• Abstract and Figures
• Public Full-text

[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 (ÄJ) 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 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1988 American Association for Cancer Research. PHOSPHOLIPIDS AND PHORBOL ESTER BINDING PDB-binding Assay. The binding assays were carried out by using intact monolayer cultures in plastic culture dishes according to a nioí)¡ticaiion of the method of Solanki and Slaga (15). The cells were washed twice with DME at 4°Cand incubated with 1 ml DME con taining 15 mM4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid and appropriate amounts of [3H]PDB at 4°Cwith gentle rotary shaking at 60 rpm. To estimate the amount of nonspecific binding, 10 ¿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 KÕvalueswere 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 /¿MEtnand then were for each experimental point.

Load more

  6

Copy Link