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On the biochemical mechanism of tumorigenesis in mouse skin
457
Biochimica et Biophysica Acta, 362 (1974) 457--468
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 27493 ON THE BIOCHEMICAL MECHANISM OF TUMORIGENESIS IN MOUSE SKIN VI. EARLY EFFECTS OF GROWTH-STIMULATING PHORBOL ESTERS ON PHOSPHATE TRANSPORT AND PHOSPHOLIPID SYNTHESIS IN MOUSE EPIDERMIS
ALLAN BALMAIN and ERICH HECKER Deutsches Krebsforschungszentrum, Institut for Biochemie, Heidelberg ( G.F.R.)
(Received November llth, 1973) (Revised manuscript received April 9th, 1974)
Summary The effect of the t u m o u r p r o m o t o r 12-O-tetradecanoyl-phorbol-13acetate on 32 Pi incorporation into the phosphatidylcholine fraction in mouse epidermis was investigated. 1. Two peaks in incorporation are observed, one at 4--6 h and one at 48 h after treatment. 2. The incorporation [3 H] thymidine into DNA of mouse epidermis is inhibited for about 1 0 h after administration of 12-O-tetradecanoylphorbol-13-acetate. At the same time, however, the incorporation of 32 Pi into epidermal DNA is rapidly stimulated, suggesting that the specific activity of the intracellular ATP pool is increased by the t u m o u r p r o m o t o r soon after its application. The first peak in 3-~Pi incorporation into phosphotidylcholine may therefore be attributed in part to pool changes. 3. After a short lag phase, the accumulation of phosphatidylcholine in mouse epidermis is stimulated. The ratio phosphatidylcholine/DNA in the epidermis reaches a m a x i m u m around 8 h after treatment. 4. Isolated mouse skin pieces pretreated with 12-O-tetradecanoyl-phorbol13-acetate are capable of incorporating 32 Pi into the phosphatidylcholine fraction in a manner which is similar to that observed in vivo. The early accumulation of phosphatidylcholine within the epidermis is therefore due to the localised stimulation of the skin microsomal enzymes responsible for its synthesis. 5. The role played by the early 12-O-tetradecanoyl-phorbol-13-acetateinduced stimulation of phospholipid metabolism in the development of tumours during initiation--promotion experiments in mouse skin is discussed.
458 Introduction
"~
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12-O-Tetradecanoyl-phorbol-13-acetate, the most active constituent of croton oil [1] is irritant and t u m o u r promoting for mouse skin [2] and causes changes in biochemical and cell kinetic parameters which are characteristic of a direct stimulation of cell proliferation. In vitro, it releases the density-dependent inhibition of growth in fibroblast cell cultures [3]. The controlled and sequential stimulation of the synthesis of phospholipids [4--6], RNA, protein and DNA [7--9], followed by a wave of mitoses [10] has been reported. This particular sequence of biochemical events exhibits striking similarities to that observed using other model systems for the study of growth control [11]. Several lines of evidence have implicated the cell membrane as being of crucial importance in the regulation of cell growth and division [12]. Treatment of cells in culture with hyaluronidase [13], trypsin [141, or other proteolytic enzymes has been shown to induce certain cell-surface alterations which lead to the initiation of DNA synthesis and cell division. Lymphocytes are susceptible to stimulation by phytohemagglutinin, a substance which is reputed to interact with specific cell-surface receptor sites [15]. In spite of the vast a m o u n t of research which has been carried out in this field, the exact mechanism by which changes in the architecture of the cell surface are manifested in the release of growth control remains to be elucidated. There are several indications in the literature that the primary site of action of 12-O-tetradecanoyl-phorbol-13-acetate is the external cell membrane. It has been reported that treatment of cells in culture with very low concentrations of 12-O-tetradecanoyl-phorbol-13-acetate can cause changes in typical membrane properties such as permeability [16] and configuration [17], and activates the plasma membrane enzyme (Na÷--K~)-dependent ATPase [18]. More recently, it was demonstrated that 12-O-tetradecanoyl-phorbol-13-acetate eliminates the normal ~-adrenergic elevation of the cyclic AMP level in mouse epidermis in vivo on injection of isoproterenol [ 19], suggesting that 12-O-tetradecanoyl-phorbol-13-acetate attacks or renders inactive certain receptor sites on the outer surface of the cell membrane. The role played by phospholipids in the amplification of a growth-stimulatory signal has received considerable attention using a number of experimental model systems. These include the stimulation of lymphocytes by phytohemagglutinin [20], treatment of rat uterus with estrogenic hormones [21], the regenerating rat liver [22] and the release of density-dependent inhibition of growth of 3T3 cells by addition of serum [23]. Preliminary investigations have shown that one of the earliest metabolic events induced by 12-O-tetradecanoyl-phorbol-13-acetate is the stimulation of [3 H] choline incorporatior, into phosphatidylcholine [4], suggesting that alterations in phospholipid metabolism could be one of the primary links in the chain of events leading to the stimulation of DNA synthesis. We have now investigated the early effects of 12-O-tetradecanoyl-phorbol13-acetate on 32 P-labelled orthophosphate incorporation into mouse epidermal phosphatidylcholine in vivo and in vitro, with a view to throwing some further light on the earliest metabolic events which occur on the initial stimulation of cell growth and proliferation. Parts of this work have appeared in preliminary reports [5,24].
459 Materials and Methods
Materials 32p-labelled orthophosphate (5--140 Ci/mg Pi) was obtained from the Radiochemical Centre, Amersham, England, and was injected intraperitoneally (500 pCi/20 g mouse). 12-O-Tetradecanoyl-phorbol-13-acetate and 4-O-methylphorbol-12,13-didecanoate were synthesised from phorbol by methods previously described [1,25]. The following were obtained from E. Merck AG, Darmstadt: ready-made Kieselgel plates (0.25 mm), which were used for all chromatographic separations unless otherwise stated; phosphatidylcholine standard, chromatographically pure; chloroform, methanol and acetone (p.a. grade), which were used without further purification. As in standardised Berenblum experiments [1], 7--8-week-old female NMRI mice were used throughout. The animals were maintained in a climatised room with an artificial inversed day--night r h y t h m , with water and food (Altromin GmbH, 4937 Lage Lippe, W. Germany) available ad libitum. The back skins of the mice (about 6 cm 2 ) were shaved 1--2 days prior to the experiment. Any animals showing evidence of wounds or hair regrowth were not used in experiments. Mouse skin pieces were incubated in Eagle's Minimum Essential Medium (Flow Laboratories, Irvine, Scotland) containing 4-fold amino acid and vitamin concentrations and at a final pH of 7.4. Methods Isolation of DNA and lipids from mouse epidermis. The shaved areas on the back skins of the mice were treated either with 0.1 ml acetone, in the case of the controls, or with 0 . l ml of acetone containing 0.02 pmole 12-O-tetradecanoyl-phorbol-13-acetate. Mice were routinely treated at 8.00 a.m. in order to avoid differences due to diurnal r h y t h m , but control experiments showed that Similar results were obtained when the mice were treated at 8.00 p.m. At various times after application groups of four mice were killed by cervical dislocation, the back skins were removed and wetted thoroughly in ice-cold 0.4 M HC104. The skins were pinned dermis side down on the convex surface of a clock glass and the epidermal cells from a defined part of the treated area were removed by scraping gently with a scalpel. The individual epidermal scrapings were then homogenised by hand in ice-cold 0.4 M HC104 in an allglass conical homogeniser. The homogenates were left to stand in ice for 30 min, centrifuged (3000 rev./min, 10 min) and the pellet further washed three times with cold 0.4 M HC104. The lipids were then thoroughly extracted with chloroform-methanol (2 : 1, v/v) at room temperature, the resulting lipid extract being washed according to Folch et al. [26]. Most of the solvent was removed under a stream of nitrogen and the residue containing the desired phospholipids was subjected to chromatographic separation as described below. The pellet remaining after extraction of the phospholipids was used for the determination of the total DNA content by the m e t h o d of Burton [27]. In experiments in which the incorporation of [32 p] orthophosphate into DNA was measured, the RNA in the pellet was first removed by hydrolysis in 0.3 M KOH for 3 h at 37 ° C. The DNA was precipitated by acidification with concen-
460 trated HC104 at 2°C and centrifugation, after which the pellet was washed with 0.4 M HC104 and the DNA content measured. Thin-layer chromatography o f lipids. The lipid extracts were spotted on to Merck ready-made plates which were developed in pre-equilibrated chromatography tanks containing chloroform--methanol--water in the ratio 65 : 25 : 4 {by vol.). The bands were visualised by spraying with iodine vapour and the phophatidylcholine band identified by comparison with an authentic standard run on the same plate. The phosphatidylcholine band was scraped off and used either for phosphate determination or for radioactivity measurement as described below. Since the procedure does not eliminate the possibility that very small amounts of other phospholipids may be present, for the purpose of the discussion this material will be referred to as the phosphatidylcholine fraction. Phosphate determination. The phosphatidylcholine bands obtained from the plates were ashed with 70% HC104 at 200 °C. Thereafter the concentration of inorganic phosphate liberated was estimated by a scaled down modification of the ascorbic acid m e t h o d of Chen et al. [28]. A standard curve was first prepared by carrying known amounts of phosphatidylcholine through the above procedure. The absorbance of the reduced phosphomolybdate complex could then be translated directly into pg phosphatidylcholine for each skin extract. Blank values were obtained using blank areas of the plate which were roughly equivalent to that containing the phosphatidylcholine band. Radioactivity measurement. The radioactivity incorporated into the phosphatidylcholine fraction was measured by scraping the bands from the plate directly into vials containing 10 ml scintillation fluid. The scintillation fluid contained: toluene (700 ml), dioxane (700 ml), ethanol (420 ml), naphthalene (147.6 g), PPO (9.2 g), dimethyl POPOP (0.1 g). Samples were counted in a Nuclear Chicago Mark II Analyser. External standardisation showed that differences in quenching between individual samples were negligible. Incorporation data are therefore presented as cpm in the phosphatidylcholine fraction/pg DNA. [32p]Orthophosphate incorporated into DNA was determined by counting 0.1-ml aliquots of the DNA hydrolysate, obtained by heating the RNA- and lipid-free pellet in 5% trichloroacetic acid in a boiling water bath for 7 min. Incubation o f mouse skin pieces in vitt'o. The back skins of shaved mice were treated with acetone or 12-O-tetradecanoyl-phorbol-13-acetate as described above. All subsequent operations were carried out at 4°C unless otherwise stated. At various time intervals after treatment, groups of four mice were killed by cervical dislocation, the back skins were quickly removed and pinned epidermis side down on cork plates. The subcutaneous tissue was gently scraped off with a scalpel, the skins were cut into small pieces (approx. 1 mm ~ ) with scissors and incubated for 1 h at 37 ° C in 3 ml of Medium A containing the radioactive orthophosphate (20 pCi/ml). The incubation was terminated by addition of ice water, the skins were homogenised and the lipids and DNA isolated as previously described. Results and Discussion Treatment of the dorsal skins of shaved mice with 0.02 pmole 12-O-tetra-
461
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Fig. 1. The effect o f 12-O-tetradecanoyl-phorbol-13-acetate (0.02 //moles/application) on the incorporat i o n o f [ 3 2 p ] o r t h o p h o s p h a t e i n t o the mouse epidermal p h o s p h a t i d y l c h o l i n e f r a c t i o n in v~vo. Mice were treated t o p i c a l l y either w i t h 0.02 //moles 12-O-tetradecanoyl-phorbol-13-acetate in 0.1 m l o f acetone or w i t h 0.1 m l a c e t o n e a l o n e . G r o u p s o f f o u r a n i m a l s w e r e killed at t h e t i m e s s h o w n . E a c h m o u s e w a s i n j e c t e d i n t r a p e r i t o n e a U y 1 h p r i o r to d e a t h w i t h 5 0 0 ~tCi [ 3 2 p ] o r t h o p h o s p h a t e . L i p i d s w e r e e x t r a c t e d f r o m t h e d o r s a l e p i d e r m i s of e a c h i n d i v i d u a l m o u s e , t h e p h o s p h a t i d y l c h o l i n e w a s i s o l a t e d a n d t h e r a d i o a c t i v i t y i n c o r p o r a t e d d e t e r m i n e d b y l i q u i d s c i n t i l l a t i o n c o u n t i n g . T h e D N A c o n t e n t of t h e e p i d e r m a l pellet w a s m e a s u r e d as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . T h e r e s u l t s are p r e s e n t e d as c p m in p h o s p h a t i d y l c h o l i n e / / / g D N A . E a c h p o i n t r e p r e s e n t s t h e m e a n v a l u e f r o m f o u r m i c e . S t a n d a r d d e v i a t i o n s are i n d i c a t e d b y t h e v e r t i c a l lines. T h e v a l u e s o b t a i n e d f r o m a c e t o n e - t r e a t e d c o n t r o l m i c e w e r e w i t h i n t h e h a t c h e d area. T h e c u r v e s h o w n w a s o b t a i n e d f r o m o n e e x p e r i m e n t in a series o f f o u r , all o f w h i c h g a v e s i m i l a r r e s u l t s . D o u b l e v a r i a n c e a n a l y s i s o f t h e l o g a r i t h m s of t h e a c t i v i t i e s i n c o r p o r a t e d b y e a c h m o u s e s h o w e d t h a t t h e d i f f e r e n c e s b e t w e e n t r e a t e d a n d c o n t r o l m i c e w e r e h i g h l y s i g n i f i c a n t (P < 0 . 0 0 1 ) , as w e r e t h e d i f f e r e n c e s b e t w e e n i n d i v i d u a l g r o u p s o f t r e a t e d m i c e (P < 0 . 0 1 ) . Single v a r i a n c e a n a l y s i s w a s also c a r r i e d o u t to c o m p a r e t h e m e a n v a l u e s o b t a i n e d at e a c h t i m e p o i n t . T h i s s h o w e d , f o r e x a m p l e , t h a t s i g n i f i c a n t d i f f e r e n c e s e x i s t b e t w e e n t h e p o i n t s at 4 a n d 8 h (P < 0 . 0 1 ) a n d at 6 a n d 8 h (P < 0 . 0 5 ) .
decanoyl-phorbol-13-acetate in 0.1 ml acetone resulted in a biphasic stimulation of the incorporation of [3: p] orthophosphate into the epidermal phosphatidylcholine fraction (Fig. 1). Application of acetone alone or of the nonirritant and non-tumour promoting 4-O-methyl-phorbol-12,13-didecanoate [25] caused no significant stimulation of incorporation, showing that the observed response was due to the active tumour promotor 12-O-tetradecanoylphorbol-13-acetate. The biphasic stimulation of incorporation, with peaks at 4--6 and at 48 h after treatment is temporally similar to that observed by Rohrschneider et al. [ 6 ] , but in our case the 48-h peak is much less prominent. This is probably due to the fact that these authors related the observed incorporation to a specific surface area of skin. We have purposely taken the total amount of DNA as our point of reference, since it is known from histological investigations that the epidermis of a 12-O-tetradecanoyl-phorbol-13-acetatetreated mouse is considerably thicker and contains at least twice as many cells as control skins around 48 h after application [29]. Indeed, measurement of the total DNA content of a specific area of treated epidermis has confirmed the increased DNA content around this time (Balmain, A., and Hecker, E.,
462
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120-
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Time 0fter treatment (hours) Fig. 2. T h e e f f e c t o f 12-O-tetradecanoyl-phorhol-13-aeetate ( 0 . 0 2 # m o l e ) o n t h e i n c o r p o r a t i o n o f [32 p] o r t h o p b o s p h a t e i n t o the acid-soluble f r a c t i o n i n m o u s e epidenmis. T r e a t m e n t o f zmce a n d i n j e c t i o n of [32p] orthophosphate as d e s c r i b e d i n t h e l e g e n d f o r F i g . 1. T h e e p i d e r m i s f r o m e a c h i n d i v i d u a l m o u s e was homogenised thoroughly in ice-cold 0.4 m HC104 (2 ml), the homogenate was centrifuged and the radioactivity in 0.1-ml aliquots of the supernatant was determined. The DNA content of the pellet was m e a s u r e d as d e s c r i b e d i n M e t h o d s . R e s u l t s a r e e x p r e s s e d as c p m / O . l - m l a l i q u o t / p g D N A i n t h e c o r r e s p o n d i n g p e l l e t . E a c h p o i n t is t h e a v e r a g e o b t a i n e d f r o m f o u r m i c e . S t a n d a r d d e v i a t i o n s a r e r e p r e s e n t e d b y t h e v e r t i c a l lines. T h e z e r o - t i m e v a l u e w a s o b t a i n e d u s i n g a c e t o n e - t r e a t e d m i c e .
unpublished). By relating the activity incorporated into the phosphatidylcholine fraction in a specific area of skin to the total a m o u n t of DNA in that area, a more realistic representation is obtained of the degree of stimulation of incorporation per cell. Several explanations for the early stimulation of 32pi incorporation into epidermal phosphatidylcholine on treatment with 12-O-tetradecanoyl-phorbol13-acetate (Fig. 1) can be envisaged: a change in the rate of uptake of phosphate from the extracellular medium, leading to an increase in the specific activity of the intracellular phosphate pool; the stimulation of the de novo phosphatidylcholine synthesis; and increased turnover of the phosphorylcholine moiety. As regards the first of these possibilities, increased cellular uptake of phosphate has been observed in several systems to be one of the early responses to a growth stimulus [23,30]. As a rough measure of the general increase in orthophosphate concentration, the effect of 12-O-tetradecanoyl-phorbol-13acetate on the incorporation of 32 Pi into the acid-soluble fraction of mouse epidermis was determined (Fig. 2). Uptake into the tissue increases rapidly to reach a plateau about 6 h after 12-O-tetradecanoyl-phorbol-13-acetate administration. However, the data presented in Fig. 2 cannot be taken as a true representation of the change in intracellular precursor concentration, since the acid-soluble fraction of the homogenised tissue will have both intracellular and extracellular contributing factors. Estimation of the latter is complicated by the increased blood flow through the skin and the dilation of the intercellular spaces in the epidermis [10] on treatment with the t u m o u r promotor. An indirect measure of the change in intracellular specific activity can be
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time after treatment(hours) Fig. 3. T h e e f f e c t o f 1 2 - O - t e t r a d e e a n o y l - p h o r b o l - 1 3 - a c e t a t e ( 0 . 0 2 # m o l e s ) o n [ 3 2 p ] o r t b o p h o s p h a t e a n d [ 3 H ] t h y m i d i n e i n c o r p o r a t i o n i n t o D N A in m o u s e e p i d e r m i s . T h e m i c e w e r e t r e a t e d a n d killed as des c r i b e d in t h e l e g e n d to Fig. 1. T h e p e l l e t o b t a i n e d a f t e r e x t r a c t i o n o f t h e lipids w a s i n c u b a t e d in 0.3 M K O H (1 m l ) f o r 3 h at 37'~C t o h y d r o l y s e t h e R N A . T h e D N A w a s p r e c i p i t a t e d b y a d d i t i o n o f c o n c e n t r a t e d H C l O 4 ( 0 . 0 6 m l ) at 0 ° C. T h e p e l l e t o b t a i n e d a f t e r c e n t r i f u g a t i o n w a s w a s h e d t w i c e w i t h cold 0.4 M H C I O 4 , t h e D N A w a s h y d r o l y s e d in 5% t r i c h l o r o a c e t i c a c i d a n d its s p e c i f i c a c t i v i t y d e t e r m i n e d as d e s c r i b e d in M e t h o d s . P o i n t s r e p r e s e n t t h e a v e r a g e v a l u e s f r o m f o u r m i c e ± S. D.. R e s u l t s are p r e s e n t e d as p e r c e n t o f t h e c o r r e s p o n d i n g i n c o r p o r a t i o n in a c e t o n e - t r e a t e d c o n t r o l m i c e .
obtained, however, by determination of the extent of 3 ~ Pi incorporation into a macromolecule whose true rate of synthesis has been measured by independent means. The effect of 12-O-tetradecanoyl-phorbol-13-acetate on the rate of 32 Pi and [3 H] thymidine incorporation into the DNA in mouse epidermis is shown in Fig. 3. It can be seen that the a m o u n t of 3 2 Pi in the epidermal DNA is increased with respect to controls as rapidly as 2--4 h after treatment. The large increase in DNA specific activity observed after 12 h is attributable to the true stimulation of the synthesis of this macromolecule, and is also reflected in the [3 H] thymidine incorporation (c.f. refs 7--10). Several previous studies have demonstrated that 12-O-tetradecanoylphorbol-13-acetate causes an initial depression, lasting some 12 h, in the incorporation of [3 H] thymidine into mouse skin DNA [7--10,31]. Direct measurement of the a m o u n t of DNA in mouse epidermis suggests that the synthesis of DNA may be stimulated somewhat earlier than 12 h after application (Baimain, A., and Hecker, E., unpublished) but in any case not sooner than 6--8 h. The fact that the specific activity of DNA labelled by [32 p] orthophosphate increases so rapidly after treatment strongly suggests that the intracellular specific activity of the ATP precursor pool is raised by the t u m o u r promotor. This early effect will also by reflected in the 32 Pi incorporation into epidermal phosphatidylcholine (Fig. 1). The literature abounds with examples of increased transport as a prelude to accelerated growth [ 23,30,32] and it has been
464
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Fig. 4. T h e a c c u m u l a t i o n o f p h o s p h a t i d y l c h o l i n e in m o u s e e p i d e r m i s a f t e r t r e a t m e n t w i t h 1 2 - O - t e t r a decanoyl-phorbol-13-acetate (0.02 #moles). Groups of 4 12-O-tetradecanoyl-phorbol-13-acetate-treated m i c e w e r e killed a t t h e t i m e s s h o w n , t h e s k i n s w e r e r e m o v e d a n d a s t a n d a r d i s e d c i r c u l a r a r e a ( a b o u t 3 c m 2) o f e p i d e r m i s w a s c a r e f u l l y s c r a p e d o f f f r o m t h e m i d d l e o f t h e t r e a t e d a r e a of e a c h skin. T h e phosphatidylcholine content of each standardised area of epidermis was determined by chromatographic s e p a r a t i o n o f t h e e x t r a c t e d p h o s p h o l i p i d s , f o l l o w e d b y q u a n t i t a t i v e m e a s u r e m e n t of t h e p h o s p h a t e c o n t e n t o f t h e p h o s p h o t i d y l c h o l i n e b a n d . R e s u l t s are p r e s e n t e d as # g p h o s p h a t i d y l c h o l i n e / s t a n d a r d s k i n area. P o i n t s r e p r e s e n t t h e m e a n v a l u e s f r o m f o u r m i c e ± S. D.. Z e r o - t i m e value w a s o b t a i n e d u s i n g a c e t o n e treated controls.
previously suggested that this may be a general phenomenon [33]. One could speculate that t h e rapid influx of specific metabolites immediately after a growth signal is responsible for the activation of enzymes required for macromolecular synthesis. In this respect it is n o t e w o r t h y that phosphate concentration has been shown to be of critical importance in the regulation of certain intracellular processes [ 34,35 ]. In order to determine the time at which a true stimulation of the synthesis of phosphatidylcholine occurs, the accumulation of phosphatidylcholine as a function of time within a specific surface area of treated mouse epidermis was measured (Fig. 4). In contrast to the results of Rohrschneider et al. [6], who observed the most rapid accumulation of phospholipid within the first 2--4 h following treatment, no increase in extractable phosphatidylcholine was seen after 2 h and only a relatively small increase was observed after 4 h. This apparent discrepancy can perhaps be explained by the fact that these authors used mouse skin rather than epidermis alone in their investigations. It can be observed that an appreciable dermal oedema develops within 4 h of treatment with a suitable dose of a t u m o u r promotor, and it could be that the rapid accumulation reported is a reflection of the resultant increase in the concentration of phosphatidylcholine in the dermis. It is noticeable that about 4 h after 12-O-tetradecanoyl-phorbol-13-acetate treatment, the epidermis can be more easily removed from the dermis by scraping with a scalpel. It is necessary therefore to exclude the possibility that the early accumulation of phosphatidylcholine within the epidermis is attributable to the fact that more cells can be scraped from the given area of epidermis. To this end, a separate experiment was carried out in which the ratio of phosphatidylcholine to DNA content in the epidermis of each treated mouse was measured (Fig. 5). Here again it can be seen that the ratio, which can be
465
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1'2 *
hme after treatment(hours) Fig. 5. T h e e f f e c t of 1 2 - O - t e t r a d e c a n o y l - p h o r h o l - 1 3 - a e e t a t e ( 0 . 0 2 # m o l e s ) o n t h e r a t i o p h o s p h a t i d y l choline c o n t e n t / D N A c o n t e n t in m o u s e e p i d e r m i s . T h e p h o s p h a t i d y l c h o l i n e c o n t e n t o f 1 2 - O - t e t r a d e c a n o y l - p h o r b o l - 1 3 - a c e t a t e - t r e a t e d m o u s e e p i d e r m i s was d e t e r m i n e d as d e s c r i b e d in t h e l e g e n d t o Fig. 4. T h e pellet r e m a i n i n g a f t e r lipid e x t r a c t i o n was f u r t h e r used for t h e d e t e r m i n a t i o n of D N A b y t h e B u r t o n m e t h o d . Points: a v e r a g e f r o m f o u r m i c e ± S.D.. Z e r o - t i m e v a l u e f r o m a c e t o n e - t r e a t e d m i c e .
taken as a measure of the phosphatidylcholine content per cell, shows an increase after 4 h, with the maximum value being reached after 8 h. Although the absolute amount of phosphatidylcholine continues to increase for 14--20 h (Fig. 4), the ratio pg phosphatidylcholine/pg DNA levels off at 8 h, owing to the appreciable increase in the DNA content of the epidermis at 12 h. One can envisage at least two possible explanations to account for the measured accumulation of phosphatidylcholine in mouse epidermis after treatment with the t u m o u r promotor: the stimulation of local phosphatidylcholine synthesis via the Kennedy pathway [36] and increased influx of phosphatidylcholine from plasma owing to induced changes in cellular permeablility. In the latter case, phosphatidylcholine synthesised primarily in the liver would be transported to the epidermis, there to accumulate owing to accelerated uptake through the cell membranes. If the former mechanism is operative, one would expect that the activation of the microsomal enzymes responsible for phosphatidylcholine synthesis would be measurable also in vitro. This possibility is verified by the experiment shown in Fig. 6. Here, the skins of mice treated in vivo with 12-O-tetradecanoyl-phorbol-13-acetate were removed at various time intervals and incubated at 37°C for 1 h in medium containing [32 p] orthophosphate. The lipids were extracted and the degree of incorporation into the phosphatidylcholine fraction was determined. Under the conditions of this experiment, any incorporation observed must have taken place within the mouse skin, since any possible contribution from phosphatidylcholine synthesised in the liver has been excluded. The fact that qualitatively similar time--response curves are obtained for incorporation in vivo and in vitro leads to the conclusion that the reaction to 12-O-tetradecanoyl-phorbol-13-acetate treatment in vivo is mainly due to localised metabolic events occurring within the epidermis. The quantitative difference between the two curves is probably due to the lower efficiency of the enzymatic synthesis of phosphatidylcholine under in vitro conditions. A very early increase in 3 2 Pi incorporation into phosphatidylinositol has been reported in lymphocytes stimulated with phytohemagglutinin [20]. The
466
5O
/~0"
z 0
30"
~
20"
10
Time a f t e r
treatment
{hours)
Fig. 6. E f f e c t of 1 2 - O - t e t r a d e c a n o y l - p h o r b o l - 1 3 - a c e t a t e ( 0 . 0 2 p m o l e s ) o n i n c o r p o r a t i o n of [ 3 2 p ] o r t h o p h o s p h a t e into p h o s p h a t i d y l c h o l i n e in m o u s e skin pieces in vitro. Mice in g r o u p s of f o u r w e r e killed at v a r i o u s t i m e s a f t e r 1 2 - O - t e t r a d e c a n o y l - p h o r b o l - 1 3 - a c e t a t e t r e a t m e n t , t h e skins w e r e r e m o v e d , c u t into small pieces a n d i n c u b a t e d i n d i v i d u a l l y for 1 h at 3 7 ° C in m e d i u m (2 m l ) c o n t a i n i n g [ 3 2 p ] o r t h o p h o s p h a t e ( 2 0 # C i / i n c u b a t i o n ) . I n c u b a t i o n s w e r e t e r m i n a t e d b y a d d i t i o n o f ice w a t e r . T h e skins w e r e t h e n h o m o g e n i s e d , t h e a c t i v i t y i n c o r p o r a t e d i n t o p h o s p h a t i d y l c h o l i n e a n d the D N A c o n t e n t w e r e d e t e r m i n e d as d e s c r i b e d in t h e l e g e n d to Fig. 1. F o r t h e p u r p o s e of d i r e c t c o m p a r i s o n of in vivo a n d in v i t r o results, s o m e o f t h e d a t a p r e s e n t e d in Fig. 1 are r e p r o d u c e d here. L~ -~, i n c o r p o r a t i o n into t h e e p i d e r m a l p h o s p h a t i d y l c h o l i n e f r a c t i o n in vivo; • . . . . . . • , i n c o r p o r a t i o n into the s a m e f r a c t i o n in skin pieces in vitro. Points: a v e r a g e value f r o m f o u r m i c e ± S. D. Z e r o - t i m e values w e r e o b t a i n e d f r o m a c e t o n e - t r e a t e d control mice.
authors attributed this effect to an increase in the rate of turnover of the inositol and phosphate groups. The possibility that an analogous turnover of the phosphorylcholine moiety of phosphatidylcholine occurs in the 12-O-tetradecanoyl-phorbol-13-acetate-stimulated epidermis can not be excluded on the evidence presently available. If this is a contributing factor, however, it is probably only of secondary importance, since most of the activity incorporated can be accounted for by pool changes, together with the stimulation of de novo phosphatidylcholine synthesis. The nature of the primary interaction of 12-O-tetradecanoyl-phorbol-13acetate with the cell membrane is at the m o m e n t completely unknown. One could imagine simply from consideration of the chemical structure of 12-0tetradecanoyl-phorbol-13-acetate, with a hydrophilic " h e a d " grouping coupled to a long hydrophobic "tail", that it could intercalate into the lipid bilayer of the cell membrane, bringing about the changes in membrane properties which are observed experimentally [16--19]. It has been established that many enzymes situated at the cell surface which are involved in the regulation of cell growth require phospholipids in order to be fully active [37]. One could speculate that a disturbance of the lipid moiety of the protein--lipid complex, conceivably the "transducer" of Rodbell [38] which is known to be susceptible to damage by detergents [39], might lead to early alterations of growth control which cultimate in the stimulation of DNA synthesis and cell division.
467 " The'~rocess of t u m o u r promotion involves the repeated treatment of " i n i t i a t e d " skin with a suitable agent, such as 12-O-tetradecanoyl-phorbol-13acetate, eventually leading to the formation of visible tumours. Hennings and Boutwell [40] have postulated that promotion may be further sub-divided into two stages, defined as "conversion", by which an initiated t u m o u r cell is converted into a " d o r m a n t t u m o u r cell", and "propagation", which involves the proliferation of d o r m a n t t u m o u r cells to macroscopic turnouts. In a recent publication, Rohrschneider and Boutwell [41] found that the early peak in 32 Pi incorporation into phosphatidylcholine is specific for tumour-promoting agents, and it was further suggested that this peak is a biochemical manifestation of the "conversion" process. These conclusions were based on the fact that non-tumour promoting hyperplastic agents and complete carcinogens did not elicit this early response in phospholipid metabolism when applied to mouse epidermis. None of these agents, however, stimulates general epidermal proliferation as rapidly or to the same extent as 12-O-tetradecanoyl-phorbol-13acetate. In addition, in the above studies no account was taken of the balance between the proliferative and possible cytotoxic effects of the hyperplastic agents used, a factor which could be of critical importance in the determination of tumour-promoting activity. We therefore think that the above conclusion is premature on the evidence presently available. We favour the interpretation that the early peak in 32 Pi incorporation is related to the pleiotypic response [42] characteristic of a stimulation of cell proliferation, and n o t necessarily to the activation of specific genetic information as would be required of the "conversion" process [40]. The 4-h peak could be considered a manifestation of a general stimulation of membrane metabolism, comprising contributions from increased permeability, de novo phospholipid synthesis and possibly elevated phosphate turnover, as a prelude to an overall acceleration of growth. The subsequent increase in the phosphatidylcholine/DNA ratio in the epidermis may be considered an expression of the proliferation of the endoplasmic reticulum in the stimulated cells, a conclusion which is supported by recent electron-microscopic observations [10]. The studies of Tata [43] have shown that accelerated growth and development is characterised by a coordinated preliferation of intracellular membranes and ribosomes, which accompanies and is necessary for the enhanced protein synthesis in the stimulated cells. References 1 H e c k e r , E. ( 1 9 7 1 ) in M e t h o d s in C a n c e r R e s e a r c h ( B u s c h , H., e d . ) Vol. VI, p p . 4 3 9 - - 4 8 4 , A c a d e m i c Press, N e w Y o r k 2 H e c k e r , E. ( 1 9 6 8 ) C a n c e r Res. 2 8 , 2 3 3 8 - - 2 3 4 9 3 Sivak, A. ( 1 9 7 2 ) J . Cell P h y s i o l . 8 0 , 1 6 7 174 4 K r e i b i c h , G., H e c k e r , E., Stiss, R . a n d Kinzel0 V. ( 1 9 7 1 ) N a t u r w i s s e n s c h a f t e n 5 8 , 3 2 3 5 B a l m a i n , A. a n d H e c k e r , E. ( 1 9 7 2 ) E . A . C . R . S y m p o s i u m o n Cellular M e m b r a n e s a n d H y b r i d i s a t i o n , J a s z o w i e c , P o l a n d , M a y 3 - - 5 , A b s t r . , in t h e p r e s s 6 Rohrschneider, L.R., O'Brien, D.H. and Boutwell, R.K. (1972) Biochim. Biophys. Acta 280, 57--70 7 B a i r d , W.M., S e d g w i c k , J . A . a n d B o u t w e l l , R . K . ( 1 9 7 1 ) C a n c e r Res. 3 1 , 1 4 3 4 - - 1 4 3 9 S P a u l , D. a n d H e c k e r , E. ( 1 9 6 9 ) Z. K r e b s f o r s c h . 7 3 , 1 4 9 - - 1 6 3 9 B r e s c h , H. a n d H e c k e r , E. ( 1 9 6 9 ) P r o c . A m . Ass. C a n c e r Res. 37 1 0 R a i c k , A . N . ( 1 9 7 3 ) C a n c e r Res. 3 3 , 2 6 9 - - 2 8 6
468 11 Mueller, G.C. ( 1 9 7 1 ) in The Cell Cycle a n d Cancer (Baserga, R., ed.), pp. 2 6 9 - - 3 0 4 , Marcel Dek~er Inc., New Y o r k 12 Pardee, A.B. ( 1 9 7 1 ) In Vitro 7, 9 5 - - 1 0 4 13 Vasiliev, J.M., Gelfand, I.M., Guelstein, V.I. a n d Fetisova, E.K. ( 1 9 7 0 ) J. Cell Physiol. 75, 3 0 5 - - 3 1 4 14 Burger, M.M. (1970) N a t u r e 2 2 7 , 1 7 0 - - 1 7 1 15 K o r n f i e l d , S., Rogers, J. a n d G r e g o r y , W. ( 1 9 7 1 ) J. Biol. Chem. 2 4 6 , 6 5 8 1 - - 6 5 8 6 16 Sivak, A., R a y , F. a n d V a n D u u r e n , B.L. ( 1 9 6 9 ) Cancer Res. 2 9 , 6 2 4 - - 6 3 0 17 Sivak, A. a n d Van D u u r e n , B.L. ( 1 9 6 7 ) Science 157, 1 4 4 3 - - 1 4 4 4 18 Sivak, A., Mossman, B.T. a n d Van D u u r e n , B.L. ( 1 9 7 2 ) Biochem. Biophys. Res. C o m m u n . 46, 605--609 19 Marks, F., G r i m m , W. a n d Krieg, L. ( 1 9 7 2 ) H o p p e Seyler's Z. Physiol. Chem. 353, 1 9 7 0 - - 1 9 7 2 20 Fisher, D.B. a n d Mueller, G.C. ( 1 9 7 1 ) Biochim. Biophys. A c t a 2 4 8 , 4 3 4 - - 4 4 8 21 S p o o n e r , P.M. a n d Gorski, J. ( 1 9 7 2 ) E n d o c r i n o l o g y 91, 1 2 7 3 - - 1 2 8 3 22 F e x , G. ( 1 9 7 0 ) Biochem, J. 1 1 9 , 7 4 3 - - 7 4 7 23 C u n n i n g h a m , D.D. a n d Pardee, A.B. ( 1 9 6 9 ) Proc. Natl. Acad. Sci. U.S. 64~ 1 0 4 9 - - 1 0 5 6 24 Balmain, A. ( 1 9 7 3 ) FEBS Lett. 3 3 , 2 7 5 - - 2 8 0 25 J a c o b i , P. ( 1 9 7 2 ) Dissertation, University of Heidelberg 26 F o l c h , J., Lees, M. a n d Sloane S t a n l e y , G.H. ( 1 9 5 6 ) J. Biol. Chem. 2 2 6 , 4 9 7 - - 5 0 9 27 B u r t o n , K.A. ( 1 9 5 6 ) Biochem. J. 6 2 , 3 1 5 - - 3 2 3 28 Chen, P.S., T o r i b a r a , T.V. a n d Warner, H. ( 1 9 5 6 ) Anal. Chem. 28, 1 7 5 6 - - 1 7 5 8 29 Bach, H. a n d G o e r t t l e r , K. ( 1 9 7 1 ) V i r c h o w s Arch. A b t . B. Zellpath. 8, 1 9 6 - - 2 0 5 30 C u n n i n g h a m , D.D. a n d Pardee, A.B. ( 1 9 7 1 ) Ciba S y m p o s i u m on G r o w t h C o n t r o l in Cell Cultures (Wolstenholme, G.E.W. a n d K n i g h t s , J., eds), pp. 2 0 7 - - 2 2 0 , Churchill-Livingstone, L o n d o n 31 Raick, A.N., T h u m m , K. a n d Chivers, B.R. ( 1 9 7 2 ) Cancer Res. 32, 1 5 6 2 - - 1 5 6 8 32 Tara, J . R . ( 1 9 6 8 ) N a t u r e 2 1 9 , 3 3 1 - - 3 3 7 33 Bhargava, P.M. ( 1 9 7 0 ) in Ciba S y m p o s i u m on C o n t r o l Processes in Multicellular O r g a n i s m s (Wolstenh o l m e , G.E.W. a n d Knights, J., eds), p p . 1 5 8 - - 1 7 7 , J. a n d A. Churchill, L o n d o n 34 Izzard, S. a n d Tedeschi, H. ( 1 9 7 3 ) A r c h . Biochem. Biophys. 154, 5 2 7 - - 5 3 9 35 Holley, R.W. ( 1 9 7 2 ) Proc. Natl. Acad. Sci. U.S. 69, 2 8 4 0 - - 2 8 4 1 36 K e n n e d y , E.P. ( 1 9 5 7 ) Fed. Proc. 1 6 , 8 4 7 37 R o t h f e l d , L. a n d R o m e o , D. ( 1 9 7 1 ) in S t r u c t u r e a n d F u n c t i o n of Biological M e m b r a n e s ( R o t h f e l d , L., ed.), p p . 2 5 1 - - 2 8 4 , A c a d e m i c Press, New Y o r k 38 Rodbell, M. ( 1 9 7 2 ) in C u r r e n t Topics in B i o c h e m i s t r y ( A n f i n s o n , C.B., G o l d b e r g e r , A.F. a n d Schechter, A.N., eds), pp. 1 8 7 - - 2 1 8 , A c a d e m i c Press, New Y o r k 39 B i r n b a u m e r , L., Pohl, S.L. a n d Rodbell, M. ( 1 9 7 1 ) J. Biol. Chem. 2 4 6 , 1 8 5 7 - - 1 8 6 0 40 Hennings, H. a n d Boutwell, P~.K. ( 1 9 7 0 ) Cancer Res. 3 0 , 3 1 2 - - 3 2 0 41 R o h r s c h n e i d e r , L.R. a n d Boutwell, R.K. ( 1 9 7 3 ) Cancer Res. 33, 1 9 4 5 - - 1 9 5 2 42 T o m k i n s , G.M. ( 1 9 7 1 ) in Effects of Drugs on Cellular C o n t r o l Mechanisms (Rabin, B.R. a n d Freedm a n , R.B., eds), p p . 1 - - 9 , MacMillan, L o n d o n 43 Tata, J . R . ( 1 9 7 1 ) in H o r m o n e s in D e v e l o p m e n t ( H a m b u r g h , M. a n d B a r r i n g t o n , E.J.W., eds), pp. 1 9 - - 3 9 , Meredith Corp., New Y o r k
Biochimica et Biophysica Acta, 362 (1974) 457--468
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 27493 ON THE BIOCHEMICAL MECHANISM OF TUMORIGENESIS IN MOUSE SKIN VI. EARLY EFFECTS OF GROWTH-STIMULATING PHORBOL ESTERS ON PHOSPHATE TRANSPORT AND PHOSPHOLIPID SYNTHESIS IN MOUSE EPIDERMIS
ALLAN BALMAIN and ERICH HECKER Deutsches Krebsforschungszentrum, Institut for Biochemie, Heidelberg ( G.F.R.)
(Received November llth, 1973) (Revised manuscript received April 9th, 1974)
Summary The effect of the t u m o u r p r o m o t o r 12-O-tetradecanoyl-phorbol-13acetate on 32 Pi incorporation into the phosphatidylcholine fraction in mouse epidermis was investigated. 1. Two peaks in incorporation are observed, one at 4--6 h and one at 48 h after treatment. 2. The incorporation [3 H] thymidine into DNA of mouse epidermis is inhibited for about 1 0 h after administration of 12-O-tetradecanoylphorbol-13-acetate. At the same time, however, the incorporation of 32 Pi into epidermal DNA is rapidly stimulated, suggesting that the specific activity of the intracellular ATP pool is increased by the t u m o u r p r o m o t o r soon after its application. The first peak in 3-~Pi incorporation into phosphotidylcholine may therefore be attributed in part to pool changes. 3. After a short lag phase, the accumulation of phosphatidylcholine in mouse epidermis is stimulated. The ratio phosphatidylcholine/DNA in the epidermis reaches a m a x i m u m around 8 h after treatment. 4. Isolated mouse skin pieces pretreated with 12-O-tetradecanoyl-phorbol13-acetate are capable of incorporating 32 Pi into the phosphatidylcholine fraction in a manner which is similar to that observed in vivo. The early accumulation of phosphatidylcholine within the epidermis is therefore due to the localised stimulation of the skin microsomal enzymes responsible for its synthesis. 5. The role played by the early 12-O-tetradecanoyl-phorbol-13-acetateinduced stimulation of phospholipid metabolism in the development of tumours during initiation--promotion experiments in mouse skin is discussed.
458 Introduction
"~
.
12-O-Tetradecanoyl-phorbol-13-acetate, the most active constituent of croton oil [1] is irritant and t u m o u r promoting for mouse skin [2] and causes changes in biochemical and cell kinetic parameters which are characteristic of a direct stimulation of cell proliferation. In vitro, it releases the density-dependent inhibition of growth in fibroblast cell cultures [3]. The controlled and sequential stimulation of the synthesis of phospholipids [4--6], RNA, protein and DNA [7--9], followed by a wave of mitoses [10] has been reported. This particular sequence of biochemical events exhibits striking similarities to that observed using other model systems for the study of growth control [11]. Several lines of evidence have implicated the cell membrane as being of crucial importance in the regulation of cell growth and division [12]. Treatment of cells in culture with hyaluronidase [13], trypsin [141, or other proteolytic enzymes has been shown to induce certain cell-surface alterations which lead to the initiation of DNA synthesis and cell division. Lymphocytes are susceptible to stimulation by phytohemagglutinin, a substance which is reputed to interact with specific cell-surface receptor sites [15]. In spite of the vast a m o u n t of research which has been carried out in this field, the exact mechanism by which changes in the architecture of the cell surface are manifested in the release of growth control remains to be elucidated. There are several indications in the literature that the primary site of action of 12-O-tetradecanoyl-phorbol-13-acetate is the external cell membrane. It has been reported that treatment of cells in culture with very low concentrations of 12-O-tetradecanoyl-phorbol-13-acetate can cause changes in typical membrane properties such as permeability [16] and configuration [17], and activates the plasma membrane enzyme (Na÷--K~)-dependent ATPase [18]. More recently, it was demonstrated that 12-O-tetradecanoyl-phorbol-13-acetate eliminates the normal ~-adrenergic elevation of the cyclic AMP level in mouse epidermis in vivo on injection of isoproterenol [ 19], suggesting that 12-O-tetradecanoyl-phorbol-13-acetate attacks or renders inactive certain receptor sites on the outer surface of the cell membrane. The role played by phospholipids in the amplification of a growth-stimulatory signal has received considerable attention using a number of experimental model systems. These include the stimulation of lymphocytes by phytohemagglutinin [20], treatment of rat uterus with estrogenic hormones [21], the regenerating rat liver [22] and the release of density-dependent inhibition of growth of 3T3 cells by addition of serum [23]. Preliminary investigations have shown that one of the earliest metabolic events induced by 12-O-tetradecanoyl-phorbol-13-acetate is the stimulation of [3 H] choline incorporatior, into phosphatidylcholine [4], suggesting that alterations in phospholipid metabolism could be one of the primary links in the chain of events leading to the stimulation of DNA synthesis. We have now investigated the early effects of 12-O-tetradecanoyl-phorbol13-acetate on 32 P-labelled orthophosphate incorporation into mouse epidermal phosphatidylcholine in vivo and in vitro, with a view to throwing some further light on the earliest metabolic events which occur on the initial stimulation of cell growth and proliferation. Parts of this work have appeared in preliminary reports [5,24].
459 Materials and Methods
Materials 32p-labelled orthophosphate (5--140 Ci/mg Pi) was obtained from the Radiochemical Centre, Amersham, England, and was injected intraperitoneally (500 pCi/20 g mouse). 12-O-Tetradecanoyl-phorbol-13-acetate and 4-O-methylphorbol-12,13-didecanoate were synthesised from phorbol by methods previously described [1,25]. The following were obtained from E. Merck AG, Darmstadt: ready-made Kieselgel plates (0.25 mm), which were used for all chromatographic separations unless otherwise stated; phosphatidylcholine standard, chromatographically pure; chloroform, methanol and acetone (p.a. grade), which were used without further purification. As in standardised Berenblum experiments [1], 7--8-week-old female NMRI mice were used throughout. The animals were maintained in a climatised room with an artificial inversed day--night r h y t h m , with water and food (Altromin GmbH, 4937 Lage Lippe, W. Germany) available ad libitum. The back skins of the mice (about 6 cm 2 ) were shaved 1--2 days prior to the experiment. Any animals showing evidence of wounds or hair regrowth were not used in experiments. Mouse skin pieces were incubated in Eagle's Minimum Essential Medium (Flow Laboratories, Irvine, Scotland) containing 4-fold amino acid and vitamin concentrations and at a final pH of 7.4. Methods Isolation of DNA and lipids from mouse epidermis. The shaved areas on the back skins of the mice were treated either with 0.1 ml acetone, in the case of the controls, or with 0 . l ml of acetone containing 0.02 pmole 12-O-tetradecanoyl-phorbol-13-acetate. Mice were routinely treated at 8.00 a.m. in order to avoid differences due to diurnal r h y t h m , but control experiments showed that Similar results were obtained when the mice were treated at 8.00 p.m. At various times after application groups of four mice were killed by cervical dislocation, the back skins were removed and wetted thoroughly in ice-cold 0.4 M HC104. The skins were pinned dermis side down on the convex surface of a clock glass and the epidermal cells from a defined part of the treated area were removed by scraping gently with a scalpel. The individual epidermal scrapings were then homogenised by hand in ice-cold 0.4 M HC104 in an allglass conical homogeniser. The homogenates were left to stand in ice for 30 min, centrifuged (3000 rev./min, 10 min) and the pellet further washed three times with cold 0.4 M HC104. The lipids were then thoroughly extracted with chloroform-methanol (2 : 1, v/v) at room temperature, the resulting lipid extract being washed according to Folch et al. [26]. Most of the solvent was removed under a stream of nitrogen and the residue containing the desired phospholipids was subjected to chromatographic separation as described below. The pellet remaining after extraction of the phospholipids was used for the determination of the total DNA content by the m e t h o d of Burton [27]. In experiments in which the incorporation of [32 p] orthophosphate into DNA was measured, the RNA in the pellet was first removed by hydrolysis in 0.3 M KOH for 3 h at 37 ° C. The DNA was precipitated by acidification with concen-
460 trated HC104 at 2°C and centrifugation, after which the pellet was washed with 0.4 M HC104 and the DNA content measured. Thin-layer chromatography o f lipids. The lipid extracts were spotted on to Merck ready-made plates which were developed in pre-equilibrated chromatography tanks containing chloroform--methanol--water in the ratio 65 : 25 : 4 {by vol.). The bands were visualised by spraying with iodine vapour and the phophatidylcholine band identified by comparison with an authentic standard run on the same plate. The phosphatidylcholine band was scraped off and used either for phosphate determination or for radioactivity measurement as described below. Since the procedure does not eliminate the possibility that very small amounts of other phospholipids may be present, for the purpose of the discussion this material will be referred to as the phosphatidylcholine fraction. Phosphate determination. The phosphatidylcholine bands obtained from the plates were ashed with 70% HC104 at 200 °C. Thereafter the concentration of inorganic phosphate liberated was estimated by a scaled down modification of the ascorbic acid m e t h o d of Chen et al. [28]. A standard curve was first prepared by carrying known amounts of phosphatidylcholine through the above procedure. The absorbance of the reduced phosphomolybdate complex could then be translated directly into pg phosphatidylcholine for each skin extract. Blank values were obtained using blank areas of the plate which were roughly equivalent to that containing the phosphatidylcholine band. Radioactivity measurement. The radioactivity incorporated into the phosphatidylcholine fraction was measured by scraping the bands from the plate directly into vials containing 10 ml scintillation fluid. The scintillation fluid contained: toluene (700 ml), dioxane (700 ml), ethanol (420 ml), naphthalene (147.6 g), PPO (9.2 g), dimethyl POPOP (0.1 g). Samples were counted in a Nuclear Chicago Mark II Analyser. External standardisation showed that differences in quenching between individual samples were negligible. Incorporation data are therefore presented as cpm in the phosphatidylcholine fraction/pg DNA. [32p]Orthophosphate incorporated into DNA was determined by counting 0.1-ml aliquots of the DNA hydrolysate, obtained by heating the RNA- and lipid-free pellet in 5% trichloroacetic acid in a boiling water bath for 7 min. Incubation o f mouse skin pieces in vitt'o. The back skins of shaved mice were treated with acetone or 12-O-tetradecanoyl-phorbol-13-acetate as described above. All subsequent operations were carried out at 4°C unless otherwise stated. At various time intervals after treatment, groups of four mice were killed by cervical dislocation, the back skins were quickly removed and pinned epidermis side down on cork plates. The subcutaneous tissue was gently scraped off with a scalpel, the skins were cut into small pieces (approx. 1 mm ~ ) with scissors and incubated for 1 h at 37 ° C in 3 ml of Medium A containing the radioactive orthophosphate (20 pCi/ml). The incubation was terminated by addition of ice water, the skins were homogenised and the lipids and DNA isolated as previously described. Results and Discussion Treatment of the dorsal skins of shaved mice with 0.02 pmole 12-O-tetra-
461
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0 Time after treatment
(hours)
Fig. 1. The effect o f 12-O-tetradecanoyl-phorbol-13-acetate (0.02 //moles/application) on the incorporat i o n o f [ 3 2 p ] o r t h o p h o s p h a t e i n t o the mouse epidermal p h o s p h a t i d y l c h o l i n e f r a c t i o n in v~vo. Mice were treated t o p i c a l l y either w i t h 0.02 //moles 12-O-tetradecanoyl-phorbol-13-acetate in 0.1 m l o f acetone or w i t h 0.1 m l a c e t o n e a l o n e . G r o u p s o f f o u r a n i m a l s w e r e killed at t h e t i m e s s h o w n . E a c h m o u s e w a s i n j e c t e d i n t r a p e r i t o n e a U y 1 h p r i o r to d e a t h w i t h 5 0 0 ~tCi [ 3 2 p ] o r t h o p h o s p h a t e . L i p i d s w e r e e x t r a c t e d f r o m t h e d o r s a l e p i d e r m i s of e a c h i n d i v i d u a l m o u s e , t h e p h o s p h a t i d y l c h o l i n e w a s i s o l a t e d a n d t h e r a d i o a c t i v i t y i n c o r p o r a t e d d e t e r m i n e d b y l i q u i d s c i n t i l l a t i o n c o u n t i n g . T h e D N A c o n t e n t of t h e e p i d e r m a l pellet w a s m e a s u r e d as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . T h e r e s u l t s are p r e s e n t e d as c p m in p h o s p h a t i d y l c h o l i n e / / / g D N A . E a c h p o i n t r e p r e s e n t s t h e m e a n v a l u e f r o m f o u r m i c e . S t a n d a r d d e v i a t i o n s are i n d i c a t e d b y t h e v e r t i c a l lines. T h e v a l u e s o b t a i n e d f r o m a c e t o n e - t r e a t e d c o n t r o l m i c e w e r e w i t h i n t h e h a t c h e d area. T h e c u r v e s h o w n w a s o b t a i n e d f r o m o n e e x p e r i m e n t in a series o f f o u r , all o f w h i c h g a v e s i m i l a r r e s u l t s . D o u b l e v a r i a n c e a n a l y s i s o f t h e l o g a r i t h m s of t h e a c t i v i t i e s i n c o r p o r a t e d b y e a c h m o u s e s h o w e d t h a t t h e d i f f e r e n c e s b e t w e e n t r e a t e d a n d c o n t r o l m i c e w e r e h i g h l y s i g n i f i c a n t (P < 0 . 0 0 1 ) , as w e r e t h e d i f f e r e n c e s b e t w e e n i n d i v i d u a l g r o u p s o f t r e a t e d m i c e (P < 0 . 0 1 ) . Single v a r i a n c e a n a l y s i s w a s also c a r r i e d o u t to c o m p a r e t h e m e a n v a l u e s o b t a i n e d at e a c h t i m e p o i n t . T h i s s h o w e d , f o r e x a m p l e , t h a t s i g n i f i c a n t d i f f e r e n c e s e x i s t b e t w e e n t h e p o i n t s at 4 a n d 8 h (P < 0 . 0 1 ) a n d at 6 a n d 8 h (P < 0 . 0 5 ) .
decanoyl-phorbol-13-acetate in 0.1 ml acetone resulted in a biphasic stimulation of the incorporation of [3: p] orthophosphate into the epidermal phosphatidylcholine fraction (Fig. 1). Application of acetone alone or of the nonirritant and non-tumour promoting 4-O-methyl-phorbol-12,13-didecanoate [25] caused no significant stimulation of incorporation, showing that the observed response was due to the active tumour promotor 12-O-tetradecanoylphorbol-13-acetate. The biphasic stimulation of incorporation, with peaks at 4--6 and at 48 h after treatment is temporally similar to that observed by Rohrschneider et al. [ 6 ] , but in our case the 48-h peak is much less prominent. This is probably due to the fact that these authors related the observed incorporation to a specific surface area of skin. We have purposely taken the total amount of DNA as our point of reference, since it is known from histological investigations that the epidermis of a 12-O-tetradecanoyl-phorbol-13-acetatetreated mouse is considerably thicker and contains at least twice as many cells as control skins around 48 h after application [29]. Indeed, measurement of the total DNA content of a specific area of treated epidermis has confirmed the increased DNA content around this time (Balmain, A., and Hecker, E.,
462
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120-
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Time 0fter treatment (hours) Fig. 2. T h e e f f e c t o f 12-O-tetradecanoyl-phorhol-13-aeetate ( 0 . 0 2 # m o l e ) o n t h e i n c o r p o r a t i o n o f [32 p] o r t h o p b o s p h a t e i n t o the acid-soluble f r a c t i o n i n m o u s e epidenmis. T r e a t m e n t o f zmce a n d i n j e c t i o n of [32p] orthophosphate as d e s c r i b e d i n t h e l e g e n d f o r F i g . 1. T h e e p i d e r m i s f r o m e a c h i n d i v i d u a l m o u s e was homogenised thoroughly in ice-cold 0.4 m HC104 (2 ml), the homogenate was centrifuged and the radioactivity in 0.1-ml aliquots of the supernatant was determined. The DNA content of the pellet was m e a s u r e d as d e s c r i b e d i n M e t h o d s . R e s u l t s a r e e x p r e s s e d as c p m / O . l - m l a l i q u o t / p g D N A i n t h e c o r r e s p o n d i n g p e l l e t . E a c h p o i n t is t h e a v e r a g e o b t a i n e d f r o m f o u r m i c e . S t a n d a r d d e v i a t i o n s a r e r e p r e s e n t e d b y t h e v e r t i c a l lines. T h e z e r o - t i m e v a l u e w a s o b t a i n e d u s i n g a c e t o n e - t r e a t e d m i c e .
unpublished). By relating the activity incorporated into the phosphatidylcholine fraction in a specific area of skin to the total a m o u n t of DNA in that area, a more realistic representation is obtained of the degree of stimulation of incorporation per cell. Several explanations for the early stimulation of 32pi incorporation into epidermal phosphatidylcholine on treatment with 12-O-tetradecanoyl-phorbol13-acetate (Fig. 1) can be envisaged: a change in the rate of uptake of phosphate from the extracellular medium, leading to an increase in the specific activity of the intracellular phosphate pool; the stimulation of the de novo phosphatidylcholine synthesis; and increased turnover of the phosphorylcholine moiety. As regards the first of these possibilities, increased cellular uptake of phosphate has been observed in several systems to be one of the early responses to a growth stimulus [23,30]. As a rough measure of the general increase in orthophosphate concentration, the effect of 12-O-tetradecanoyl-phorbol-13acetate on the incorporation of 32 Pi into the acid-soluble fraction of mouse epidermis was determined (Fig. 2). Uptake into the tissue increases rapidly to reach a plateau about 6 h after 12-O-tetradecanoyl-phorbol-13-acetate administration. However, the data presented in Fig. 2 cannot be taken as a true representation of the change in intracellular precursor concentration, since the acid-soluble fraction of the homogenised tissue will have both intracellular and extracellular contributing factors. Estimation of the latter is complicated by the increased blood flow through the skin and the dilation of the intercellular spaces in the epidermis [10] on treatment with the t u m o u r promotor. An indirect measure of the change in intracellular specific activity can be
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time after treatment(hours) Fig. 3. T h e e f f e c t o f 1 2 - O - t e t r a d e e a n o y l - p h o r b o l - 1 3 - a c e t a t e ( 0 . 0 2 # m o l e s ) o n [ 3 2 p ] o r t b o p h o s p h a t e a n d [ 3 H ] t h y m i d i n e i n c o r p o r a t i o n i n t o D N A in m o u s e e p i d e r m i s . T h e m i c e w e r e t r e a t e d a n d killed as des c r i b e d in t h e l e g e n d to Fig. 1. T h e p e l l e t o b t a i n e d a f t e r e x t r a c t i o n o f t h e lipids w a s i n c u b a t e d in 0.3 M K O H (1 m l ) f o r 3 h at 37'~C t o h y d r o l y s e t h e R N A . T h e D N A w a s p r e c i p i t a t e d b y a d d i t i o n o f c o n c e n t r a t e d H C l O 4 ( 0 . 0 6 m l ) at 0 ° C. T h e p e l l e t o b t a i n e d a f t e r c e n t r i f u g a t i o n w a s w a s h e d t w i c e w i t h cold 0.4 M H C I O 4 , t h e D N A w a s h y d r o l y s e d in 5% t r i c h l o r o a c e t i c a c i d a n d its s p e c i f i c a c t i v i t y d e t e r m i n e d as d e s c r i b e d in M e t h o d s . P o i n t s r e p r e s e n t t h e a v e r a g e v a l u e s f r o m f o u r m i c e ± S. D.. R e s u l t s are p r e s e n t e d as p e r c e n t o f t h e c o r r e s p o n d i n g i n c o r p o r a t i o n in a c e t o n e - t r e a t e d c o n t r o l m i c e .
obtained, however, by determination of the extent of 3 ~ Pi incorporation into a macromolecule whose true rate of synthesis has been measured by independent means. The effect of 12-O-tetradecanoyl-phorbol-13-acetate on the rate of 32 Pi and [3 H] thymidine incorporation into the DNA in mouse epidermis is shown in Fig. 3. It can be seen that the a m o u n t of 3 2 Pi in the epidermal DNA is increased with respect to controls as rapidly as 2--4 h after treatment. The large increase in DNA specific activity observed after 12 h is attributable to the true stimulation of the synthesis of this macromolecule, and is also reflected in the [3 H] thymidine incorporation (c.f. refs 7--10). Several previous studies have demonstrated that 12-O-tetradecanoylphorbol-13-acetate causes an initial depression, lasting some 12 h, in the incorporation of [3 H] thymidine into mouse skin DNA [7--10,31]. Direct measurement of the a m o u n t of DNA in mouse epidermis suggests that the synthesis of DNA may be stimulated somewhat earlier than 12 h after application (Baimain, A., and Hecker, E., unpublished) but in any case not sooner than 6--8 h. The fact that the specific activity of DNA labelled by [32 p] orthophosphate increases so rapidly after treatment strongly suggests that the intracellular specific activity of the ATP precursor pool is raised by the t u m o u r promotor. This early effect will also by reflected in the 32 Pi incorporation into epidermal phosphatidylcholine (Fig. 1). The literature abounds with examples of increased transport as a prelude to accelerated growth [ 23,30,32] and it has been
464
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z,, G 8 14 timeofter t r e a t m e n t ( h o u r s )
2X
Fig. 4. T h e a c c u m u l a t i o n o f p h o s p h a t i d y l c h o l i n e in m o u s e e p i d e r m i s a f t e r t r e a t m e n t w i t h 1 2 - O - t e t r a decanoyl-phorbol-13-acetate (0.02 #moles). Groups of 4 12-O-tetradecanoyl-phorbol-13-acetate-treated m i c e w e r e killed a t t h e t i m e s s h o w n , t h e s k i n s w e r e r e m o v e d a n d a s t a n d a r d i s e d c i r c u l a r a r e a ( a b o u t 3 c m 2) o f e p i d e r m i s w a s c a r e f u l l y s c r a p e d o f f f r o m t h e m i d d l e o f t h e t r e a t e d a r e a of e a c h skin. T h e phosphatidylcholine content of each standardised area of epidermis was determined by chromatographic s e p a r a t i o n o f t h e e x t r a c t e d p h o s p h o l i p i d s , f o l l o w e d b y q u a n t i t a t i v e m e a s u r e m e n t of t h e p h o s p h a t e c o n t e n t o f t h e p h o s p h o t i d y l c h o l i n e b a n d . R e s u l t s are p r e s e n t e d as # g p h o s p h a t i d y l c h o l i n e / s t a n d a r d s k i n area. P o i n t s r e p r e s e n t t h e m e a n v a l u e s f r o m f o u r m i c e ± S. D.. Z e r o - t i m e value w a s o b t a i n e d u s i n g a c e t o n e treated controls.
previously suggested that this may be a general phenomenon [33]. One could speculate that t h e rapid influx of specific metabolites immediately after a growth signal is responsible for the activation of enzymes required for macromolecular synthesis. In this respect it is n o t e w o r t h y that phosphate concentration has been shown to be of critical importance in the regulation of certain intracellular processes [ 34,35 ]. In order to determine the time at which a true stimulation of the synthesis of phosphatidylcholine occurs, the accumulation of phosphatidylcholine as a function of time within a specific surface area of treated mouse epidermis was measured (Fig. 4). In contrast to the results of Rohrschneider et al. [6], who observed the most rapid accumulation of phospholipid within the first 2--4 h following treatment, no increase in extractable phosphatidylcholine was seen after 2 h and only a relatively small increase was observed after 4 h. This apparent discrepancy can perhaps be explained by the fact that these authors used mouse skin rather than epidermis alone in their investigations. It can be observed that an appreciable dermal oedema develops within 4 h of treatment with a suitable dose of a t u m o u r promotor, and it could be that the rapid accumulation reported is a reflection of the resultant increase in the concentration of phosphatidylcholine in the dermis. It is noticeable that about 4 h after 12-O-tetradecanoyl-phorbol-13-acetate treatment, the epidermis can be more easily removed from the dermis by scraping with a scalpel. It is necessary therefore to exclude the possibility that the early accumulation of phosphatidylcholine within the epidermis is attributable to the fact that more cells can be scraped from the given area of epidermis. To this end, a separate experiment was carried out in which the ratio of phosphatidylcholine to DNA content in the epidermis of each treated mouse was measured (Fig. 5). Here again it can be seen that the ratio, which can be
465
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hme after treatment(hours) Fig. 5. T h e e f f e c t of 1 2 - O - t e t r a d e c a n o y l - p h o r h o l - 1 3 - a e e t a t e ( 0 . 0 2 # m o l e s ) o n t h e r a t i o p h o s p h a t i d y l choline c o n t e n t / D N A c o n t e n t in m o u s e e p i d e r m i s . T h e p h o s p h a t i d y l c h o l i n e c o n t e n t o f 1 2 - O - t e t r a d e c a n o y l - p h o r b o l - 1 3 - a c e t a t e - t r e a t e d m o u s e e p i d e r m i s was d e t e r m i n e d as d e s c r i b e d in t h e l e g e n d t o Fig. 4. T h e pellet r e m a i n i n g a f t e r lipid e x t r a c t i o n was f u r t h e r used for t h e d e t e r m i n a t i o n of D N A b y t h e B u r t o n m e t h o d . Points: a v e r a g e f r o m f o u r m i c e ± S.D.. Z e r o - t i m e v a l u e f r o m a c e t o n e - t r e a t e d m i c e .
taken as a measure of the phosphatidylcholine content per cell, shows an increase after 4 h, with the maximum value being reached after 8 h. Although the absolute amount of phosphatidylcholine continues to increase for 14--20 h (Fig. 4), the ratio pg phosphatidylcholine/pg DNA levels off at 8 h, owing to the appreciable increase in the DNA content of the epidermis at 12 h. One can envisage at least two possible explanations to account for the measured accumulation of phosphatidylcholine in mouse epidermis after treatment with the t u m o u r promotor: the stimulation of local phosphatidylcholine synthesis via the Kennedy pathway [36] and increased influx of phosphatidylcholine from plasma owing to induced changes in cellular permeablility. In the latter case, phosphatidylcholine synthesised primarily in the liver would be transported to the epidermis, there to accumulate owing to accelerated uptake through the cell membranes. If the former mechanism is operative, one would expect that the activation of the microsomal enzymes responsible for phosphatidylcholine synthesis would be measurable also in vitro. This possibility is verified by the experiment shown in Fig. 6. Here, the skins of mice treated in vivo with 12-O-tetradecanoyl-phorbol-13-acetate were removed at various time intervals and incubated at 37°C for 1 h in medium containing [32 p] orthophosphate. The lipids were extracted and the degree of incorporation into the phosphatidylcholine fraction was determined. Under the conditions of this experiment, any incorporation observed must have taken place within the mouse skin, since any possible contribution from phosphatidylcholine synthesised in the liver has been excluded. The fact that qualitatively similar time--response curves are obtained for incorporation in vivo and in vitro leads to the conclusion that the reaction to 12-O-tetradecanoyl-phorbol-13-acetate treatment in vivo is mainly due to localised metabolic events occurring within the epidermis. The quantitative difference between the two curves is probably due to the lower efficiency of the enzymatic synthesis of phosphatidylcholine under in vitro conditions. A very early increase in 3 2 Pi incorporation into phosphatidylinositol has been reported in lymphocytes stimulated with phytohemagglutinin [20]. The
466
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Fig. 6. E f f e c t of 1 2 - O - t e t r a d e c a n o y l - p h o r b o l - 1 3 - a c e t a t e ( 0 . 0 2 p m o l e s ) o n i n c o r p o r a t i o n of [ 3 2 p ] o r t h o p h o s p h a t e into p h o s p h a t i d y l c h o l i n e in m o u s e skin pieces in vitro. Mice in g r o u p s of f o u r w e r e killed at v a r i o u s t i m e s a f t e r 1 2 - O - t e t r a d e c a n o y l - p h o r b o l - 1 3 - a c e t a t e t r e a t m e n t , t h e skins w e r e r e m o v e d , c u t into small pieces a n d i n c u b a t e d i n d i v i d u a l l y for 1 h at 3 7 ° C in m e d i u m (2 m l ) c o n t a i n i n g [ 3 2 p ] o r t h o p h o s p h a t e ( 2 0 # C i / i n c u b a t i o n ) . I n c u b a t i o n s w e r e t e r m i n a t e d b y a d d i t i o n o f ice w a t e r . T h e skins w e r e t h e n h o m o g e n i s e d , t h e a c t i v i t y i n c o r p o r a t e d i n t o p h o s p h a t i d y l c h o l i n e a n d the D N A c o n t e n t w e r e d e t e r m i n e d as d e s c r i b e d in t h e l e g e n d to Fig. 1. F o r t h e p u r p o s e of d i r e c t c o m p a r i s o n of in vivo a n d in v i t r o results, s o m e o f t h e d a t a p r e s e n t e d in Fig. 1 are r e p r o d u c e d here. L~ -~, i n c o r p o r a t i o n into t h e e p i d e r m a l p h o s p h a t i d y l c h o l i n e f r a c t i o n in vivo; • . . . . . . • , i n c o r p o r a t i o n into the s a m e f r a c t i o n in skin pieces in vitro. Points: a v e r a g e value f r o m f o u r m i c e ± S. D. Z e r o - t i m e values w e r e o b t a i n e d f r o m a c e t o n e - t r e a t e d control mice.
authors attributed this effect to an increase in the rate of turnover of the inositol and phosphate groups. The possibility that an analogous turnover of the phosphorylcholine moiety of phosphatidylcholine occurs in the 12-O-tetradecanoyl-phorbol-13-acetate-stimulated epidermis can not be excluded on the evidence presently available. If this is a contributing factor, however, it is probably only of secondary importance, since most of the activity incorporated can be accounted for by pool changes, together with the stimulation of de novo phosphatidylcholine synthesis. The nature of the primary interaction of 12-O-tetradecanoyl-phorbol-13acetate with the cell membrane is at the m o m e n t completely unknown. One could imagine simply from consideration of the chemical structure of 12-0tetradecanoyl-phorbol-13-acetate, with a hydrophilic " h e a d " grouping coupled to a long hydrophobic "tail", that it could intercalate into the lipid bilayer of the cell membrane, bringing about the changes in membrane properties which are observed experimentally [16--19]. It has been established that many enzymes situated at the cell surface which are involved in the regulation of cell growth require phospholipids in order to be fully active [37]. One could speculate that a disturbance of the lipid moiety of the protein--lipid complex, conceivably the "transducer" of Rodbell [38] which is known to be susceptible to damage by detergents [39], might lead to early alterations of growth control which cultimate in the stimulation of DNA synthesis and cell division.
467 " The'~rocess of t u m o u r promotion involves the repeated treatment of " i n i t i a t e d " skin with a suitable agent, such as 12-O-tetradecanoyl-phorbol-13acetate, eventually leading to the formation of visible tumours. Hennings and Boutwell [40] have postulated that promotion may be further sub-divided into two stages, defined as "conversion", by which an initiated t u m o u r cell is converted into a " d o r m a n t t u m o u r cell", and "propagation", which involves the proliferation of d o r m a n t t u m o u r cells to macroscopic turnouts. In a recent publication, Rohrschneider and Boutwell [41] found that the early peak in 32 Pi incorporation into phosphatidylcholine is specific for tumour-promoting agents, and it was further suggested that this peak is a biochemical manifestation of the "conversion" process. These conclusions were based on the fact that non-tumour promoting hyperplastic agents and complete carcinogens did not elicit this early response in phospholipid metabolism when applied to mouse epidermis. None of these agents, however, stimulates general epidermal proliferation as rapidly or to the same extent as 12-O-tetradecanoyl-phorbol-13acetate. In addition, in the above studies no account was taken of the balance between the proliferative and possible cytotoxic effects of the hyperplastic agents used, a factor which could be of critical importance in the determination of tumour-promoting activity. We therefore think that the above conclusion is premature on the evidence presently available. We favour the interpretation that the early peak in 32 Pi incorporation is related to the pleiotypic response [42] characteristic of a stimulation of cell proliferation, and n o t necessarily to the activation of specific genetic information as would be required of the "conversion" process [40]. The 4-h peak could be considered a manifestation of a general stimulation of membrane metabolism, comprising contributions from increased permeability, de novo phospholipid synthesis and possibly elevated phosphate turnover, as a prelude to an overall acceleration of growth. The subsequent increase in the phosphatidylcholine/DNA ratio in the epidermis may be considered an expression of the proliferation of the endoplasmic reticulum in the stimulated cells, a conclusion which is supported by recent electron-microscopic observations [10]. The studies of Tata [43] have shown that accelerated growth and development is characterised by a coordinated preliferation of intracellular membranes and ribosomes, which accompanies and is necessary for the enhanced protein synthesis in the stimulated cells. References 1 H e c k e r , E. ( 1 9 7 1 ) in M e t h o d s in C a n c e r R e s e a r c h ( B u s c h , H., e d . ) Vol. VI, p p . 4 3 9 - - 4 8 4 , A c a d e m i c Press, N e w Y o r k 2 H e c k e r , E. ( 1 9 6 8 ) C a n c e r Res. 2 8 , 2 3 3 8 - - 2 3 4 9 3 Sivak, A. ( 1 9 7 2 ) J . Cell P h y s i o l . 8 0 , 1 6 7 174 4 K r e i b i c h , G., H e c k e r , E., Stiss, R . a n d Kinzel0 V. ( 1 9 7 1 ) N a t u r w i s s e n s c h a f t e n 5 8 , 3 2 3 5 B a l m a i n , A. a n d H e c k e r , E. ( 1 9 7 2 ) E . A . C . R . S y m p o s i u m o n Cellular M e m b r a n e s a n d H y b r i d i s a t i o n , J a s z o w i e c , P o l a n d , M a y 3 - - 5 , A b s t r . , in t h e p r e s s 6 Rohrschneider, L.R., O'Brien, D.H. and Boutwell, R.K. (1972) Biochim. Biophys. Acta 280, 57--70 7 B a i r d , W.M., S e d g w i c k , J . A . a n d B o u t w e l l , R . K . ( 1 9 7 1 ) C a n c e r Res. 3 1 , 1 4 3 4 - - 1 4 3 9 S P a u l , D. a n d H e c k e r , E. ( 1 9 6 9 ) Z. K r e b s f o r s c h . 7 3 , 1 4 9 - - 1 6 3 9 B r e s c h , H. a n d H e c k e r , E. ( 1 9 6 9 ) P r o c . A m . Ass. C a n c e r Res. 37 1 0 R a i c k , A . N . ( 1 9 7 3 ) C a n c e r Res. 3 3 , 2 6 9 - - 2 8 6
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