Tumor Promoter Stimulation of Phosphatidylcholine Turnover in Hela Cells1

[CANCER RESEARCH 42, 1980-1985, May 1982] 0008-5472/82/0042-OOOOS02.00 Tumor Promoter Stimulation of Phosphatidylcholine Turnover in HeLa Cells1

Graeme R. Guy and Andrew W. Murray2

School of Biological Sciences, Flinders University. Bedford Park, South Australia 5042

ABSTRACT from an mtracellular pool. An alternative explanation is that the phosphorylated derivative was a product of a phospholipase C Incubation of HeLa cells for 24 hr with [3H]choline resulted enzyme utilizing labeled phosphatidylcholine as substrate. in extensive labeling of the phosphorylcholine and phospha- Some evidence for this was obtained by Weinstein et al. (26), tidylcholine pools. 12-O-Tetradecanoylphorbol-13-acetate who reported that TPA stimulated the release of [3H]choline (TPA), other phorbol ester tumor promoters, and mezerein and [3H]phosphorylcholine from C3H10T1/2 cells in which stimulated the release of [3H]choline and [3H]phosphorylcholine the phospholipid pool had been prelabeled by incubation with from such prelabeled cells. The release was accompanied by [3H]choline for 24 hr. In the present paper, we have character decreased radioactivity in the phosphorylcholine pool, raising ized the promoter-stimulated release of choline metabolites the possibility that the released materials were derived by from prelabeled HeLa cells. Evidence is presented that the leakage from this pool. However, TPA did not induce the stimulation results from the activation of a phospholipase C- release of radioactivity from cells containing a prelabeled nu- type enzyme. cleotide pool. Similarly, the TPA-stimulated release of radio activity from prelabeled cells closely paralleled the label pres ent in the phospholipid pool rather than the phosphocholine MATERIALS AND METHODS pool. Consequently, it is suggested that the primary source of Materials. TPA, phorbol-12,13-didecanoate. 4o-phorboldideca- the released material is phosphatidylcholine acting as a sub noate, and 4-O-methyl TPA were obtained from P-L Biochemicals, strate for a phospholipase C enzyme. TPA also stimulated the incorporation of [3H]choline into phospholipids, but a time- Milwaukee, Wis. Phorbol 12,13-dibutyrate, pnorbol-l2,13-dibenzoate, phorbol-l2,13-diacetate, mezerein, and phorbol were from Life Sys course study indicated that phospholipase C activation pre tems, Newton, Mass. Resiniferotoxin was a generous gift from Dr. F. ceded this event. This was supported by the observation that incorporation of [3H]choline was also stimulated by exoge- Evans, Department of Pharmacognosy, University of London. Phosphorylcholine, phosphatidylcholine, lysophosphatidylcholine, nously added phospholipase C. sphingomyelin, choline kinase, alkaline phosphatase (type HIS), phos pholipase C (Clostridium perfringens and Bacillus cereus), phospholi INTRODUCTION pase A2 (Crotaius adamanteus), and phospholipase D (cabbage) were all obtained from Sigma Chemical Co., St. Louis, Mo. Current evidence obtained with cultured mammalian cells TFP and the calcium ionophore A23187 were gifts from Smith Kline suggests that the cell surface is the initial site of action of the & French Laboratories, Australia, and Eli Lilly and Co., Indianapolis, phorbol diester tumor promoters. Specific, high-affinity binding Ind., respectively. MEM and fetal calf serum were obtained from the Commonwealth Serum Laboratories, Melbourne, Australia. Supple sites have been characterized on many cells (3, 5, 7, 16, 21, mented MEM contained a 50% increase in essential amino acids, 23), and several of the early effects of promoters involve glutamine, and vitamins and a 100% increase in nonessential amino modulation of membrane functions (2, 4, 25). One such effect acids and 1 HIM sodium pyruvate. is a promoter-induced enhancement of phospholipid synthesis [mef/jy/-3H]Thymidine (specific activity, 24 Ci/mmol), [8-'4C]hypo- and deacylation. Thus, TPA3 has been shown to stimulate the xanthine (specific activity, 0.44 Ci/mmol), [mef/iy/-3H]choline chloride incorporation of [3H]choline into the phospholipids of lympho (specific activity, 77 Ci/mmol), and [mefhy/-14C]choline chloride (spe cytes (28, 29) and HeLa cells (10, 18). In both cases, the cific activity, 58 mCi/mmol) were obtained from the Radiochemical majority of the label was incorporated into phosphatidylcholine. Centre, Amersham, United Kingdom. HeLa cells were maintained on 35-mm plastic dishes (Lux) in MEM In HeLa cells at least, this involves the sequential action of supplemented with 10% fetal calf serum and antibiotics (penicillin, 100 choline kinase, CTP:phosphorylcholine cytidylyltransferase, Ill/ml; streptomycin, 100 /ig/ml; complete medium). Incubations were and CDP-choline:diacylglycerol choline phosphotransferase. carried out at 37Â° in a humidified atmosphere of 5% CO? in air, and Paddon and Vance (18) concluded that the TPA enhancement cultures were used at cell densities of 1.5 to 2 x 10e cells/dish. of choline incorporation in HeLa cells resulted from activation Methods. The measurement of radioactivity release from [3H]cho- of the cytidylyltransferase enzyme. These authors also re line-prelabeled cells was carried out as follows. HeLa cells were ported that TPA stimulated the release of phosphorylcholine incubated for 20 to 24 hr in 2 ml supplemented MEM containing 10% into the medium, which they implied arose by direct extrusion fetal calf serum and 6 to 8 /iCi [metfiy/-3H]choline. The medium was removed, and the cells were washed with prewarmed PBS (4x2 ml). 1 This study was supported by the Australian Research Grants Committee, the Complete medium (2 ml) containing the test substance was added, and the dishes were incubated for an appropriate time at 37Â°. The dishes National Health and Medical Research Council, and the Universities of South Australia Anti-Cancer Foundation. were placed on ice, and the medium was collected and centrifuged (20 2 To whom requests for reprints should be addressed. min, 4Â°, 200 x g). An aliquot (1 ml) was removed, and radioactivity 3 The abbreviations used are: TPA. 12-O-tetradecanoylphorbol-13-acetate; TFP, trifluoperazine; MEM, Eagle's minimal essential medium; PBS, Oulbecco's was measured using a Triton:xylene scintillation fluid (1). The measurement of [3H]choline incorporation into HeLa cell phos phosphate-buffered saline; DMSO, dimethyl sulfoxide. Received August 17. 1981; accepted February 8, 1982. pholipids was carried out as described before (6).

1980 CANCER RESEARCH VOL. 42

Analysis of the distribution of radioactivity in various choline metab ated with lysophosphatidylcholine (<5%) and sphinogomyelin olites released into the medium and in cellular metabolites derived from Â«3%). the acid-soluble and -insoluble fractions of the cells utilized the Chro As shown in Chart 1, the addition of 10"7 M TPA to such matographie procedures described by Wertz and Mueller (28). Choline, prelabeled cells resulted in a rapid and sustained stimulation phosphorylcholine, and CDP-choline were measured as described (24), of radioactivity released into the medium. The nature of the and phosphatidylcholine was estimated by the method of Raheja ef al. released material over a 2-hr period was analyzed by thin-layer (19). chromatography as described in "Materials and Methods." In For the detection of metabolite "leakage," HeLa cells were incu bated for 3 hr in complete medium containing 0.1 fiCi [14C]hypoxan- control cultures, 94% of the released radioactivity was asso thine. The cells were washed (5x2 ml) with PBS at 37Â°and further ciated with choline, while in the presence of 10~7 M TPA, 41 % incubated for 2 hr in complete medium in the presence or absence of was associated with phosphorylcholine and the remainder, with 10~7 M TPA. An aliquot of the medium was taken for scintillation choline (data not shown). Chart 2 shows a dose response for counting, and radioactivity associated with the acid-soluble pool of the the TPA-stimulated release, and Chart 3 summarizes the effects cells was measured as described (14). To determine the extent of cell of a range of promoting and nonpromoting compounds. There loss during incubation, cells were prelabeled by incubation with was a good correlation between the potency of a compound as [mefriy/-3H]thymidine (1 fiCi/ml) in complete medium for 24 hr. The cells were washed (5x2 ml) with PBS at 37Â°and further incubated a tumor promoter (see Ref. 10) and its ability to release choline for 2 hr in complete medium in the presence or absence of 10~7 M metabolites from prelabeled HeLa cells. TPA. An aliquot of the medium was taken for radioactivity measure An analysis of radioactivity associated with the medium and ment. The cells were washed with ice-cold PBS and solubilized in 1 ml with the acid-soluble and phospholipid pools after a 2-hr ex of 1% sodium dodecyl sulfate, and radioactivity was determined by posure to DMSO or TPA is shown in Table 1. TPA induced a scintillation counting. The counting efficiency for the system used was decrease in radioactivity associated with acid-soluble materials 43.6%. (mainly phosphorylcholine) and increased release into the me TPA and other promoters were added as solutions in DMSO; control dium. Clearly, the released material could have arisen by direct incubations contained DMSO alone (0.1%). leakage from the intracellular pool. Alternatively, the material could have arisen by activation of phospholipase C- and D-type RESULTS enzyme(s) followed by enhanced phosphatidylcholine synthe sis leading to a secondary depletion of the internal [3H]phos- Preliminary experiments established that incubation of HeLa phorylcholine pool. The following experiments were designed cells with [3H]choline for 24 hr resulted in extensive labeling of to test these possibilities. the phospholipid pool. Typically, between about 57 and 65 x To determine whether TPA made the cell membrane leaky to 103 dpm/106 cells were associated with phospholipids, and phosphorylated derivatives, its effect on the release of nucleo- between about 50 and 55 x 103 dpm/106 cells were associ tides into the medium was examined. HeLa cells were prela ated with phosphorylcholine; no [3H]choline was detectable. beled with [14C]hypoxanthine for 3 hr (see "Materials and The majority of the phospholipid radioactivity was associated with phosphatidylcholine (>91%) with small amounts associ- o x (0

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44 O (A t * I CONTROL

30 60 (O 120 o 10 10 10 10 io "x"1 Time (min ) TPA CONCI (M) Chart 1. Effect of TPA on the release ot radioactivity from [3HJcholine-prela- Chart 2. Dose-response curve for the TPA-stimulated release of radioactivity beled HeLa cells. Cells were prelabeled as described in "Materials and Methods" from [3H]choline-prelabeied HeLa cells. Cells were incubated for 2 hr at each and incubated in the presence of DMSO (A) or 10 ' M TPA (A). The results are concentration (CONCA/.) of TPA. Points, mean of 5 replicate determinations; given as radioactivity released per 10e cells. bars, S.E.

MAY 1982 1981

Methods"), and the effect of 10 7 M TRA on the release of rylcholine, CDP-choline, and phosphatidylcholine, after incu radioactivity over a 2-hr period was measured (Table 2). The bation in the presence and absence of TPA, is shown in Table promoter had no effect on radioactivity release, indicating that 3. The pool sizes of all choline metabolites were unaffected by the cells had not become generally leaky. In addition, experi TPA. In the case of phosphatidylcholine, the pool size remained ments with [3H]thymidine-prelabeled cells indicated that TPA constant for up to 6 hr of incubation in the presence of TPA. did not induce an increase in cell loss over a 2-hr incubation During this period of incubation, TPA resulted in the extensive period (Table 2). release of radioactivity. Consequently, if the released material An analysis of the absolute pool sizes of choline, phospho- arises by the action of phospholipases C and D, the cells must have an efficient mechanism for maintaining a constant con centration of this phospholipid. In relation to this point, chick embryo muscle cells have been shown to maintain a constant 400 phosphatidylcholine pool during incubation with exogenously added phospholipase C (9, 22). In a further series of experiments, [3H]choline-prelabeled cells were incubated for 1, 12, or 24 hr in unlabeled complete 300 JL JL medium. At these time points, the release of radioactivity over a 2-hr period and the radioactivity present in the acid-soluble and phospholipid pools were determined in the presence of 200 TPA or of exogenously added C. perfringens phospholipase C (Table 4). It can be seen that, over the 24-hr incubation period, the radioactivity associated with the acid-soluble pool de creased by 46%, implying a similar decrease in phosphocho- H 100 JL line specific activity, while that associated with phospholipid decreased by only 9%. The TPA-stimulated release of radio activity remained essentially constant. This result is consistent with the phospholipid pool representing the primary source of the released material. If the TPA stimulation of release was a consequence of phosphocholine leakage, approximately twice the absolute amount must have been released at 24 hr com Chart 3. Effect of a range of phorbol diesters, mezerein, and resiniferotoxin on the release of radioactivity from [3H)choline-prelabeled HeLa cells. Prelabeled pared with 1 hr. A similar result was observed when the cells cells were incubated for 2 hr with a 10~7 M concentration of each compound. were incubated with phospholipase C, where it can be assumed POD. phorbol-12.13-didecanoate; MEZ, mezerein; PDBu, phorbol 12,13-dibu- that the majority of the released radioactivity arises from the tyrate; POBe, phorbol-12,13-dibenzoate; PDAc. phorbol-12,13-diacetate; RES, direct hydrolysis of phosphatidylcholine. resiniferotoxin; 4-O-Me TPA, 4-O-methyl TPA; 4a POD, 4a-phorboldidecanoate. In a further experiment, cells were prelabeled with [14C]- Points, mean of 5 replicate determinations; bars. S.E. choline for 24 hr and then pulsed with [3H]choline for 8 min. This labeling procedure resulted in incorporation of tritium into Table 1 the acid-soluble pool, which had an initial ratio of MC to 3H Effect of TPA on the distribution of radioactivity between the medium and the cellular acid-soluble and phospholipid pools radioactivity of about 1.2 (Table 5). However, little tritium was HeLa cells prelabeled with [3H]choline were incubated for 2 hr with DMSO present in the phospholipid pool, which had an initial 14C:3H (control) or with 10 " M TPA. Radioactivity associated with the medium and the 2 intracellular pools was determined as described in "Materials and Methods." ratio of 11.5 (Table 5). The radioactivity released into the medium in the presence of TPA after short incubation times Radioactivity/10e cells (dpm) had a high ratio of 14C to 3H, clearly establishing that the

Treatment Medium Acid soluble Phospholipid phospholipid pool was the primary source of the released Control material. As expected, the ratio decreased with increased time TPAOICT7M)4.166 1 5.676Â± Â±147"14257,11848,862Â± Â±9591.99052,952 53,669Â± Â±1,478818 of incubation as more of the tritium in the acid-soluble pool was '' Mean Â± S.E. of 5 separate determinations. incorporated into phosphatidylcholine. These data are not con-

Table 2 Effect of TPA on the release of radioactivity from HeLa cells prelabeled with Â¡''Cihypoxanthine or pHJthymidine HeLa cells were prelabeled as described in "Materials and Methods," washed, and then incubated for a further 2 hr in the presence of DMSO (control) or 10"' M TPA. Radioactivity released into the medium was measured by liquid scintillation counting. cells)Treatmentr'C|-Hypoxanthme Radioactivity (cpm 10'

soluble24,716 cell associated prelabeled Â±208a DMSO Â± 2,861 TPAMedium2,802 2.614 Â±223Acid 23,818 Â± 2,593Total

("C]Thymidine prelabeled DMSO 595 Â± 67 185,087 Â±31,558 TPA 615 Â± 43 193,113 Â±18.437 " Mean Â±S.E. of 5 replicate determinations.

1982 CANCER RESEARCH VOL. 42

Table 3 Effect of TPA on the pool sizes of choline and choline metabolites in HeLa cells Determinations were carried out after appropriate incubation times as de 9 scribed in "Materials and Methods." n I Incuba 2 8 tion X 10" time DMSO(nmol/107 M TPA (nmol/ IO7 cells) "> 7 Compound

Table 4 Effect of TPA and phospholipase C on the release of radioactivity from prelabeled cells Cells were prelabeled with [ lH|cholin<> as described in "Materials and Meth ods," washed, and incubated in unlabeled complete medium for a further 1,12, or 24 hr. At each of these times, dishes were incubated for a further 2 hr in the presence of DMSO (control), TPA <10~7 M), or C. perfringens phospholipase C 10 15 20 25 30 (0.05 unit/ml), and the distribution of radioactivity was determined. Time (Min) Radioactivity (10~3 x dpm/10e cells) following incu bation time in unlabeled medium B 1 hr 12 hr 24 hr S 28 7 "o DMSOMediumAcid ~ 24 a solublePhospholipid1 I Â£ 20 0-7 MTPAMediumAcid I 16 solublePhospholipidPhospholipase Â«5 | o 0) o 5 CMediumAcid S 8

solublePhospholipid6.2"64.350.815.748.953.724.143.550.54.946.654.615.635.455.825.336.547.13.034.546.112.624.843.122.421.732.9 I o 8 Mean of 3 replicate determinations. JJ I IO 25 30 ^ Table 5 Effect of TPA on the release of choline and phosphocholine from HeLa cells Time (Mh) prelabeled with r'CJcholine and fHJcholine Chart 4. Time course for the effect of TPA on the release of radioactivity from HeLa cells were prelabeled with 114C|choline ( 11 /iCi/ml) for 24 hr as described [3H]choline-prelabeled HeLa cells and on the incorporation of [3H)choline into the in "Materials and Methods." The cells were further incubated with [3H]choline acid-soluble and phospholipid pools of HeLa cells. A. radioactivity release in the presence of DMSO (A) or 10~7 M TPA (O); B, [3HJcholine incorporation into the (20 Ci/ml) for 8 min, washed with PBS, and incubated in complete medium containing DMSO or 10"' M TPA. At various times, the ratio of "C to 3H acid-soluble (â€¢.A) and phospholipid (O, A) fractions of HeLa cells in the presence and absence of 10"' M TPA, respectively. radioactivity released into the medium and in the acid-soluble and phospholipid fractions was measured as described in "Materials and Methods." '4C: 'H radioactivity ratio

Medium Acid soluble Phospholipid pase C, either directly or by an alteration in substrate orienta tion or availability. An alternative is that TPA stimulates the Time (min) TPA DMSO TPA DMSO TPA DMSO cytidylyltransferase enzyme and that the release is a secondary Zerotime2.557.5153060909.67.38.06.75.75.24.72.21.82.51.62.12.01.82a.2.2.2.2.2.3.31.21.21.21.21.21.21.21.211.511.510.910.79.58.56.75.211.511.511.511.510.510.28.98.3consequence of phospholipase C acting to maintain a constant phospholipid pool size. In an attempt to resolve this question, a detailed time course was determined for the TPA stimulation of both the release of radioactivity from prelabeled cells and the incorporation of [3H]choline into phospholipids (Chart 4). Stimulation of release 8 Mean of 3 replicate determinations. was detectable after 5 min of incubation with no significant lag period. However, TPA stimulation of [3H]choline incorporation sistent with phosphocholine leakage being the source of the into phospholipid occurred only after a lag of more than 10 TPA-stimulated release. min. Incorporation of the label into the acid-soluble pool was The above experiments strongly suggest that the released rapid in both the presence and absence of TPA (Chart 4) and radioactivity is derived from the action of phospholipase C on is therefore unlikely to be rate limiting. These data provide labeled Phosphatidylcholine. However, it has not been estab direct evidence that TPA stimulation of radioactivity release lished that the primary effect of TPA is to activate phospholi- precedes the enhancement of choline incorporation.

MAY 1982 1983

Table 6 stimulated by TPA closely parallels the label present in the Effect of various treatments on the release of radioactivity from prelabeled phospholipid pool rather than that in the phosphorylcholine HeLa cells and on the uptake of pHjcholine into HeLa cell phospholipids pool (Tables 4 and 5). The presence of [3H]choline as well as Experiments were carried out as described in "Materials and Methods." and [3H]phosphorylcholine in the released material raises the pos all incubations were for 2 hr. incor sibility of a phospholipase D-type activity as well as phospho re porated into phos- lipase C. However, this is not believed likely, as preliminary leased (dpm/106 pholipid (dpm/106 experiments indicated that a proportion of exogenously added TreatmentExperiment cells)2,187 cells)2.015 [3H]phosphorylcholine was degraded to [3H]choline by HeLa 1DMSOTPAOCT'M)lonophore cell cultures.4 Other work has shown that HeLa cell plasma Â±6,839 Â±10,294 Â±1 Â±22,337 membranes contain an alkaline phosphatase activity (17). We /iM)TPA A23187 (2 1,967Â±23,102 Â±45.537 consequently propose that the majority of the released radio +ionophoreTFP Â±2,253 Â±1.278 (25UM)TPA Â±2,357 Â±1 activity arises as a result of phospholipase C activity. +TFPlonophore Â±2,308 .945Â±1 Although promoters cause both an enhanced release of TFPExperiment+ Â±5,083 ,039Â±4,905 radioactivity from prelabeled cells and an enhanced incorpo ration of [3H]choline incorporation into phospholipid, the time 2DMSOTPAdCT'M)Phospholipase Â±15,333 Â±1 course of these changes suggests that release precedes in Â±4,953 7,485Â±4,685 creased phospholipid biosynthesis. This conclusion is sup Â¡ig!ml)PhospholipaseA,, (SO Â±5.045 Â±4.795 ported by the observation that [3H]choline incorporation was /ig/ml)PhospholipaseD (50 Â±21,218 Â±29.383 stimulated by incubation of HeLa cells with C. perfringens (C.perfringens.C Â±15,189 Â±5,125 0.05units/ml)Phospholipase phospholipase C. A similar observation has been made with cultured embryonic chick muscle cells (22). In this case, phos Â±1874235071,324896745873879313748995*cereus.0.05C (B. Â±107a23538752783103133109432125232.034128[3H]Cholinepholipase C treatment caused a redistribution of the rate-limit units/mlRadioactivity Mean Â±S.E. of 5 replicate determinations. ing enzyme CTP:phosphocholine cytidylyltransferase from the cytosolic fraction to the membrane fraction, a process accom panied by activation of the enzyme. In additional experiments, the effect of the calcium ionophore There is ample evidence that modulation of membrane lipids A23187 and of various exogenously added phospholipase can affect both the initial ligand-receptor interaction and the enzymes on both release and incorporation was examined subsequent molecular events triggered by this interaction (12). (Table 6). Both phenomena were enhanced by the ionophore In particular, exogenously added phospholipase C has been and by TPA, and the stimulations were prevented by the shown to modify the binding of several hormones to their phenothiazine antipsychotic agent TFP. This compound is a cellular receptors (12) and also to decrease the apparent potent inhibitor of reactions involving the calcium-binding pro number of [3H]phorbol 12,13-dibutyrate-binding sites on epi tein Calmodulin (17) and has also been reported to interfere dermal cells.5 The close association between receptors and with phospholipid-protein interactions (13, 20). Interpretation phospholipids suggests that interaction of many ligands with of the inhibitory effect of TFP on the TPA responses is compli their receptors could cause a conformation change in both the cated by the fact that TFP has been shown to inhibit the binding receptor and the specific annulus of surrounding phospholip of promoters to their surface receptors (15). Release and ids. Such phospholipid perturbation may then, for example, incorporation were also both stimulated by C. perfringens provide substrate in the correct configuration for attack by phospholipase C but not by the enzyme from B. cereus or by phospholipase C, which would lead to further local alteration in phospholipases A2 and D (Table 6). The data in Table 6 also the phospholipid pools. Subsequent membrane changes could support the suggestion that TPA induces turnover of phospha- be triggered by these alterations or by some product of phos tidylcholine. It is apparent that, after 2 hr of incubation, there pholipase activity. For example, the immediate product, 1,2- was an extensive TPA stimulation of [3H]choline incorporation diacylglycerol, has been shown to activate a Ca2+-dependent, into phosphatidylcholine as reported by others (10, 18, 28, phospholipid-activated protein kinase from human platelets (8) 29). However, after this time, TPA did not increase the amount and rat brain (11). of radioactivity associated with phosphatidylcholine in cells that had been prelabeled with [3H]choline and had a labeled A model in which an initial activation of phospholipase C by tumor promoters precedes subsequent membrane changes is phosphocholine pool (Tables 1 and 4). This strongly implies supported by the observation that several promoter-induced that TPA stimulates both the synthesis and breakdown of events can also be induced by incubating cells with exogenous phosphatidylcholine. phospholipase C. Examples include [3H]choline incorporation into phospholipids (Table 6) (9, 22), activation of arachidonic DISCUSSION acid release, and stimulation of ornithine decarboxylase activ ity.4 The present results confirm that perturbation of membrane phospholipid metabolism is an early consequence of the inter REFERENCES action of promoters with HeLa cells. Thus, promoters stimu lated the release of choline and phosphorylcholine from cells 1. Anderson. L. E., and McClure, W. O. An improved scintillation cocktail of prelabeled with [3H]choline. The cells did not become generally high solubilizing power. Anal. Biochem., 51: 173-179. 1973. 2. Blumberg, P.M. In vitro studies on the mode of action of the phorbol esters, leaky in the presence of promoters, suggesting that phospha potent tumor promoters: part 1. CRC Crit. Rev. Toxicol.. 8:153-197, 1980. tidylcholine acting as a substrate for a phospholipase enzyme 3. Delclos, K. B.. Nagle. D. S., and Blumberg, P. M. Specific binding of phorbol was the primary source of the released materials. This is ' G. R. Guy and A. W. Murray, unpublished data. strongly supported by the finding that radioactivity release 5 M. J. Jones and A. W. Murray, manuscript in preparation.

1984 CANCER RESEARCH VOL. 42

ester tumor promoters to mouse skin. Cell, 79: 1025-1032. 1980. in HeLa cells. Biochim. Biophys. Acta. 488: 181-189, 1977. 4. Diamond. L., O'Brien. T. Q., and Baird. W. M. Tumor promoters and the 18. Paddon, H. B., and Vance, D. E. Tetradecanoyl-phorbol acetate stimulates mechanism of tumor promotion. Adv. Cancer Res.. 32: 1-74. 1980. phosphatidylcholine biosynthesis in HeLa cells by an increase in the rate of 5. Driedger, P. E., and Blumberg, P. M. Specific binding of phorbol ester tumor the reaction catalysed by CTP: phosphocholine cytidylyltransferase. promoters. Proc. Nati. Acad. Sei. U. S. A., 77: 567-571, 1980. Biochim. Biophys. Acta. 620: 636-640. 1980. 6. Froscio, M.. Guy. G. R., and Murray, A. W. Calmodulin inhibitors modify cell 19. Raheja, R. K., Jaur. C., Singh, A., and Bhatia. I. S. New colorimetrie method surface changes triggered by a tumor promoter. Biochem. Biophys. Res. for the quantitative estimation of phospholipids without acid digestion. J. 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MAY 1982 1985

Graeme R. Guy and Andrew W. Murray

Cancer Res 1982;42:1980-1985.

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