Molecular and Cellular Biochemistry56, 137 144 (1983). © 1983, Martinus Nijhoff Publishers, Boston. Printed in The Netherlands.

The phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate and stimulation of 3H-choline incorporation into endoplasmic reticulum membranes and other subcellular fractions of Krebs II ascites cells during in vitro incubation R. Lillehaug Department of Biochemistry, Preclinical Institute, University of Bergen, ~frstadveien 19, N-5000 Bergen, Norway

Anders Fjose, Ian F. Pryme* and J o h a n

Summary After transfer of Krebs II ascites cells from the mouse peritoneum to suspension culture addition of the phorbol ester 12-0-tetradecanoyl-phorbol-I 3-acetate (TPA) causes an early stimulation of 3H-choline incorporation into phosphatidylcholine (PC). Choline transport into the treated cells, however, was unaffected, Within 30 min of T P A treatment 3H-choline incorporation was almost 300% above the control level. During a 5 hr period of suspension culture the overall patterns of 3H-choline incorporation were similar in TPA-treated and control cultures though the rate was greatly accentuated by the presence of the phorbol ester. Incubation of cells with cycloheximide prior to incubation with T P A did not result in an inhibition of the TPA-directed 3H-choline incorporation. After 3 hr incubation with T P A there were large increases in radioactivity in all subcellular fractions. At 20 hr, however, the values were not far from those of the control. During the first 3 hr of incubation with T P A the incorporation of 3H-choline into light rough (LR) and smooth (S) membranes was stimulated to levels of 400% and 320% respectively above control values. At later times the profiles of radioactivity in membrane subfractions in TPA-treated and control cultures were similar, The results illustrate an early effect of T P A on PC biosynthesis in Krebs II ascites cells while at later times of incubation the stimulatory effect was virtually abolished.

Introduction The endoplasmic reticulum is quite different from other organelles in that it has a potential to undergo dynamic alteration, for example, certain drugs elicit dramatic changes in both total membrane mass and molecular composition (1,2). Studies on the dynamic changes which probably occur in the endoplasmic reticulum (ER) during the process of cellular transformation may provide further insight into the mechanism of carcinogenesis. Treatment of normal cells with tumor-promoting phorbol esters alone results in many cases in the induction of several characteristics similar to those found in transformed cells. If transformed cells are

* To whom correspondence should be addressed.

exposed to phorbol esters then the expression of these features is further accentuated (3-5). Subfractionation studies performed on the ER membranes isolated from several transformed cell lines have shown the presence of three ER membrane subfractions in such cells - heavy rough (HR), light rough (LR) and smooth (S) membranes (6-10). Cells from normal tissues such as liver, pancreas, kidney, spleen, heart, lung and bone marrow were found to contain only LR and S membranes (8, 9). H R membranes, however, were identified in a non-tumorigenic cell line (C3H/10Tl/2 mouse fibroblasts) upon treatment with the tumor promoter T P A (9). The H R and LR subfractions have been best characterized in MPC-11 cells where the following differences have been reported: a) R N A / protein and RNA/phospholipid ratios are higher in H R than in LR membranes (7), b) they exhibit

138 different polysome profiles (7), c) the H R polysomes contain four times as many nascent light chain immunoglobulin polypeptides as the LR polysomes (2), d) the H R fraction does not appear in gradients when cells are disrupted by nitrogen cavitation in a buffer containing 100 mM KC1 (6), and e) the respective amounts of H R and LR membranes vary according to phase of the cell cycle (11). These results have been taken to suggest a compartmentation of the ER system (2). In the accompanying paper (10) we have shown that the transfer of Krebs II ascites cells from the mouse peritoneum to in vitro suspension culture results in a time-dependent alteration in the distribution profile of ER membrane subfractions. Although negligible amounts of 3H-choline radioactivity appeared in the H R region of gradients after 1/2 hr of in vitro incubation, large amounts were found at 18 hr. In light of these observations and because of the fact that H R membranes appear to be characteristic for transformed cells, it was of interest to investigate the behaviour of ER membrane subfractions in Krebs II ascites cells upon treatment of cells with the phorbol ester TPA. Materials and methods

Cell line and growth conditions Krebs II ascites cells were grown and cultured as described in the accompanying paper (10).

Treatment of cultures with 12-O-tetradecanoylphorbol-13-acetate (TPA) A stock solution of T P A (l X 10 3 M) was prepared in acetone and stored in the dark at -20 ° C. T P A in acetone was added to culture medium at least 24 hr before use and an equal volume of acetone was added to medium to be used for feeding control cultures. Freshly harvested, washed Krebs II ascites cells were resuspended directly in culture medium containing the appropriate T P A concentration, or an identical volume of acetone.

Labeling of cells with methylJ H-choline chloride, cell disruption by nitrogen cavitation and isolation of endoplasmic reticulum subfractions The labeling of cells, disruption by nitrogen cavitation and ER membrane subfractionation was per-

formed as indicated in the Materials and methods section of the accompanying paper (10). All experiments were performed at least twice and individual assays were done in duplicate.

Results

Influence of TPA concentration on the incorporation of 3H-choline into phosphatidylcholine The synthesis of phosphatidylcholine (PC), a major component of all cellular membranes (12), is c o m m o n l y used as a parameter in the study of membrane biosynthesis. More than 90% of radioactivity from labeled choline appears as PC in acid insoluble material (13). In many cell types PC synthesis has been shown to be stimulated by treatment of cells with phorbol esters (13-16). One of the commonly used phorbol esters is T P A which has been used at a final concentration of 1.7 X 10 7 M (14-16). The optimal concentration of T P A required in order to obtain maximal PC biosynthesis in Krebs II ascites cells was ascertained by measuring the incorporation of 3H-choline into TCA insoluble material at different concentrations of the phorbol ester (from i0 8 to 10 6 M). Several different aspects of T P A concentration-dependent PC biosynthesis are shown in Fig. 1: (a) at 1 hr the response is greatly dependent on T P A concentration; (b) no significant difference in 3H-choline incorporation was observed in cultures treated with T P A concentrations of 1.7 )< 10-7 M (Fig. 1C) and 1.7 X 10-6 M (Fig. 1E) for 1 hr indicating that m a x i m u m stimulation is reached at 1.7 X 10 7 M; (c) at 4 hr of incubation the difference in 3H-choline incorporation in cells treated with high (1.7 X 10-6 M) and low (1.7 X 10-8 M) concentrations of T P A is small (60%) relative to the difference in the response at 1 hr (200%); (d) cell viability tests showed that T P A at a concentration of !.7 × 10 6 M was apparently toxic to the cells during 20 hr in vitro incubation since viability was reduced to about 75% in these cultures in comparison to a value of 92% in control cultures and those treated with 1.7 X 10 7 M TPA. As a result of the data in Fig. 1 a concentration of 1.7 X 10-7 M T P A was chosen for all future experiments where Krebs II ascites cells were to be incubated in vitro with the phorbol ester. The incorporation of 3H-choline into PC was

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Fig. 1.3H-choline incorporation into Krebs 11 ascites cells treated with different concentrations of TPA: (A) 1.7 X l0 -8 M, (B) 8.5 X 10 8 M, (C) 1.7 X 10 7 M, (D) 3.4 X 10,7 M, and (E) 1.7 X 10_6 M. Cells obtained from a single mouse were divided into six cultures. Five were treated with various concentrations of TPA and one served as a control. 3H-choline was added to each at the commencement of the in vitro culture period (10) and cell samples were removed at 1 hr and 4 hr of incubation. The total amount of radioactivity in TCA insoluble material was determined and compared to that in the control (100%). Radioactivities in the 1 hr and 4 hr controls were 19215 cpm and 154 195 cpm respectively.

A

followed in TPA-treated and untreated cultures during a 6 hr period of in vitro incubation. A significant stimulation was observed in the TPA-treated culture especially at early times where incorporation after 30 rain was almost 300% above the control (Fig. 2A). As shown in Fig. 2B the incorporation rate of 3H-choline into PC in both treated and untreated cells was maximal at 90 min. It would appear that the initial rate increase observed in the control (first 30 min), probably caused by the favourable in vitro conditions, is accentuated by TPA. It is possible that T P A may have some structural resemblance to endogenous growth-promoting substances and thereby cause increased PC biosynthesis during a specific time interval when cells are sensitive to such stimulation. Although T P A caused an increase in the incorporation of 3H-choline into PC this was not merely a direct result of increased choline uptake as demonstrated in the section below. Choline transport into TPA-treated and untreated Krebs H ascites cells

Carrier-mediated transport of choline has been described in several cell types (17-19). Studies on the uptake of free choline by isolated perfused rat liver (20) showed that at concentrations below 40

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Time (hrs) Fig. 2. A comparison of 3H-choline incorporation in TPA-treated and untreated Krebs I1 ascites cells during the first 6 hr of in vitro culture: (A) incorporation of ~H-choline in TPA-treated cells related to that in control cells (100% at each time point), (B) 3H-choline incorporation rates in TPA-treated and untreated control cells.

140 # M transport was mostly carrier-mediated. At higher concentrations, however, a significant fraction was transported through a non-saturable mechanism (probably free diffusion). It has been reported that T P A binds to specific cell surface receptors (21, 22), however, an additional non-specific binding also occurs (23) due to the lipophilic properties of TPA. Fluorescence polarization studies of membranes have indicated that T P A elicits a change in the physical properties of the membranes probably reflected by an increase in membrane fluidity (24, 25). Thus T P A could influence carrier-mediated choline transport either by binding directly to the choline carrier or indirectly by causing changes in membrane fluidity, and furthermore, alteration in membrane fluidity could cause changes in the diffusion coefficient by which choline penetrates the cell membrane. The time course of 3H-choline uptake was investigated in both TPA-treated and untreated cultures of Krebs II ascites cells during a 2 hr period of in vitro incubation. The results shown in Fig. 3 do not indicate a TPA-induced stimulation of choline uptake. The medium used for in vitro culture of Krebs I

1

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II ascites cells contained 27.4 #M choline, a concentration at which choline transport is predominantly carrier-mediated. TPA, therefore, does not seem to affect the carrier responsible for choline uptake and transport in these cells. TPA-stimulated PC biosynthesis in the presence o f cycloheximide The rapid stimulation of PC biosynthesis by T P A (Fig. 2A) was attributed to either a direct activation of pre-existing enzyme systems by the phorbol ester or to an indirect mechanism first requiring protein synthesis. In order to examine these possibilities the effect of T P A on the stimulation of PC biosynthesis was tested in the presence of cycloheximide. Krebs II ascites cells, freshly harvested from the mouse peritoneum, were suspended in medium and divided into four aliquots consisting of two TPA-treated cultures and two untreated controls. Cycloheximide was added to a final concentration of 1 X 10 5 M to one TPA-treated and one control culture. This concentration inhibited protein synthesis by about 90% (15, and unpublished results). After 1 hr in vitro incubation 3Hcholine was added to all four cultures and the cells were incubated for a further 2 hr. The results of a typical experiment are expressed in Table 1 and it is

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Time ( h r ) Fig. 3. Time course of 3H-choline uptake in TPA-treated and untreated control cells. Cultures of TPA-treated and untreated Krebs II ascites cells were incubated with 3H-choline (10) and cell samples were removed from both cultures at various times during a 2 hr period of in vitro incubation. The radioactive content of whole cells was determined at the various time points. 0 - - 0 , TPA-treated; o - - o , control cells.

Culture

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Control Control + cycloheximide (1 × 10 s M) TP A TP A + cycloheximide (1 X 10 5 M)

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Krebs 11 ascites cells were divided into four aliquots: two control and two TPA-treated (1.7 X 10 7 M) cultures. Cyeloheximide was added to one control and one TPA-treated culture and incubation was performed for 1 hr. 3H-choline was added to all cultures and incubation was continued for a further 2 hr. Total radioactivity was determined in aliquots of cells from the four cultures.

evident that the inhibition of protein synthesis did not influence T P A stimulation of 3H-choline incorporation into PC. One can conclude, therefore, that pre-existing enzyme systems involved in PC biosyn-

14l thesis must be directly stimulated by T P A and thus produce the effects observed in Fig. 2. The same conclusion has been drawn by others from similar experiments on several other cell lines (15).

Distribution of 3H-choline incorporation into subcellular fractions of TPA-treated Krebs H ascites cells TPA-treated and untreated Krebs II ascites cells were incubated with 3H-choline and aliquots were harvested at 3 and 20 hr. The cells were disrupted by nitrogen cavitation and subcellular fractions were prepared. Figure 4 shows the amount of radioactiv-

Distribution of 3H-choline incorporation into ER membrane subfractions of TPA-treated Krebs H ascites cells and untreated cells Cell cultures exposed to T P A were harvested at several stages during a 20 hr incubation. Labeled cells were disrupted and ER membrane subfractions were separated on discontinuous sucrose gradients. The gradient profiles from TPA-treated and untreated cells are shown in Fig. 5 and the amounts L.

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Fig. 4. The incorporation of ~H-choline into PC in subcellular fractions of TPA-treated Krebs I1 ascites cells relative to untreated cells; (A) total cell homogenate, (B) nuclear fraction, (C) mitochondria, (D) ER membranes, (E) cytosol. TPA-treated and untreated Krebs lI ascites cells were incubated in the presence of 3H_choline. Aliquots of cells harvested after 3 hr and 20 hr of in vitro incubation were disrupted by nitrogen cavitation (10) and the a m o u n t of radioactivity in each subcellular fraction was determined and compared with that in the corresponding control (100% in each case).

ity in TCA-precipitable material in subcellular fractions of TPA-treated cells relative to untreated cells. At 3 hr, T P A caused a total stimulation of ]H-choline incorporation to a level about 200% above the untreated control. The degree of stimulation was approximately 200% in both the nuclear fraction and the mitochondrial fraction, while in the ER membrane fraction it was 350% and in the cytosol about 100%. After 20 br of incubation, however, the levels of radioactivity in all subcellular fractions were much closer to the control values.

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Fig. 5. Profiles of 3H-choline incorporation into PC in ER membrane subfractions in TPA-treated and untreated Krebs lI ascites cells; (A) control: 3 hr, (B) control: 8 hr, (C) control: 20 hr, (D) TPA-treated: 3 hr, (E) TPA-treated: 8 hr, (F) TPAtreated: 20 hr. TPA-treated and untreated Krebs II ascites cells were incubated in the presence of 3H-choline and cells were harvested from each culture after 3, 8 and 20 hr. Cells were disrupted by nitrogen cavitation and ER membrane subfractions were isolated on discontinuous sucrose density gradients (10). Radioactivity in TCA-insoluble material in the gradient fractions was determined.

of radioactivity appearing in subfractions is quantitated in Table 2. During the first 3 hr T P A stimulated 3H-choline incorporation into LR and S membranes to the extent of 400% and 320% respectively

142 Table 2.3 H-choline radioactivityin E R membrane subfractions isolated from untreated (control) and TPA-treated Krebs II ascites cells at various times of in vitro incubation.

Incubation

3 hr control 3 hr TPA 8 hr control 8 br TPA 20 hr control 20 hr TPA

3H-choline radioactivity (cpm) Heavy rough

'Intermediary heavy rough'

Light rough

Smooth

Total

1 170 1 170 7 280 2 340 43 600 64 140

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5 070 20 150 14 040 61 880 14 080 20 800

2 600 8 300 8 320 16 900 11 440 28 080

9 360 29 270 37 440 85 020 71 460 118 480

The data is obtained from the ER membrane distribution profiles presented in Fig. 5: HR = fractions 1-6, 'Intermediary HR'= fractions 7-9, LR = fractions 10-14, and S = fractions 15-20.

a b o v e c o n t r o l values, while at 20 hr these values were r e d u c e d to 148% and 245% (Table 2). Despite the high degree of 3H-choline i n c o r p o r a t i o n in the L R a n d S subfractions occurring in the T P A - t r e a t ed culture at 8 hr, r a d i o a c t i v i t y in H R m e m b r a n e s in T P A - t r e a t e d cells at this time was only twice t h a t in the 3 hr cells. This was in c o n t r a s t to the u n t r e a t ed cells where a six-fold increase was observed. D u r i n g a f u r t h e r 12 hr p e r i o d of in v i t r o i n c u b a t i o n there o c c u r r e d a great r e d u c t i o n in the a m o u n t of r a d i o a c t i v i t y in L R m e m b r a n e s in T P A - t r e a t e d cultures c o m p a r e d to u n t r e a t e d c o n t r o l s (Fig. 5E a n d F, and B and C). S i m u l t a n e o u s l y the H R subfraction showed a 50% higher i n c o r p o r a t i o n of 3H-cho line t h a n the u n t r e a t e d c o n t r o l cells (Table 2). A c o m p a r i s o n of the E R m e m b r a n e profiles at 8 hr (Fig. 5E) a n d 20 hr (Fig. 5F) w o u l d suggest a migration of r a d i o a c t i v i t y f r o m the L R region of the g r a d i e n t at 8 hr into the H R r e g i o n at 20 hr of i n c u b a t i o n . A similar b e h a v i o u r , however, was n o t o b s e r v e d in the u n t r e a t e d cells (Fig. B a n d C). These results m a y suggest differences in the b i o s y n t h e t i c p a t h w a y s of H R a n d L R m e m b r a n e s in u n t r e a t e d and T P A - t r e a t e d cells. The early features of T P A a c t i o n on Krebs II ascites cells thus consisted of s t i m u l a t e d 3H-choline i n c o r p o r a t i o n into L R and S m e m b r a n e s but not H R . The E R m e m b r a n e profiles in c o n t r o l a n d T P A - t r e a t e d cells were therefore quite different at 3 a n d 8 hr of in v i t r o incubation. A t a later time (20 hr), however, the profiles were basically similar t h o u g h the E R m e m b r a n e s in T P A - t r e a t e d cells c o n t a i n e d a higher level of label.

Discussion P h o r b o l esters exert a variety of effects on the cell m e m b r a n e . A m o n g the early events occurring after t r e a t m e n t with T P A are the following: altered cell a d h e s i o n p r o p e r t i e s , increased u p t a k e of 2 - d e o x y glucose, i n h i b i t i o n of the b i n d i n g of e p i d e r m a l g r o w t h f a c t o r to cell surface receptors, altered lipid m e t a b o l i s m , a n d i n d u c t i o n of p r o s t a g l a n d i n synthesis a n d a r a c h i d o n i c acid release (3). W e have previously established the presence of three s u b f r a c t i o n s of E R m e m b r a n e s in transf o r m e d cell lines ( H R , L R a n d S m e m b r a n e s ) while only L R a n d S m e m b r a n e s could be identified in n o r m a l cells (6-10). H o w e v e r , t r e a t m e n t of C3H/10T1/2 cells with T P A resulted in the a p p e a r ance of 3H-choline label in H R m e m b r a n e s (9). Using the s a m e cell line we have s h o w n t h a t T P A t r e a t m e n t results in an early release of 3H-choline label f r o m previously labeled cells (26) c o n f i r m i n g the results of M u f s o n et al. (27). We were able to d e m o n s t r a t e using in v i t r o e x p e r i m e n t s t h a t the release of r a d i o a c t i v i t y occurs a l m o s t exclusively f r o m the n u c l e a r - a s s o c i a t e d ER, a n d was d e p e n d ent on the presence of divalent cations suggesting that T P A activates a p h o s p h o l i p a s e in the membrane. In the same study it b e c a m e a p p a r e n t that P C biosynthesis was s t i m u l a t e d earlier in the N E R t h a n in the E R m e m b r a n e f r a c t i o n in response to t r e a t m e n t of cultures with T P A . We have therefore c o n c l u d e d that T P A does not merely have a general effect on all m e m b r a n e c o m p a r t m e n t s of the cell but rather a m o r e specific one (26).

143 When normal cells are treated with T P A several characteristic features of transformed cells are induced and if transformed cells are similarly treated then m a n y of these features are further accentuated (3-5). In light of this and earlier results using C3H/10TI/2 cells (9, 26) it was of interest to study the effects of T P A on the appearance of 3H-choline in ER membrane subfractions in a transformed cell line. For these studies Krebs II ascites cells were chosen because the behaviour of these cells under in vitro culture conditions had been previously studied (10), and a dynamic alteration in the appearance of ER membrane fractions had been observed as a function of time. Experiments on a variety of cell lines have been performed using a T P A concentration of 1.7 X 10-7 M (14-16). This concentration was also found to be optimal for stimulation of Krebs II ascites cells as measured by the incorporation of 3H-choline into PC (Fig. 1C). A ten-fold lower concentration was insufficient to give significant stimulation while a ten-fold higher concentration resulted in a loss of cell viability during a 20 hr incubation. It was notable that the differences in 3H-choline incorporation observed at 1 hr and 4 hr were similar in cells treated with T P A concentrations within the range 1.7 X 10 7 M - 1.7 X 10-6 M while at concentrations below 1.7 X 10 -7 M the differences were much smaller. The transport of 3H-choline into Krebs II ascites cells was not stimulated by treatment with T P A and this observation was in agreement with results from HeLa cells (28). Though the cellular uptake of choline was apparently not stimulated the incorporation of 3H-choline into PC was about 70% above the control value already after 5 min of T P A treatment rising to a b o u t 300% at 30 min. After the initial burst of incorporation during the first hour of incubation it fell slowly from then onwards though after 6 hr it was still 100% above the control value. A rapid stimulation of 3H-choline into phospholipids caused by phorbol esters has also been observed in HeLa cells (13, 15), bovine lymphocytes (29) and in the HL-60 promyelocytic leukemia cell line (30, 31). The stimulation of PC biosynthesis by T P A in Krebs II ascites cells is not prevented by prior incubation of cells with cycloheximide (Table 1) and similar results have been obtained in HeLa cells (15) and bovine lymphocytes (29). It is thus apparent that phorbol esters activate phospho-

lipid synthesis through stimulation of pre-existing enzyme systems and Paddon and Vance (28) have provided evidence that in HeLa cells the reaction stimulated is that catalyzed by the enzyme CTP: phosphocholine cytidyltransferase. Stimulation in Krebs II ascites cells, however, would seem to be only transient and appears to be complete within the space of 1 hr. The stimulation of the rate of 3H-choline incorporation into PC occurs in a similar time-dependent manner both in TPA-treated and untreated cells, the difference being that the process was greatly accentuated by the presence of TPA. One can therefore speculate that two phenomena are operating simultaneously, firstly an early stimulation by T P A of PC synthesizing enzymes, and secondly, an effect of T P A acting as a growth promoting factor. As shown in Fig. 4 T P A causes a general increase in the incorporation of 3H-choline into all subcellular fractions investigated. The greatest increase observed at 3 hr was in the ER membrane fraction while the smallest was seen in the cytosol. At 20 hr, however, differences were small between the amounts of radioactivity in subcellular fractions isolated from control and TPA-treated cells. In similar subfractionation experiments performed with C3H/10TI/2 cells (26) we found that T P A caused greatest stimulation of 3H-choline incorporation into ER membranes and NER after 2 hr incubation. In the accompanying paper (10) we demonstrated the presence of three ER membrane subfractions in Krebs II ascites cells (HR, LR and S) after a period of in vitro incubation following removal of cells from the mouse peritoneal cavity. It was therefore of interest to investigate in what manner T P A affected the appearance of PC in ER subfractions during in vitro incubation. After 3 and 8 hr of T P A treatment there were large increases in the amount of radioactivity in both the LR and S subfractions. H R membranes, on the other hand, appeared at 8 hr in untreated cells but not in cells treated with TPA. The explanation of this observation may lie in some cell cycle phenomenon and preliminary results with Krebs II ascites cells suggest that progression through G l phase is slowed down by TPA. At 20 hr the relative distribution of radioactivity in ER membrane subfractions was similar, again suggesting that T P A elicits changes at early times in phospholipid metabolism. It is likely, therefore,

144 that Krebs II ascites cells respond to T P A treatment by inducing the synthesis of an enzyme which metabolizes the phorbol ester such that T P A would exhibit a maximal effect during the first few hours of incubation. Kreibich et al. (32) have shown that in L-cell cultures, unlike HeLa cells, T P A and other phorbol derivatives are metabolized to a great extent within 12 hr of incubation. The present study has clearly demonstrated that not only lipid metabolism in the plasma membrane is affected by T P A treatment but also that in membranes of the ER system.

Acknowledgements Mrs Anny Knudsen is thanked for excellent technical assistance. This work was partly supported by grants from the Norwegian Research Council for Science and the Humanities (NAVF) and the Norwegian Research Council for Science and Technology (NTNF).

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