Corticosteroids and Phosphatidylcholine Biosynthesis in Microsomal Fractions from L5178Y Lymphoma1

[CANCER RESEARCH 36, 1545-1550, May 1976] Corticosteroids and Phosphatidylcholine Biosynthesis in Microsomal Fractions from L5178Y Lymphoma1

George Melnykovych,2 Mary M. Standaert, Edna R. Matthews, and Susan L. Gray

Research Service, United States veterans Administration Hospital, Kansas City, Missouri 64128 (0. M., M. M. S., S. L. G.j, and Department of Microbiology, University of Kansas Medical School, Kansas City, Kansas 66103 1G. M., E. R. M.J

SUMMARY pe?iod was required for the growth inhibition to become manifest or that a limiting nutrient in the medium had to be Microsomal fractions from mouse lymphoma L5178Y and exhausted before the cells became susceptible to the toxic from rat thymocytes were used to follow incorporation of effects of the hormone. A search among several alternate rad iolabel from cytid me d iphosphate-[methyl-'4C]cho line possibilities led to the observation that incorporation of into microsornal lipids. Dexamethasone, at concentrations choline into PL' was inhibited by glucocorticoids in either ranging from 2.8 x 10@M to 2.8 x 10@M, partially inhibited synchronized or logarithmically growing cells, provided the this transfer reaction. Microsomes prepared from freshly latter were first depleted of choline (22). Neither choline isolated thymocytes were more sensitive to the effects of uptake nor its phosphorylation appeared to be involved in dexamethasone showing inhibition at concentrations of the steroid effect. Further studies of this system (16) have steroid as low as 2.8 x 10@ M. The inhibitory effect did not shown that microsomal fractions from L5178Y cells could depend on the amount of the available endogenous diglyc incorporate choline by 2 alternate pathways including the enides and was not related to a possible stimulation of â€œdirectâ€•Ca2@-stimulatedexchange of choline and the so cytidine diphosphate choline transferase back reaction by called Kennedy pathway. The latter involves phosphoryla the steroid. tion of choline to phosphorylcholine followed by the forma The survey of a broad selection of different steroids re tion of CDP-choline with the subsequent transfer of choline vealed a lack of correlation between the known lymphocyto to a diglyceride acceptor yielding PL and CMP (10). Prelimi lytic properties of steroids and their effects on cytidine nary results utilizing microsomal fraction from cells ex diphosphate choline transferase. Dexamethasone was the posed to steroids during growth indicated that CDP-choline only steroid of the glucocorticoid group that inhibited this transferase (CDP-choline:1 ,2-diglycenide choline phospho reaction in microsomal fractions of L5178Y lymphoma. The transferase, EC 2.7.8.2) was inhibited while the choline ex structural requirement for the inhibitory effect was related change reaction was unaffected (20). This work has now to the absence of oxygen functions in positions 11 and 17 of been extended to include experiments in which steroids the steroid and, possibly, to the presence of both C-20 and have been added directly to the reaction mixture without C-21 on the side chain. previous treatment of intact cells.

INTRODUCTION MATERIALS AND METHODS

For the most part, recent studies on the mode of action of Cells and Media. The origin and the procedure for grow steroids toward lymphatic cells have been concerned with ing L5178Y cells have been described in detail in an earlier identification of steroid receptors (1, 2, 12). Attempts to ex paper (21). For these experiments the cells were grown in plain the emergence of steroid-resistant clones among pop suspension in Fischer's medium (Grand Island Biological ulations of susceptible cells have led to the proposal that Co., Grand Island, N. Y.) supplemented with 10% horse the resistance to the cytolytic effects is related to the ab serum; penicillin G, 100 units/mI; and streptomycin sulfate, sence of specific steroid-binding molecules (18). Neverthe 200 j.@g/ml.The cells were monitored regularly for Myco less, this important finding does not change the fact that, in plasma and were found to be free of contamination. biochemical terms, there is very little information about the Growth Experiments. For growth measurement, the mechanism of steroid-induced lymphocytolysis. Some time L5178Y cells were inoculated at the density of 5 x 10@cells/ ago we reported (21) that, in synchronous cultures of ml into 50-mI Erlenmeyer flasks containing 10 ml of growth L5178Y lymphoma, the growth-inhibitory effect could not be medium and were grown on a rotary shaker at 75 rpm at 37Â° seen unless the cells have been exposed to the hormone for in 5% CO2 in air. The incubation times varied from 12 to 90 at least 1 growth cycle. This suggested that either a long lag hr. In some experiments delipidized serum proteins from whole sera according to the procedure of G. Rothblat (per I This investigation was supported in part by USPHS Research Grant CA 08315 from the National Cancer Institute. sonal communication) were used. At the end of each incu 2 To whom requests for reprints should be sent, at: Research Service (151), Veterans Administration Hospital, 4801 Linwood Boulevard, Kansas City, Mo. 3 The abbreviations used are: PL phosphatidylcholine; CDP-choline, cyti 64128. dine diphosphate choline; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethane Received October 28, 1975; accepted January 16, 1976. sulfonic acid; DEX, dexamethasone.

MAY 1976 1545

bation period cells were enumerated in a Model B Coulter activity. At the end of the incubation period, 1-ml portions of counter. the incubation mixture were added to 8 ml of metha Thymocytes. Adrenalectomized male rats weighing ap nol:chloroform (1:1). The mixture was kept in the cold room proximately 150 g were obtained from Charles River Farms, overnight and then extracted according to a modified pro North Wilmington, Mass., and were sacrificed 10 days post cedure of Folch et a!. (8) as previously described (16). adrenalectomy. The thymocyte suspensions were prepared Briefly, the method involved filtration of the extract through according to the method of Munck (17). glass wool, reextraction with chloroform, and washing the Preparation of Microsomal Fractions. Freshly harvested extract 6 times to remove CDPcholine. After the final wash, L5178Y cells or freshly isolated thymocytes were washed in the lower phase was evaporated to dryness in scintillation 0.85% NaCl solution and were suspended in 1 mM phos vials and dissolved in a toluene-based scintillation fluid (5 g phate buffer, pH 7.4, that contained 1 mM MgCI2 and 0.32 M PPO and 0.12 g POPOP per liter of toluene). The samples sucrose (14). They were disrupted in the cold by homogeni were counted in a Packard Tni-Carb scintillation spectrome zation and then subjected to a differential centnifugation ten, and the results were expressed as dpm/mg of microso procedure as previously described (16). The clean microso mal protein as determined by the procedure of Lowry et a!. mal pellet isolated from the final centnifugation at 111,000 x (13). g was resuspended in 0.27 M sucrose. The preparations For the study of the back reaction of CDP-choline trans were stored at â€”20Â°andwere found to retain their activity ferase, the microsomal fractions from L51 78Y cells that had for at least 1 month. been grown in the presence of [methy!-'4C]choline were CDP-Choline Transferase. The reaction mixture con incubated at 37Â°for30 mm in the reaction mixture contain tamed o.s to 0.8 mg microsomal protein, 20 mM HEPES ing 20 mM HEPES buffer (pH 7.5), 34 mM sucrose, 1 mM buffer (pH 7.5), 34 mM sucrose, 1 mM MgCI2 and 0.12 @Ci MgCI2, and 4 x 1O@MCMP. The radioactivity in the microso CDP-[methyl-'4C]choline chloride in a final volume of 2.4 ml. mal lipid fraction was determined as described above. The preparations were incubated at 37Â°forvarious times. As Steroids. The complete list of steroids used is shown in shown previously (16), the reaction proceeded linearly with Table 1. They were dissolved in absolute ethanol. Appropni time for at least 30 mm. This was the usual time used to ate ethanol controls were included with all experiments. determineeffectofsteroidson CDP-cholinetransferase Radioactive Isotopes. The CDP-[methy!-â€˜4C]choline(spe

Table 1 Effects of selectedsteroids on CDP-cholinetransferasein microsomalpreparations from L5178Ycells Cellswere grown in Fischer'smediumsupplementedwith 10%horseserum.The microsomalfractions from cells grown for 72 hr at 37Â° were incubated in a mixture containing, in a final volume of 2.4 ml, 20 mM HEPES buffer pH 7.5, 34 mM sucrose, 1 mM MgCI2,0.12 j@CiCOP [methyl-'4C]cholinechloride, and the indicated concentrations of steroids. The radioactivity in the lipid fraction was determined after incubation at 37Â°for 30 mm. COP-cholinetransferaseRelative activity of (% of control) at fol concentrationsTrivial lowing steroid M2.8. 1O@ nameSteroid chemical namex 0.10Deoxycorticosterone21-Hydroxy-4-pregnene-3,20-dione22 1.0 0.28

76Progesterone4-Pregnene-3,20-dione48 35 58 785a-Pregnanedione5a-Pregnan-3,20-dione77 64 88 106Pregnenolone3j3-Hydroxy-5-pregnen-20-one26 55 72 56Dexamethasone9a-Fluoro-1 12 40 88Corticosterone1 1/3,17,21 -trihydroxy-1 6co-methyl-1 ,4-pregnadiene-3,20- 56 84 dione47 95Cortexolone17a,21-Oihydroxy-4-pregnene-3,20-dione981f3,21-Dihydroxy-4-pregnene-3,20-dione96 1175f3-Pregnanedione5/3-Pregnan-3,20-dione72 1101 100 104 7a-Pregnenolone3f3,l7cs-Oihydroxy-5-pregnen-20-one93f3-Methasone9a-Fluoro-1 1f3,17,21-trihydroxy-16f3-methyl-1,4-Pregnadiene-3,20- dione95Etiocholanolone3a-Hydroxy-5f3-androsten-1 7-one101Cortisol1 104Contisol 1f3,17a,21-Trihydroxy-4-pregnene-3,20-dione96 7a,21-Trihydroxy-4-pregnene-3,20-dione-21-acetate100Cortisoneacetate1 1 @S,1 7a,21-Dihydroxy-4-pregnene-3,1 1,20-trione 103 Cortisone103Prednisolone11 acetate1 17a,21-Dihydroxy-4-pregnene-3,1 1,20-tnione-21-acetate96 102Prednisolone f3,17a,21-Trihydroxy-1,4-pregnadiene-3,20-dione96 ,4-pregnadiene-3,20-dione-21-acetate97Methylprednisolone1acetate1 1,6,17a,21-Tnihydroxy-1 ,4-pregnadiene-3,20-dione93Tniamcinolone9a-Fluoro-11 @3,17a,21-Tnihydroxy-1 6a-methyl-1 ,4-pregnadiene-3,20-dione97Triamcinolone1/3,16a,17,21-tetrahydroxy-1 acetonide9a-Fluoro-1 1/3,21-dihydroxy-16a,17a-isopropylidenedioxy-1,4-pregna diene-3-20-dione97Aldosterone4-Pregnene-1 97@3-Estradiol1 1/3-21-dihydroxy-3,18,20-trione1 06 7f.@-diol101Testosterone1 ,3,5-Estratriene-3,1 7f3-Hydroxy-4-androsten-3-one93

1546 CANCER RESEARCH VOL.36

Phosphatidylcholine and Corticosteroids

cific activity, 40 Ci/mole) and [methy!-'4C]choline (specific activity, 41.3 Ci/mole) were purchased from New England Nuclear, Boston, Mass.

RESULTS

Inhibition of CDP-Choline Transferase and Availability of Microsomal Lipids. In Chart 1 are shown the results of a comparison between microsomes prepared from cells incu bated in whole calf serum or in delipidized calf serum. In this experiment L5178Y cells were incubated for 24 hr in NO TREATMENTPLCCONTROL PLC Fischer's medium containing 1 of the 2 serum supplements Chart 2. Effect of pretreatment with phospholipase C from Clostridium at the protein level equivalent to 8% whole serum. The welchii on the ability of L5178Y microsomes to incorporate label from CDP microsomes were then prepared from each batch of cells as [methyl-'4Cjcholine into their phospholipid fraction. The microsomes were preincubated for 2 mm either with or without 5 units of phospholipase C, described in â€œMaterialsand Methods.â€•It is evident that the washed, and assayed for COP-choline transferase as described in â€˜â€˜Materials â€œlipid-depletedâ€•preparations incorporated considerably and Methods.â€•Microsomes receiving no treatment were assayed directly without exposure to the phospholipase C procedure. The values are based on less label than the microsomes made from cells grown in 3 samples from each of 3 assays Â±S.D. whole serum. In addition, the degree of inhibition of CDP choline transferase was also affected by incubating cells in delipidized serum. At both levels of DEX, the relative reaction was stopped with ethylene glycol bis(f3-aminoethyl amount of inhibition by DEX was somewhat diminished in ether)-N,N'-tetraacetic acid at a final concentration of 15 microsomes prepared from cells grown in delipidized serum mM. The microsomes were washed and CDP-choline trans as compared to those prepared from cells grown in corn ferase reaction was measured as previously described. The plete serum. In the former, the relative inhibition was 39% at results (Chart 2) showed that, as expected, preincubation 2.8 x 10-s M DEX and 4% at 2.8 x 106 M DEX, while in the with phospholipase C markedly increased the incorporation control (whole serum) the respective values were 49 and of label into the microsomal lipids. Nevertheless, the relative 11%. When the same experiment was repeated using the inhibition by DEX remained unchanged. direct choline exchange incubation system (19), no effect of CDP-Choline Transferase Back Reaction. One possible steroidcouldbe demonstrated. explanation for the inhibitory effect of DEX on CDP-choline Following these results, we attempted to assay CDP-cho transferase might be the stimulation of the back reaction, line tnansferase activity under the conditions of increased i.e., generation of CDP-choline from PL and CMP. It has availability of diglycenide acceptor. The procedure of Mc been studied extensively by BjOnnstad and Bremer (4) and, Murray (14), based on the release of diglycerides from en more recently, by Kanoh and Ohno (9). The L5178Y cells dogenous PL, was used. The microsomal fractions were were labeled during growth with [methy!-'4C]choline, and suspended in 0.27 M sucrose in 5 mM HEPES buffer, pH 7.5. the isolated microsomal fractions were incubated in a reac To this mixture were added 5 mM CaCl2 and 5 units of tion mixture similar to the one used for the forward reaction. phospholipase C from C!ostridium we!chii (Sigma Chemical The steroids used in this case were either progesterone or Co., St. Louis, Mo.). The incubation was 2 mm at 37Â°.The prednisolone. The former, as will be shown below, inhibits COP-choline transferase whereas the latter does not. The results of such experiments demonstrated little, if any, ac 20 WHOLESERUM f DELIPIDIZED tivity of the back reaction regardless of the presence or @ Control iO@ SERUM absence of steroids in the reaction mixture. Modifications of 1O@6@f 0 the procedure by addition of glutathione and potassium @@ 15 fluoride as suggested by Kanoh and Ohno (9) were, simi z larly, without effect. 0 Thymocytes. In order to relate the effects of DEX on CDP

0) choline transferase to the lympholytic effects of specific E @@ N Control @@-o steroids, the inhibition would have to be demonstrated at 0@ low concentration in a system that is known to be very 3 3 sensitive to steroid effects. In terms of growth inhibition, L5178Y lymphoma is relatively resistant to some steroids of this group (21) and therefore is not well suited for such r@ testing. This is in contrast to isolated thymocytes that have â€˜- --â€”.-@- I DEX Mx2.78, DEX Mx 2.78@ been reported to respond to cortisol by showing a de creased glucose uptake at concentrations as low as 10@ M Chart 1. Effects of DEX on the incorporation of CDP-[methyl-'4C]choline into L5178Y microsomes. The microsomes were prepared from cells preincu (17). The results of a typical assay of microsomal thymocyte bated in the presence of either whole or delipidized calf serum in Fischer's fractions are shown in Chart 3. They demonstrate the higher medium for 24 hr. Whole serum medium microsomes, 0.66 mg microsomal sensitivity of the thymocyte system to DEX in comparison to protein per assay; delipidized serum medium microsomes, 0.55 mg protein per assay. The incubation time was 30 mm. The values represent means from the L5178Y system. At the highest concentration of steroid 3 samples from each of 2 assay tubes Â±S.D. used (2.8 x 10-s M), CDP-choline transferase was inhibited

MAY 1976 1547

microsomal fractions prepared from freshly isolated rat thy mocytes. In the lymphoma strain, the inhibition may be b15@ detected at 2.8 x 10-6 M DEX. The microsomes from thymo z cytes exhibit greater sensitivity down to the relatively low LU level of 2.8 x 10@M DEX. The effect in L5178Y microsomes does not appear to be dependent on the availability of endogenous diglyceride acceptor nor does it involve stimu lation of back reaction of the COP-choline transferase. Whether such inhibition is in any way related to the antiin flammatory activity of glucocorticoids is not certain. The requirement for relatively high concentrations of steroid would argue against it. Moreover, there appears to be a lack DEX M x 2.78) of correlation between the steroid structure that is charac tenistic of antiinflammatony steroids and the configuration Chart 3. Effects of DEX on the incorporation of CDP-(methyl-'4Clcholine into microsomal preparations from rat thymocytes. The microsomal prepara associated with the partial inhibition of COP-choline trans tions were made from thymocytes isolated from thymuses of 5 to 10 adrenal ferase. On the basis of a survey of a selected number of ectomized rats. CDP-choline transferase was assayed as described in â€œMate rials and Methodsâ€•.Amount of microsomal protein, 0.53 mg/assay. The representative compounds, steroids that inhibit COP-cho values represent means from 3 samples each from 2 assay tubes Â±S.D. line transferase resemble those steroids that are associated with labilization of lysosomal membranes as determined by by almost 71%, and the inhibition appeared to be significant de Ouve et a!. (6) and by Weissman (25). There are, however, at as low a concentration as 2.78 x 10Â°M. some important differences between the 2 systems point Steroid Specificity and Growth Inhibition. Additional evi ing, in the case of microsomal COP-choline transferase, to a dence for a correlation between lympholytic activity and the greater structural specificity. This is not surprising since the inhibition of CDP-choline transferase would have to depend susceptibility to Iabilizing effects of steroids might vary on the demonstration that C21steroids with 11/3,17a,21- among different membrane preparations. It appears that in trihydroxy structure are active as CDP-choline transferase our system both C-20 and C-21 might be required since inhibitors while other steroids of this series are not. To this testosterone, etiocholanolone, and estradiol were all mac end a screening of steroids was carried out to determine tive although they are known to have labilizing effects on such structure-function relationships. As shown in Table 1, lysosomes. The presence of oxygen function at position 11 there was no correlation between the typical glucocorticoid and, particularly, at position 17 abolishes the inhibitory ef structure and the ability to inhibit COP-choline transferase. fect on COP-choline transferase. Therefore, most glucocorti Although the number of inhibitory steroids tested was small, coids tested, including fluorinated and methylated deniva they did have certain common characteristics that include tives, were inactive. In comparison to other antiinflamma lack of oxygen function in positions 11 and 17 and the tory steroids, OEX, perhaps the most active synthetic deniva presence of C-20 to C-21 side chain. DEX constituted an tive in this group, behaved in an anomalous way towards exception since it has a hydroxy group in both positions 11 COP-choline transferase by inhibiting its activity. It appears and 17 but had considerable inhibitory activity toward COP that the presence of a-methyl group in position 16 counter choline transferase. In contrast to DEX, which has a 16a- acts in some fashion the activity of the OH group in position methyl radical, f3-methasone with a 16f3-methyl configura tion was inactive, in common with other glucocorticoids. 2Growth Table Lack of correlation between lympholytic activity was not steroidsTheinhibition of L5178Ycells by selected as clear when growth inhibition of L5178Y cells was exam x10@ suspensioncultures were started from an inoculum of 5 med under 2 growth conditions (Table 2). When cells were mediumwascell/mI and were incubated at 37Â°for55 hr. Fischer's supplementedwith 8% calf serum or with the equivalent con grown on whole serum 2 glucocorticoids, i.e., DEX and centration of delipidized serum proteins. Final cell density of con cortisol, were considerably more toxic than such active trolwhole-serum-supplementedmedium cultures was6.25 x 10@cells/miin COP-choline transferase inhibitors as deoxycorticosterone numbersrepresentand 7.3 x 10@cells/mI in delipidized serum. The and progesterone. However, when the cells were main meansof 3cultures.Growth tamed on delipidized serum-containing medium and did not (% of control) at following steroid con divide rapidly, the difference in relative growth inhibition by MWhole centrations x 10-Â° DEX and progesterone became negligible. This indicates that no lysis by steroids can take place unless the cells are serumSteroid serum Delipidized dividing rapidly or that the presence of exogenous lipid is 0.28Dexamethasone28 2.8 0.28 28 2.8 necessary for the cytolytic effect by both types of steroids. 42.0Cortisol 3.2 6.0 4.0 28.8 41.0 61.6Cortisone 3.4 6.4 20.8 41.0 52.0 DISCUSSION 78.1Deoxycorticos-17.4 30.7 45.4 41.1 68.5 89.0teroneProgesterone30.0 81.0 These results show that DEX significantly inhibits COP 95.5Estradiol 9.9 25.8 74.5 28.4 54.8 choline transferase activity in microsomal preparations of 100.0Testosterone33.0 98.0 L5178Y lymphoma. The effect is also evident in similar 69.0 87.0 93.0

1548 CANCERRESEARCHVOL.36

17, making DEX active as an inhibitor of COP-choline trans PL biosynthesis could be along the line of the proposed fenase. This assumption is supported by the observation model of Tunnell et a!. (24). They have demonstrated an that f@-methasone, whose only structural difference from increase in free fatty acids in steroid-treated P1798S lym DEX is the configuration of the 16-methyl radical in the f3- phosancoma and have attributed the cytolytic effects of position, was inactive. An inconsistent behavior of DEX, these compounds to the stimulation of lipolysis. Lamberts which is used frequently as a typical representative of an et a!. (11) have demonstrated increased lipolysis in isolated tiinflammatory steroids, has been observed in other sys epididymal cells incubated with both epinephnine and DEX. tems. In the original study of de Duve et a!. (6), DEX showed If such effects exist in the L5178Y lymphoma, the liberated only marginal lysosome-protecting activity in spite of its free fatty acids could affect the activity of COP-choline very high glucocorticoid potency. It has also been reported transfenase since this enzyme appears to be membrane that DEX does not bind to some cortisol-binding proteins (1, bound and is known to be affected by compounds with 23). detergent-like activity (10). Inhibition of COP-choline transferase cannot be associ ated with other specific types of activity since phanmacolog ically different steroids such as deoxycorticosterone, pro ACKNOWLEDGMENTS gesterone, and DEX all inhibited activity of this micnosomal enzyme. We should like to thank Dr. George Rothblat from Wistar Institute, Phila delphia, Pa., for the donation of samples of delipidized sera and for providing The results of this study bring out an important fact that us with the method for serum delipidization. must always be considered when attempting to construct a Technical help of Ida C. Lopez is gratefully acknowledged. biochemical model for a mode of action of closely related chemical compounds. This includes, in this case, C21ste roids of the glucocorticoid series. During recent years, most REFERENCES of the work concerned with these compounds has revolved 1. Beato, M., and Feigelson, P. Glucocorticoid-binding Proteins of Rat Liver around the receptor model with little attention focused on Cytosol. I. Separation and Identification of the Binding Proteins. J. Biol. possible â€˜â€˜direct'â€˜effectson various biochemical processes Chem., 247: 7890-7896, 1972. that do not involve transcriptional or translational controls. 2. Bell, P. A., and Munck, A. Steroid-binding Properties and Stabilization of Cytoplasmic Glucocorticoid Receptors from Rat Thymus Cells. Biochem. Such mOdulation of metabolic functions may, in terms of J., 136: 97-107, 1973. steroid structure, correspond to a specific pharmacological 3. Berliner, 0. L. Studies of the Mechanisms by Which Cells Become Resist on physiological effect such as antiinflammatony or thymoly ant to Corticosteroids. Cancer Res., 25: 1085-1095, 1965. 4. BjOrnstad, P., and Bremer, J. In Vitro Studies on Pathways for the Biosyn tic activity. This seems to be the case with the elevation of thesis of Lecithin in the Rat. J. Lipid Res., 7: 38-45, 1966. alkaline phosphatase in HeLa cells by 11f3,17a,21-tnihydroxy 5. Cristofalo, V. J. Cellular Senescence: Factors Modulating Cell Prolifera tion in Vitro. Monographie Inst. Nat. Sante Rech. Med., 27: 65-92, 1973. steroids of the glucocorticoid series (15). On the other hand, 6. de Ouve, C., Wattiaux, R., and Wibo, M. Effects of Fat-soluble Com steroids involved in an in vitro effect might not reflect any pounds on Lysomes in Vitro. Biochem. Pharmacol., 9: 97-116, 1962. particular type of biochemical activity in vivo. Perhaps the 7. English, J., Chakraborty, J., and Marks, V. The Metabolism of Dexametha sone in the Ratâ€”Effectof Phenytoin. J. Steroid Biochem., 6: 65-68, 1975. most clean-cut case is that of the stimulation of DNA synthe 8. Folch, J., Lees, M., and Stanley, G. H. S. A Simple Method for the Isolation sis in Wl-38 cells (5). Although the original observation was and Purification of Total Lipids from Animal Tissue. J. Biol. Chem., 226: made with cortisol, this property is shared with aldosterone, 497-508, 1957. 9. Kanoh, H., and Ohno, K. Studies on 1,2-Oiglycerides Formed from Endog corticosterone, and 11/3-hydroxyprogesterone. In contrast, enous Lecithins by the Back-Reaction of Rat Liver Microsomal COP prednisolone is inactive in this system. Evidently, the results choline: 1,2-Diacylglycerol Choline Phosphotransferase. Biochim. Bio of our experiments on the inhibition of CDP-choline trans phys. Acta, 326: 17-25, 1973. 10. Kennedy, E. P. Metabolism of Lipids. Ann. Rev. Biochem., 26: 119-148, fenase represents another example of such â€œnonspecificâ€• 1957. activity. 11. Lamberts, S. w. J., Timmermans, H. A. T., Kramer-Blankenstijn, M., and Birkenhager, J. C. The Mechanism of the Potentiating Effect of Glucocor The inconsistency between the previously reported inhi ticoids on Catecholamine-induced Lipolysis. Metab. Clin. Exptl., 24: 681- bition of PL biosynthesis in L5178Y cells exposed to gluco 689, 1975. corticoids (21) and lack of such specific effect in the micro 12. Lippman, M. E., Perry, S., and Thompson, E. B. Cytoplasmic Glucocorti coid-binding Proteins in Glucocorticoid-unresponsive Human and somal preparations remains to be elucidated. It appears Mouse Leukemic Cell Lines. Cancer Res., 34: 1572-1576, 1974. unlikely that L5178Y cells are capable of converting these 13. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem., 193: 265- steroids into inhibitory derivatives. It is known that steroid 275, 1951. catabolism in vitro results in the formation of highly polar 14. McMurray, W. C. Lecithin Biosynthesis in Liver Mitochondrial Fractions. metabolites (3) that would be even less likely to affect syn Biochem. Biophys. Res. Commun., 58: 467-474, 1974. 15. Melnykovych, G. Effects of Corticosteroids on the Formation of Alkaline thesis of PL by the micnosomal COP-choline transfenase Phosphatase in HeLa Cells. Biochem. Biophys. Res. Commun., 8: 81-86, than active glucocorticoids. Nevertheless, this point must 1962. be investigated particularly in the case of DEX whose catab 16. Melnykovych, G., and Standaert, M. M. Choline Incorporation into Phos pholipids by Microsomal Fractions from L5178Y Lymphoma. Arch. Bio olism has not been evaluated in detail in a cell culture chem. Biophys., 164: 674-681, 1974. system. Recent studies in rats by English et a!. (7) have 17. Munck, A. Steroid Concentration and Tissue Integrity as Factors Deter mining the Physiological Significance of Effects of Adrenal Steroids in shown that only slightly over 50% of administered steroid Vitro. Endocrinology, 77: 356-360, 1965. can be accounted for in feces and urine of experimental 18. Sibley, C. H., and Tomkins, G. M. Mechanisms of Steroid Resistance. Cell, animals. The fate of the remaining DEX and the identity of 2: 221-227, 1974. 19. Soto, E. F., Pasquini, I. M., and Krawiec, L. In Vitro Incorporation of its putative metabolites remains unknown. [3H]Methylcholine into Phosphatidylcholine of Rat Liver Subcellular The alternate explanation of the effects of corticoids on Fractions. Arch. Biochem. Biophys., 150: 362-370, 1972.

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20. Standaert, M. M., and Melnykovych, G. Incorporation of Choline into phoma Cells, L5178Y, in Vitro. Cancer Res., 33: 2872-2877, 1973. Phospholipids by the Microsomal Fraction from L5178Y Cells; Effects of 23. Suyemitsu, T., and Terayma, H. Specific Binding Sites for Natural Gluco Calcium and Prednisolone (Abstract). In Vitro, 8: 424, 1973. corticoids in Plasma Membranes of Rat Liver. Endocrinology, 96: 1499- 21. Story, M. T., and Melnykovych, G. Growth Inhibition of Mouse Lymphoma 1508, 1975. Cells, L5178Y, in Vitro, by Glucocorticoids; Alterations in Choline Metab 24. Turnell, R. W., Clarke, L. H., and Burton, A. F. Studies on the Mechanism olism. Exptl. Cell Res., 77: 437-449, 1973. ofCorticoid-induced Lymphocytolysis. Cancer Res.,33: 203-212, 1973. 22. Story, M. T., Standaert, M. M., and Melnykovych, G. Glucocorticoid 25. Weissman, G. Labilization and Stabilization of Lysosomes. Federation induced Alterations in Phosphatidylcholine Metabolism in Mouse Lym Proc.,23:1038-1044,1964.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1976 American Association for Cancer Research. Corticosteroids and Phosphatidylcholine Biosynthesis in Microsomal Fractions from L5178Y Lymphoma

George Melnykovych, Mary M. Standaert, Edna R. Matthews, et al.

Cancer Res 1976;36:1545-1550.

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