 Bound (pmol/mg protein) protein (Fig. 2). The specific bindings were saturable and also increased with increasing concentrations of radioactive PDB.

The results of two experiments are shown in Fig. 2. The amount 0.16 of PDB bound/mg protein at saturation tended to be higher (IS to 20%) in Etn-plus cells than in Etn-minus cells. The 0.12 Scatchard analyses of the binding data (using only concentra tion ranges of PDB up to 100 DM)yielded straight lines from 0.08 t all samples tested, indicating that both Etn-plus and -minus 0.04 cells had one species of binding sites for PDB under our experimental conditions. Two representative Scatchard plots s 12345 from two sets of Etn-plus and -minus cells are shown in Fig. 3. ' Bound (pmol/mg protein) The binding data of Etn-plus cells shown in Fig. ,1-fyielded a single slope corresponding to a A',,for 32.9 IIMPDB, and at Fig. 3. Scatchard plots of PDB binding. The results shown in Figure 2 were plotted according to Scatchard (17) to yield Ads and number of binding sites at saturation 2.8 pmol of PDB were bound/mg protein or 4.1 x saturation. Data obtained by PDB concentrations up to 100 nM were used to 10s molecules of PDB/cell. In Etn-minus cells, the Scatchard calculate the above parameters, x, Etn-plus cells; O, Etn-minus cells. analysis yielded a A'.,S4.2 n\r and 2.9 pmol bound/mg protein Table 2 Comparison of PDB binding parameters between Etn-plus and or 5.0 x 10s molecules/cell. The results of five similar experi -minus cells ments are summarized in Table 2. Although the difference in Binding sites/cell Ad values between the two types of cells were at most 2-fold, K., (nM) (xlO~5) they were reproducibly different and the difference was statis Experiment!2345AvEtn-plus32.332.932.022.946.834.0 tically significant: the average A,,for Etn-plus cells was smaller than that of Etn-minus cells. In contrast, the number of PDB- binding sites per mg of protein was literally the same between the two cell types. Etn-minus cells contained a significantly higher number of binding sites per cell since Etn-minus cells had more protein per cell than the plus cells (5.8 x K)'1cells/ Â±3.9Â°PÂ±7.50.01Etn-plus2.14.13.93.93.83.6Â±0.4Etn-minus7.75.03.75.25.25.40.60.02 Â± 0.05' =Etn-minus75.954.278.437.761.061.4< P < mg protein for Etn-minus cells versus8 x IO6cells/mg protein Mean Â±SE. for Etn-plus cells).

S o I 6 I Â£4 i 2345 PDB (ng/ml) Fig. 4. Effect of PDB on growth of 64-24 cells. 64-24 cells were plated at 5 x 10' cells/60- x id mm plate in triplicate in DME with 2% fetal calf serum in the presence or absence of 10 JIM Etn with varied amounts of PDB. The control plates received dimethyl sulfoxide without PDB. The concentration of dimethyl 50 100 ISO sulfoxide was 1.5% in all samples. After 4 days of growth the cells were harvested Free (3HJ PDB (nM) by trypsin treatment and counted in a Coulter Counter. Triplicate samples were Fig. 2. Dose saturation binding of |'II|PI)B to Etn-plus and -minus 64-24 counted for each PDB concentration. â€¢,Etn-plus cells; O, Etn-minus cells. cells. Etn-plus and -minus cells were prepared in the manner described in Fig. 1, except that they were plated in a serum-free medium after preconditioning the The KÃ•valuesfor Etn-plus and -minus cells were verified by cells in DME containing 2% fetal calf serum with or without Ein. Varying concentrations of [3H]PDB, ranging from 1 to 230 nM, were added to the plates calculating K, values from competition analysis of [3H]PDB and the plates were incubated at 4'C for 80 min. Duplicate samples were taken binding against nonradioactive PDB. The average A',for Etn- for total and nonspecific bindings at each PDB concentration. The samples were plus cells was 65 nM and that of Etn-minus cells was 150 nM. treated as described in Fig. 1. The results of two representative experiments are These values were 2 to 3 times higher than the A"dsobtainedby shown in .I and B. , â€¢A, Etn-plus cells; , O A, Etn-minus cells. â€¢ and O, specific binding; A and A, nonspecific binding. the Scatchard analysis. The cause of the discrepancy could be 1530

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1988 American Association for Cancer Research. PHOSPHOLIPIDS AND PHORBOL ESTER BINDING Table 3 Effect qfPDB on growth of Etn-nonresponsive cells some membrane-associated functions. The present study was V-79 cells (5 X IO4cells/plate) and 22-1 cells (2 x 104cells/plate) were plated in the presence or absence of 10 JIMEtn with varied amounts of I'I Mi. After 4 undertaken in order to test the above hypothesis. The results days of growth the cells were counted as described in "Materials and Methods." indicated that the binding properties of PDB in Etn-plus and of -minus cells were indeed different, although not in a striking PDB(10 of cell no. stimulation100.0167.7166.2100.0169.6151.8100.0141.9100.0171.4manner. This small but significant difference is probably CelllineV-7922-1EtnMM)(ng/ml)+ (day 4/day0)6.5 enough to affect the cascades of functions caused by PDB upon 025100025100+ Â±0.6Â°10.9 binding to the cell membrane. Indeed, Etn-plus and -minus cells Â±0.710.8 Â±0.35.6 exhibited different growth responses to PDB. Â±0.19.5 The most conspicuous difference in the properties of Etn- Â±0.88.5 plus and -minus cells is found in their membrane phospholipid Â±0.817.2 composition (2, 5, 6). Therefore, it is very likely that the PDB 01000100Ratio Â±1.024.4 binding in these cells is influenced by the characteristic property Â±0.817.1 Â±1.129.3 of their membrane phospholipid. Alteration of the membrane Â±1.8Degree phospholipid can be artificially achieved by feeding tissue cul â€¢MeanÂ±SE. ture cells choline analogues in place of choline (19). Such cells have been shown to modulate the binding property of PDB that all of the binding reactions having varied amounts of PDB (12). Delclos et al. (20) found that a photoaffinity-labeled did not reach equilibrium during the incubation period (80 min phorbol ester specifically bound to PS and PE when mouse at 4Â°C).Once again, however, a roughly 2-fold difference be brain membranes were utilized for the binding. Phorbol esters tween the two cell types was observed. are known to bind to protein kinase C in the presence of Effect of PDB on Cell Growth. The effect of PDB on growth phospholipids. PS is necessary for the binding of the kinase was examined in Etn-plus and -minus cells in order to determine and phorbol esters. In addition, PE potentiates, while PC whether the observed difference in the binding characteristics inhibits the enzyme activity (8, 11, 20). These results indicate of PDB to these two types of cells is reflected in their subsequent that PE certainly plays an important role in binding PDB to intracellular events. Fig. 4 shows an example of such experi the cells and activating the kinase C. The 64-24 cells grown in ments. Etn-plus and -minus cells responded in a significantly the presence or absence of Etn contain essentially the same different fashion to PDB when the cells were given PDB at amount of PS, but the content of PE is considerably lower and 0.25 to 25 ng/ml. Growth of Etn-plus cells was clearly stimu that of PC higher in Etn-minus versus Etn-plus cells (6). It is lated by PDB, whereas that of Etn-minus cells was not. From therefore reasonable to assume that binding of PDB to the experiment to experiment the dose-response patterns in both kinase C on the cell membrane is affected when the membrane Etn-plus and -minus cells varied to some extent. However, a becomes PE deficient and PC excess. KAvalues obtained from distinct difference between the two cell types was usually ob 64-24 cells having normal membrane phospholipid were about served. After correcting the concentrations of PDB in the cells 4 times higher than those of mouse epidermal cells or rat for partitioning, half-maximum dose of PDB that effected the embryo fibroblasts (15, 21); however, the total number of sat growth of Etn-plus cells ranged from 1.5 to 10 HM.The above urable binding sites per cell was 3 to 4 times higher. These results indicate that the effects of PDB on 64-24 cells seem to differences may arise from the characteristic properties of 64- be more sensitive in growth stimulation than in binding affinity. 24 cells or a particular binding assay procedure used in the The difference may have resulted from the fact that the growth present study. experiment was carried out at 37Â°C,whereas the binding ex The altered properties of the PE-deficient membrane seem periment was done at 4Â°C.At higher concentrations of PDB to be manifested in intracellular events subsequent to the bind such as 100 ng/ml, growth of both types of cells was inhibited, ing of PDB. This was indicated by the manner in which PDB but to a different extent. PDB did not cause any morphological affected the growth of the Etn-plus and -minus cells. When alteration in either Etn-plus or -minus cells at concentrations epithelial cells are cultured in an ordinary culture medium, such tested. Another phorbol ester tumor promoter, 12-0-tetra- as DME with an optimal amount of serum but without Etn, decanoylphorbol-13-acetate, at 100 ng/ml, also exhibited a the cells grow quite well, but their phospholipid compositions growth-inhibitory effect similar to PDB on 64-24 cells, while are significantly altered from those of their in vivo counterparts non tumor promoter phorbol esters, 4a-phorbol 12,13-dideca- (2). The results obtained in the present study suggest that noate and 4a-phorbol, did not affect growth of these cells. The membrane-associated functions in these cells are likely to be effect of PDB was also tested on the proliferation of Etn- altered from those of the in vivo state. nonresponsive cells, a Chinese hamster lung fibroblast cell line, V-79, and a rat mammary carcinoma cells line, 21-1. In contrast REFERENCES to Etn-responsive cells, growth of these cells was stimulated by PDB at 100 ng/ml, and their growth seemed to be affected to 1. Kano-Sueoka, T., Cohen, D. 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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1988 American Association for Cancer Research. Effects of Phosphatidylethanolamine and Phosphatidylcholine in Membrane Phospholipid on Binding of Phorbol Ester in Rat Mammary Carcinoma Cells

Tamiko Kano-Sueoka and David M. King

Cancer Res 1988;48:1528-1532.

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