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Epithelial migration in organ culture
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Experimental Cell Research 80 (1973) 281-290
EPITHELIAL Role of Protein
MIGRATION
IN ORGAN CULTURE
Synthesis as Determined
by Metabolic
Inhibitors
J. R. GIBBINS Department
of Pathology,
University
of Sydney, Sydney, Australia
SUMMARY The role of protein synthesis in epithelial migration in the first 24 h after injury was assessedby exposing explants of rat palatal mucosa to the inhibitors puromycin, cycloheximide and Sbromodeoxyuridine (BUdR). Epithelial migration was determined by morphological examination of fixed and sectioned explants and the extent of migration was estimated by counting the number of nuclei that had moved beyond the line of incision. The effects of these inhibitors on epithelial migration and on the relevant biochemical pathways were correlated by the use of dual label radioactive tracer technique. With puromycin and cycloheximide it was found that a significant depression of protein synthesis (greater than 50 % of the control) was required before epithelial migration was completely inhibited. BUdR had no significant effect on the extent of epithelial migration or on protein synthesis at any concentration tested but significantly depressed thymidine incorporation at the higher concentrations of inhibitor (7.5 and 75 ,ug/ml). The results of these experiments are interpreted as indicating that ‘new’ protein synthesis is not required for the initiation of epithelial migration following injury and alternative mechanisms are discussed.
Migration of stratified squamous epithelial cells following injury can be demonstrated readily both in vivo [l] and in vitro [2]. Despite the central importance of this cell movement in the process of wound healing its mechanism is not understood. The traditional view of stratified squamous epithelium proposes that in normal, undamaged adult tissue the cells are individually immobile and that their unrelenting progress from the basal layers towards the surface with eventual desquamation is the result of population pressurefrom the continuously dividing cells in the lower layers. After injury, or some other similar stimulus, these otherwise immobile cells are ‘mobilized’ and begin to migrate [3]. If this view that has grown out of histological and tissue culture studies is correct then there is no good reason to assume that, prior to the injury, the epithelial cells should 19-731804
possess an organised migratory apparatus similar to that found in other actively motile cell types, such as fibroblasts [4] and polymorphs [5]. Accordingly it is logical to propose that in order to convert the static epithelial cells into mobile cells the injury must stimulate the synthesis of a ymigratory apparatus in the epithelial cells; i.e. induce the synthesis of a ‘new’ protein complex. The experiments reported here were designed to investigate the role of protein synthesis in the epithelial migration that occurs in the 24 h after injury, To this end, the effects of the inhibitors of protein synthesis, puromycin and cycloheximide, and of the thymidine analogue 5-bromodeoxyuridine (BUdR) have been studied in an organ culture system. The results obtained do not support the hypothesis that synthesis of a ‘new’ protein is necessaryfor epithelial migration. Exptl Cell Res 80 (1973)
282
J. R. Gibbins
MATERIALS AND METHODS Organ culture technique The basic organ culture technique has been described in detail earlier [2, 61 but in these experiments Eagle Basal Medium (BME) based on Earle balanced salt solution replaced the Medium 199 based on Hanks balanced salt solution used earlier. No essential differences in the behaviour of the cells in these two media were found.
Preparation of inhibitors Puromycin, cycloheximide and BUdR were obtained as dry powders from Sigma Chemical Corp., St Louis, MO. Stock solutions were prepared at a concentration of 1 mg/ml in BME sterilized by Milipore filtration and either used immediately or frozen and stored. The stock solution was diluted with BME containing 20 % horse serum to the final incubating concentrations.
Fixation, processing and examination Techniques used for the morphological aspects of this study have been described earlier [6].
Radioactive tracer experiments Dual label technique: Radioactively labelled tracers, (amino acids and thymidine) of high spec. act. containing either 14C or tritium were obtained from the Radi&hemical Centre, Amersham. The amino acids used were SH-cystine (2 Ci/mmol), ‘*C-cystine (335 mCi/mmol), 3H-glycine (2.2 Ci/mmol), 14C-glytine (108 mCi/mmol), aH-leucine (17 Ci/mmol) and W-leucine (330 mCi/mmol). SH-thymidine (5 Ci/ mmol) and 14C-thymidine (57 mCi/mmol) were the radioactively labelled nucleoside tracers. As far as possible the concentrations of the tracers in each experiment were matched to produce a level during incubation of approx. 1 ,uCi/ml. If they could not be matched, the less energetic isotope (tritium) was at the higher concentration. Explants of rat palatal mucosa were selected at random from the pooled explants obtained from a number of animals and placed on the rafts. The subsequent procedures are summarized below with the elapsed time in hours from the start of the experiment indicated before each procedure.
0. Eauilibrate for 1 h in media without inhibitor; 1, p&se& with tritium labelled tracer in media with&t inhibitor; 2. chase in media with inhibitor plus 100 x concentr&bn of non-radioactive compound; 3, incubate in media with inhibitor; 22, pulse with laClabelled tracer in media with inhibitor; 23, chase in media with inhibitor plus 100 x concentration of nonradioactive compound; 24, terminate experiment. The control rafts were treated identically except that no inhibitor was added to the cultures at any stage. Exptl
Cell Res 80 (1973)
At the end of the second chase, i.e. 24 h after explanting, the rafts were removed and treated according to one of the following regimens. Amino acid uptake into acid-precipitable material: The explants were removed from the rafts, placed in distilled water and freeze-thawed 3 times by immersing in liauid air and thawing at room temperature. The distilled water was then made up to 7 %irichforoacetic acid (TCA) and placed in a water-bath at 90°C for 30 min. At the end of this period the supernatant containing the hot TCA-soluble material was discarded and the explants washed twice in Dulbecco phosphate buffer and transferred to 1 ml of digestion medium containing pronase (0.05 %) collagenase (0.05 %) and trypsin (0.05 %) and incubated overnight at 37°C. The digest was then transferred to liquid scintillation vials. Nucleoside uptake into acid-soluble material: The same procedure as above was followed until the end of the freeze-thaw step. At that point the explants were placed in 0.3 M perchloric acid (PCA) at 4°C and extracted for 1 h. The explants were then washed in dist. water and digested overnight in the enzyme mixture. Next day the digest was precipitated with hot (90°C) 1.6 M PCA, extracted for 1 h and centrifuged. The supernatant containing the extracted material was transferred to liquid scintillation vials. scintillation analysis: Fifteen millilitres of tolu&e-Triton Xl00 sciniillant [7] was added to each of the liauid scintillation vials and the vials were counted in a Philips Liquid Scintillation Analyser in two channels simultaneously. Quench correction was performed automatically by the analyser utilizing the external standard channels ratio with computer derived correction co-efficients that were determined before each experiment with W-hexadecane and 3H-water standards prepared from Amersham standard solutions. Over the entire range of quenching anticipated in the experiments the correction error was less than 5 % and within any given experiment the vials were counted to an error of 1 %. Counts per minute (cpm) were automatically corrected for background and converted to decays per minute (dpm) using the computer-derived correction co-efficients. Liquid
of data: The dpm values of the tritium and 14C obtained from the experiments were used to calculate the ratio: “C/SH (dpm) for the control and each concentration of inhibitor in the experiment. An analysis of variance was performed on the results from each experiment and the results are expressed as means plus or minus the 95 % confidence limits based on the standard error derived from the analysis of variance and tabulated values of t. The ratios obtained at different concentrations of inhibitors were then compared with the ratios obtained in the control culture thus indicating the effect of the inhibitor over 22 h of the experimental period. This experimental procedure allowed a correlation with the morphological studies where the effect of the inhibitor was assessedat the end of a 24-h experimental period. Presentation
Protein synthesis in migrating epithelium 283
Fig. I. The effect of puromycin on epithelial migration in organ culture. (a) Control; (b) 1 pg/ml puromycin; (c) 2 pg/ml puromycin; (d) 3 pg/ml puromycin; (e) 5 pg/ml puromycin. A progressive reduction in extent of epithelial migration has occurred with increasing concentration of puromycin. At 5 pg/ml the cells have failed to migrate. The method used to estimate the extent of epithelial migration in each inhibitor is also indicated. The original line of incision was taken as corresponding to a line extending from the cut-edge of the comified layer and intersecting approximately at right angles with the epithelial-connective tissue interface. The number of nuclei that had moved beyond this point in a number of explants were then counted and used to obtain the results presented in the tables. The heterogeneity in the cellular composition of the connective tissue components of the explants that required the use of the dual label technique to assessthe metabolic effects of the inhibitors is also illustrated. A large nerve is evident in the control (a) and an artery in one of the explants incubated in inhibitor (c) whereas the other explants illustrated are devoid of similar structures. Toluidine blue stain of 0.5 pm sections of glutaraldehyde-fixed, epoxy resin-embedded tissue. Magnification approx. 250 x . Exptl Cell Res 80 (1973)
284
J. R. Gibbins
RESULTS The effect of puromycin and cycloheximide on epithelial migration Puromycin. Incubation of the explants in the presence of puromycin for a period of 24 h resulted in a reduction in the extent of migration that appeared to be concentration-dependent (fig. 1). As the concentration of puromycin was increased from 1 ,ug/ml (fig. lb) to 2 ,ug/ml (fig. 1c) and 3 pug/ml (fig. 1d) the distance that the epithelial cells migrated was obviously reduced progressively when compared with the control (fig. 1a). At 5 pg/ ml (fig. le) no migration was evident. A rough estimation of the extent of migration was obtained by counting the number of nuclei distal to a line drawn from the cut edge of the cornified layer and intersecting approximately at right angles the epithelialconnective tissue interface (black line on fig. 1a-d). As can be seenin table 1 at 1 ,ug/ml an obvious reduction (approx. 30%) had occurred. At higher concentration of the inhibitor the number of nuclei was progressively less until at 5 pg/ml no nuclei were counted. At 5 ,ug/ml most of the cells in the basal and suprabasal layers appeared morphologically intact but some were necrotic. At 7 pg/ml the number of necrotic cells had increased while at 10 pug/mlmost of the basal and suprabasal
Table 1. Estimate of the extent of epithelial migration in puromycin Treatment
No. of nuclei
Total
Mean
Control 1 &ml 2 ,&ml 3 Hz/~ 5 ks/ml 7 ,&ml 10 &ml
64, 36, 83, 59 43, 42, 17, 39 16, 20, 32 21, 16, 13 0, 0, 0, 3 Basal cell necrosis Total necrosis
242 205 68 50 3
60.3 41 22.6 16.6
Expt I Cell Res 80 (1973)
-
Table 2. Estimate of the extent of epithelial migration in cycloheximide Treatment
No. of nuclei
Total
Mean
Control 1 i4ml 5pglml 10 pg/mI 20 yg/ml
73, 16, 54, 69 4, 5, 8, 5, 10, 14, 10, 8 0 0 0
272 64
68 8 -
cells were dead. No migration was evident at 7 or 10 pg/ml. At concentrations of puromycin that prevented migration at 24 h the cells failed to recover if they were transferred to medium without inhibitor for a second 24 h. Cycloheximide. Exposure of explants to cycloheximide for 24 h also resulted in the inhibition of migration but the morphological picture was different from that with puromycin. At 1 pg/ml migration was still evident but much reduced in extent (table 2) when compared with the control. At 5 ,ug/ml no migration had occurred nor was it evident at 10 or 20 ,ug/ml. In contrast to the effect of puromycin however, cellular necrosis in the basal and suprabasal layers was not a feature with cycloheximide. The cells of the basal and suprabasal layers had a surprisingly normal appearance while the cells of the spinous layer above them had the ‘washed out’ appearance that was found in most explants in which migration had not occurred. When explants that had been exposed to inhibiting concentrations of cycloheximide were placed in recovery medium they failed to migrate but still appeared morphologically normal. Effects of puromycin and cycloheximide on amino acid uptake
Amino acid uptake into TCA-precipitable material was assessedby a dual label ratio technique that relies on the ratio of the dpm
Protein synthesis in migrating epithelium 285 When expressed as a percentage of the control, incorporation at 5 pg/ml of inhibitor for both cystine and glycine was nearly the same (70%): i.e. there was an apparent depression below the control of approx. 30~;. Migration was no longer evident (fig. 1e).
T
Cycloheximide. In the case of cycloheximide T
0
000 tI 21
.:
(
3
I
4
c-3 1 6I 5
0 I 7
I
8
I
9
0 ,;,
Fig. 2. Abscissa: puromycin concentration (pg/ml); ordinate: ratio of 14Cdpm to 3H dpm. Effect of puromycin on uptake of radioactive cystine into hot TCA-precipitable material. Mean i95 % confidence interval of the estimate of the mean. Relative to the control, a significant effect on amino acid uptake was not observed at concentrations of puromycin below 5 fig/ml. With increasing concentration, the depression of incorporation relative to the control increased. Signs within circles indicate presence or absence of epithelial migration as determined by morphological examination at the corresponding concentrations of inhibitor.
only one amino acid, glycine, was used. A significant depression in the incorporation ratio was found at all concentrations tested (fig. 4). Expressing the ratios obtained as a percentage of the control, at 1 pug/ml the incorporation ratio was depressed approx. 78 %. At 5 ,ug/ml the depression had increased to 93 % and did not increase significantly at 10 ,ug/ml when the degree of depression was 96%. In the presence of cycloheximide when amino acid incorporation was reduced 78 :‘,
04
obtained from one label in the absence of the inhibitor to the dpm at a second, energetically different label in the presence of the inhibitor.
Puromycin. The effect of puromycin was assessed in separate experiments with the amino acids cystine and glycine. The results of these experiments are llustrated graphically in figs 2 and 3. In both cases the ratio obtained in the presence of the inhibitor was not depressed below the control level at inhibitor concentrations of 1 and 2 pg/ml. In the case of glycine (fig. 3) the ratios were significantly greater at 1 and 2 pg/ml than in the control thus appearing to indicate an accelerated rather than depressed rate of incorporation. At 5 pg/ml the incorporation ratio was reduced significantly below the control in both cystine and glycine and, as the concentration of puromycin was increased, the degree of depression continued to increase.
03
O2 -1 o,
0
0
0
0 Y
0
1
2
5
10
Fig. 3. Abscissa: puromycin concentration (fig/ml): ordinate: ratio of r4C dpm to 3H dpm. Effect of puromycin on uptake of radioactive glytine into hot TCA-precipitable material. Mean + 95 % confidence interval. A similar effect to that illustrated in fig. 2 is apparent with a significant depression of uptake relative to the control at 5 pg/ml puromycin and a corresponding inhibition of migration. At low concentrations of puromycin (1 and 2 pg/ml) a significant increase in the incorporation ratio relative to the control is evident. This apparent increase results from the effect of puromycin which at low concentrations causes premature release of peptidyl-puromycin from polyribosomes. Signs within circles indicate presence or absence of epithelial migration. Exptl Cell Res 80 (1973)
286
J. R. Gibbins
Fig. 4. Abscissa: cycloheximide concentration &g/ml); ordinate: ratio of 14Cdpm to 3H dpm. Effect of cycloheximide on uptake of radioactive glycine into hot TCA-precipitable material. Mean t95 % confidence interval. A highly significant depression of amino acid uptake (approx. 78 % of control ratio) is evident at the lowest concentration tested (1 pg/ml). Signs in circles indicate presence or absence of epithelial migration.
Fig. 5. Abscissa: BUdR (5-bromodeoxyuridine) concentration @g/ml); ordinate: ratio of 14C dpm to 3H dpm. Effect of BUdR on uptake of radioactive leucine into hot TCA-precipitable material. BUdR did not produce a significant alteration in the uptake of radioactive amino acid at any of the concentrations used in these experiments. Epithelial migration occurred at all concentrations.
was still occurring though markedly reduced in amount (table 2).
Effect of BUdR on uptake of radioactive amino acid
migration
Effect of BUdR on epithelial migration When sections obtained from explants exposed to BUdR at concentrations of 1 x 1O-6 M, (0.3 ,ug/ml), 2.8 x 10~~ M (0.7 kg/ ml), 2.8 x 10~~ M (7.5 pug/ml), 1 x’ 1O-4 M (30 pug/ml) and 2.8 x 1O-4 M (75 ,ug/ml) were compared there was no morphological difference between the control and experimental incubations (table 3). The BUdR did not significantly reduce the extent of migration even at the highest concentration tested.
At each of the BUdR concentrations tested for its effect on epithelial migration the uptake of radioactively labelled leucine was determined by the dual label technique. As can be seen in fig. 5 the ratios of incorporation into hot TCA-precipitable material obtained at various concentrations of BUdR did not differ significantly from the control ratio during the incubation period. At the highest concentration of BUdR tested (2.8 x 10-4, 75 pg/ml) there was no significant difference from the control. These results indicate that
Table 3. Estimate of the extent of epithelial migration in BUdR Treatment
Number of nuclei
Total
Mean
S.E.
Control 0.7 pg/ml 7.5 pg/ml 75 ,alml
51, 39, 77, 67, 116, 0, 27, 21 34, 79, 44, 47 64, 56, 63, 19, 47, 41 22, 65, 35, 10, 35, 47, 51, 56, 62, 33
398 204 290 416
49.75 51 48.33 41.6
12.91 9.73 6.92 5.61
Exptl Cell Res 80 (1973)
Protein synthesis in migrating epithelium 281 DISCUSSION Analysis of effects of inhibitors
oi
75
Fig. 6. Abscissa: BUdR (5bromodeoxyuridine) concentration in pg/ml; ordinate: ratio of 14Cdpm to “H dpm. Effect of BUdR on the uptake of radioactive thymidine into the hot PCA-soluble fraction. Mean +95 % confidence interval. A significant denression of the incorporation ratio relative to the control is evident at concentrations of 1.5 and 15 pg/ml BUdR. At 0.1 ,ug/ml there is no significant difference from the control. Epithelial migration occurred at all concentrations of BUdR tested.
the BUdR is not having any effect on total protein synthesis in the organ culture system. Effect of BUdR on uptake of radioactive thymidine The uptake of labelled thymidine into acidextractable material was investigated using the dual label technique at BUdR concentrations of 2.8 x 1O-6(0.7 pg/ml), 2.8 x 1O-5(7.5 pg/ml) and 2.8 x 10u4M (75 ,ug/ml) (fig. 6). At 0.7 ,ug/ml the mean of the observations on the incorporation ratio was less than the mean of the control but the difference was not significant at the 5 % level. At 7.5 ,ug/ml the incorporation ratio was significantly less than the control and it was depressed still further at 75 pg/ml. At these last two concentrations BUdR was demonstrably competing with radioactive thymidine for incorporation into acid extractable material and undoubtedly being incorporated into cellular DNA.
In this study the effects on epithelial migration of three inhibitors was assessedby their effect firstly on cellular migration under the controlled conditions of a simple organ culture system and secondly on the uptake of specific metabolic precursors in same system. An attempt was made to ensure that observations on the two effects of each inhibitor could be correlated as far as possible. This correlation of the effects of the inhibitors in the organ culture systemwas madepossible by the use of a radioactive dual-label technique. Earlier attempts with a single radioactive tracer did not provide reproducible results mainly because there was no sound basis of comparison within or between experiments. Despite care in their preparation there is variation between explants in the amount of connective tissue, heterogeneity in the cellular composition of the connective tissue and variation in the relative numbers of epithelial and connective tissue cells (fig. 1). Clearly if the weight of tissue or the amount of cellular DNA were used as a basis for comparison between explants it would prove to be inadequate. The use of a dual label technique overcametheseproblems in large part. By using the ratio between the uptake of radioactive label in the absence and in the presence of the inhibitor these inherent variations were internally compensated in any one experiment. In addition to reducing systematic error, the dual label technique also allowed the relative effect of the inhibitor to be assessedover the prolonged period of exposure (24 h) that was necessary to correlate the biochemical and the morphological studies.
Exptl Cell Res 80 (1973)
288
J. R. Gibbins
Effects of puromycin and cycloheximide Puromycin and cycloheximide are both inhibitors of protein synthesis at the level of the ribosome but their modes of action at this site are quite different. Puromycin acts as an analogue of aminoacyl-t RNA for which it substitutes on the growing polypeptide chain. Peptidyl-puromycin is thus formed and incomplete peptide chains are released that are precipitable with TCA [8]. At low concentrations of puromycin this mode of action results in an accelerated release of peptidyl-puromycin and an apparent stimulation of protein synthesis as measured by amino acid incorporation into acid-precipitable material [9]. As the concentration of puromycin is increased inhibition of amino acid uptake becomes apparent and the peptides released are shorter and acid soluble. Cycloheximide on the other hand appears to exert its effect byinhibiting the’ formation” of the peptide chain probably by an action on transfer factors [lo] and by ‘freezing’ the growing peptide on the polysome and preventing peptide bond formation [l I]. Its action therefore is to stop the incorporation of amino acids into TCAprecipitable material and to inhibit the release of incomplete peptides from the polysomes. These two different effects on the incorporation of radioactive amino acids into TCAprecipitable material are evident in this study, especially in the case of glycine (figs 3, 4). With puromycin at 1 and 2 lug/ml there is an increase in incorporation relative to the control (fig. 3), whereas in the case of cycloheximide at 1 pg/ml there is a definite depression relative to the control (fig. 4). Despite this apparent stimulation of amino acid uptake that occurs at low puromycin concentration it is reasonable to assume that the peptidyl-puromycin is functionally defective. From the point of view of synthesis of functional protein the real depression produced by Exptl Cell Res 80 (1973)
puromycin at 5 pug/ml is almost certainly greater than the apparent depression of amino acid uptake measured in the experiment. The correlation of the results obtained from both morphology, and analysis of amino acid uptake indicate that a large disruption of protein synthesis is necessary before a cessation of epithelial migration is apparent. With cycloheximide, migration still occurs in the presence of a 78 “/o reduction of amino acid uptake. In both puromycin and cycloheximide the concentrations of inhibitor needed to prevent migration are also toxic to the cells because in both cases the cells failed to recover when the inhibitor was removed. Clearly these results indicate that protein synthesis is not a critical and sensitive step in the initiation of epithelial migration following injury. Effect of BUdR Since both puromycin and cycloheximide inhibit protein synthesis at the level of the ribosome, all the proteins being synthesized in the cell at the time of exposure to inhibitor will be affected. The investigation reported in this paper was directed mainly at the synthesis of ‘new’proteins that may be specificforepithelial migration and in order to study this question of specific protein synthesis more precisely, the effect of BUdR (5-bromodeoxyuridine) was investigated. BUdR is an analogue of thymidine that is incorporated into DNA during replication. It has been shown in a number of systems in vitro that BUdR exerts an effect on the synthesis of specific proteins characteristic of certain differentiated cells without affecting either the ability of these cells to synthesize other proteins required for their maintenance or their ability to divide. This effect of BUdR has now been demonstrated in a wide range of cells; chicken embryo myogenic cells [12, 131, chicken am-
Protein synthesis in migrating epithelium 289 nion cells [14, 151, mouse mammary glands [ 161, chicken embryo yolk sac erythropoietic cells [17] and mouse melanoma cells [18]. It appeared reasonable therefore to apply BUdR to the organ culture system used here to investigate the role of ‘new’ protein synthesis in epithelial migration. At all the concentrations tested BUdR was without effect on either the extent of migration, the morphology of the migrating cells or total protein synthesis over the 24 h period of the experiment. An objection may be raised, however, that over this period an insufficient number of cells will have passed through a mitotic cycle for the BUdR to be incorporated into DNA and to express its effect in the daughter cells. This objection can be met only in part but the following points lend support to the above interpretation. The depression of thymidine incorporation into acidsoluble material indicates that BUdR was being incorporated into the DNA replicated during the experimental period. Those cells that have undergone mitosis during this period would contain BUdR and one would expect that if these cells were affected by the BUdR then the number of cells migrating would have been reduced accordingly. However, when the extent of migration was estimated by counting the number of nuclei that moved beyond the incision point, no significant difference was detected between the control and the experimental incubations (table 3) thus indicating that the BUdR was not affecting epithelial migration.
Role of protein synthesis in epithelial migration The combination of all the results of these experiments appears to indicate that the induction and synthesis of a ‘new’ protein is not an essential requirement for epithelial migration after injury. The facts that epithelial cells can continue to migrate in the face
of a significant reduction in amino acid uptake into protein, and are unaffected by BUdR over a wide range of concentrations during the period in which the stimulus to migrate is being expressed, require that the original hypothesis be rejected and alternatives proposed. The most obvious alternative hypothesis is that the epithelial cells that migrate already possess a metabolic machinery for motility and that the injury merely gives this machinery an opportunity to move the cells in a preferred direction, that is, into the wound. Another hypothesis that would fit the observations is that the components of the migratory apparatus already exist in the cells in some other structural or functional form which may be disassembled into smaller intermediate units and then reassembled into the migratory apparatus following the stimulus of injury. A precedent for this type of disassembly and reassembly of structural and functional components of the cytoplasm in response to changes in the demands on a cell is to be found in the microtubular system [19]. Irrespective of what is the final mechanism of epithelial ‘migration, it is clear that these superficial cells that are so frequently exposed to traumatic insult are adapted to respond rapidly to such insults with minimal alteration to their metabolic machinery at the level of protein synthesis. Expert technical assistance has been provided by Mrs Jennifer Parcel]. The computer programmes used to calibrate the Liquid Scintillation Analyser and to process the data obtained from it were written in Algol in this laboratorv bv Mr E. Smvth. This work was supported by research grants from the National Health and Medical Research Council of Australia and the University of Sydney Cancer Research Fund.
REFERENCES 1. Gibbins, J R, Am j path01 53 (1968) 929. 2. -Pathology 1 (1969) 225. 3. Abercrombie, M, Wound healing (ed C Illingworth) p. 61. Churchill, London (1966). Exptl Cell Res 80 (1973)
290 J. R. Gibbins 4. Hoffman-Berling. H. Biochim bionhvs - . acta 19 (1956) 453. -’ 5. Shibata. N. Tatsumi. N. Tanaka. K. Okamura. Y & Senda, N, Biochim’biophys acta 256 (1972) 565. 6. Gibbins, J R, Exptl cell res 71 (1972) 329. 7. Patterson, M S & Greene, R C, Anal them 37 (1965) 854. 8. Nathans, D, Proc natl acad sci US 51 (1964) 585. 9. Stenesh, J & Shen, P Y, Biochem biophys res corn 37 (1969) 873. 10. Siegel, M R & Sisler, H D, Biochim biophys acta 87 (1964) 83. 11. Felicetti, L, Colombo, B & Baglioni, C, Biochim biophys acta 199 (1966) 120.
Exptl Cell Res 80 (1973)
12. Coleman, J R, Coleman, A W & Hartline, E J H, Dev biol 19 (1969) 537. 13. Bischoff, R & Holtzer, H, J cell bio144 (1970) 134. 14. - Exptl cell res 66 (1971) 224. 15. Mayne, R, Sanger, J W & Holtzer, H, Dev biol 25 (1971) 547. 16. Turkington, R W, Majumder, G C & Riddle, M, J biol them 246 (1971) 1814. 17. Miura, Y & Wilt, F H, J cell biol 48 (1971) 523. 18. Silagi, S & Bruce, S A, Proc natl acad sci US 66 (1970) 72. 19. Tilney, L G, Dev biol, suppl. 2 (1968) 63. Received December 5, 1972
Experimental Cell Research 80 (1973) 281-290
EPITHELIAL Role of Protein
MIGRATION
IN ORGAN CULTURE
Synthesis as Determined
by Metabolic
Inhibitors
J. R. GIBBINS Department
of Pathology,
University
of Sydney, Sydney, Australia
SUMMARY The role of protein synthesis in epithelial migration in the first 24 h after injury was assessedby exposing explants of rat palatal mucosa to the inhibitors puromycin, cycloheximide and Sbromodeoxyuridine (BUdR). Epithelial migration was determined by morphological examination of fixed and sectioned explants and the extent of migration was estimated by counting the number of nuclei that had moved beyond the line of incision. The effects of these inhibitors on epithelial migration and on the relevant biochemical pathways were correlated by the use of dual label radioactive tracer technique. With puromycin and cycloheximide it was found that a significant depression of protein synthesis (greater than 50 % of the control) was required before epithelial migration was completely inhibited. BUdR had no significant effect on the extent of epithelial migration or on protein synthesis at any concentration tested but significantly depressed thymidine incorporation at the higher concentrations of inhibitor (7.5 and 75 ,ug/ml). The results of these experiments are interpreted as indicating that ‘new’ protein synthesis is not required for the initiation of epithelial migration following injury and alternative mechanisms are discussed.
Migration of stratified squamous epithelial cells following injury can be demonstrated readily both in vivo [l] and in vitro [2]. Despite the central importance of this cell movement in the process of wound healing its mechanism is not understood. The traditional view of stratified squamous epithelium proposes that in normal, undamaged adult tissue the cells are individually immobile and that their unrelenting progress from the basal layers towards the surface with eventual desquamation is the result of population pressurefrom the continuously dividing cells in the lower layers. After injury, or some other similar stimulus, these otherwise immobile cells are ‘mobilized’ and begin to migrate [3]. If this view that has grown out of histological and tissue culture studies is correct then there is no good reason to assume that, prior to the injury, the epithelial cells should 19-731804
possess an organised migratory apparatus similar to that found in other actively motile cell types, such as fibroblasts [4] and polymorphs [5]. Accordingly it is logical to propose that in order to convert the static epithelial cells into mobile cells the injury must stimulate the synthesis of a ymigratory apparatus in the epithelial cells; i.e. induce the synthesis of a ‘new’ protein complex. The experiments reported here were designed to investigate the role of protein synthesis in the epithelial migration that occurs in the 24 h after injury, To this end, the effects of the inhibitors of protein synthesis, puromycin and cycloheximide, and of the thymidine analogue 5-bromodeoxyuridine (BUdR) have been studied in an organ culture system. The results obtained do not support the hypothesis that synthesis of a ‘new’ protein is necessaryfor epithelial migration. Exptl Cell Res 80 (1973)
282
J. R. Gibbins
MATERIALS AND METHODS Organ culture technique The basic organ culture technique has been described in detail earlier [2, 61 but in these experiments Eagle Basal Medium (BME) based on Earle balanced salt solution replaced the Medium 199 based on Hanks balanced salt solution used earlier. No essential differences in the behaviour of the cells in these two media were found.
Preparation of inhibitors Puromycin, cycloheximide and BUdR were obtained as dry powders from Sigma Chemical Corp., St Louis, MO. Stock solutions were prepared at a concentration of 1 mg/ml in BME sterilized by Milipore filtration and either used immediately or frozen and stored. The stock solution was diluted with BME containing 20 % horse serum to the final incubating concentrations.
Fixation, processing and examination Techniques used for the morphological aspects of this study have been described earlier [6].
Radioactive tracer experiments Dual label technique: Radioactively labelled tracers, (amino acids and thymidine) of high spec. act. containing either 14C or tritium were obtained from the Radi&hemical Centre, Amersham. The amino acids used were SH-cystine (2 Ci/mmol), ‘*C-cystine (335 mCi/mmol), 3H-glycine (2.2 Ci/mmol), 14C-glytine (108 mCi/mmol), aH-leucine (17 Ci/mmol) and W-leucine (330 mCi/mmol). SH-thymidine (5 Ci/ mmol) and 14C-thymidine (57 mCi/mmol) were the radioactively labelled nucleoside tracers. As far as possible the concentrations of the tracers in each experiment were matched to produce a level during incubation of approx. 1 ,uCi/ml. If they could not be matched, the less energetic isotope (tritium) was at the higher concentration. Explants of rat palatal mucosa were selected at random from the pooled explants obtained from a number of animals and placed on the rafts. The subsequent procedures are summarized below with the elapsed time in hours from the start of the experiment indicated before each procedure.
0. Eauilibrate for 1 h in media without inhibitor; 1, p&se& with tritium labelled tracer in media with&t inhibitor; 2. chase in media with inhibitor plus 100 x concentr&bn of non-radioactive compound; 3, incubate in media with inhibitor; 22, pulse with laClabelled tracer in media with inhibitor; 23, chase in media with inhibitor plus 100 x concentration of nonradioactive compound; 24, terminate experiment. The control rafts were treated identically except that no inhibitor was added to the cultures at any stage. Exptl
Cell Res 80 (1973)
At the end of the second chase, i.e. 24 h after explanting, the rafts were removed and treated according to one of the following regimens. Amino acid uptake into acid-precipitable material: The explants were removed from the rafts, placed in distilled water and freeze-thawed 3 times by immersing in liauid air and thawing at room temperature. The distilled water was then made up to 7 %irichforoacetic acid (TCA) and placed in a water-bath at 90°C for 30 min. At the end of this period the supernatant containing the hot TCA-soluble material was discarded and the explants washed twice in Dulbecco phosphate buffer and transferred to 1 ml of digestion medium containing pronase (0.05 %) collagenase (0.05 %) and trypsin (0.05 %) and incubated overnight at 37°C. The digest was then transferred to liquid scintillation vials. Nucleoside uptake into acid-soluble material: The same procedure as above was followed until the end of the freeze-thaw step. At that point the explants were placed in 0.3 M perchloric acid (PCA) at 4°C and extracted for 1 h. The explants were then washed in dist. water and digested overnight in the enzyme mixture. Next day the digest was precipitated with hot (90°C) 1.6 M PCA, extracted for 1 h and centrifuged. The supernatant containing the extracted material was transferred to liquid scintillation vials. scintillation analysis: Fifteen millilitres of tolu&e-Triton Xl00 sciniillant [7] was added to each of the liauid scintillation vials and the vials were counted in a Philips Liquid Scintillation Analyser in two channels simultaneously. Quench correction was performed automatically by the analyser utilizing the external standard channels ratio with computer derived correction co-efficients that were determined before each experiment with W-hexadecane and 3H-water standards prepared from Amersham standard solutions. Over the entire range of quenching anticipated in the experiments the correction error was less than 5 % and within any given experiment the vials were counted to an error of 1 %. Counts per minute (cpm) were automatically corrected for background and converted to decays per minute (dpm) using the computer-derived correction co-efficients. Liquid
of data: The dpm values of the tritium and 14C obtained from the experiments were used to calculate the ratio: “C/SH (dpm) for the control and each concentration of inhibitor in the experiment. An analysis of variance was performed on the results from each experiment and the results are expressed as means plus or minus the 95 % confidence limits based on the standard error derived from the analysis of variance and tabulated values of t. The ratios obtained at different concentrations of inhibitors were then compared with the ratios obtained in the control culture thus indicating the effect of the inhibitor over 22 h of the experimental period. This experimental procedure allowed a correlation with the morphological studies where the effect of the inhibitor was assessedat the end of a 24-h experimental period. Presentation
Protein synthesis in migrating epithelium 283
Fig. I. The effect of puromycin on epithelial migration in organ culture. (a) Control; (b) 1 pg/ml puromycin; (c) 2 pg/ml puromycin; (d) 3 pg/ml puromycin; (e) 5 pg/ml puromycin. A progressive reduction in extent of epithelial migration has occurred with increasing concentration of puromycin. At 5 pg/ml the cells have failed to migrate. The method used to estimate the extent of epithelial migration in each inhibitor is also indicated. The original line of incision was taken as corresponding to a line extending from the cut-edge of the comified layer and intersecting approximately at right angles with the epithelial-connective tissue interface. The number of nuclei that had moved beyond this point in a number of explants were then counted and used to obtain the results presented in the tables. The heterogeneity in the cellular composition of the connective tissue components of the explants that required the use of the dual label technique to assessthe metabolic effects of the inhibitors is also illustrated. A large nerve is evident in the control (a) and an artery in one of the explants incubated in inhibitor (c) whereas the other explants illustrated are devoid of similar structures. Toluidine blue stain of 0.5 pm sections of glutaraldehyde-fixed, epoxy resin-embedded tissue. Magnification approx. 250 x . Exptl Cell Res 80 (1973)
284
J. R. Gibbins
RESULTS The effect of puromycin and cycloheximide on epithelial migration Puromycin. Incubation of the explants in the presence of puromycin for a period of 24 h resulted in a reduction in the extent of migration that appeared to be concentration-dependent (fig. 1). As the concentration of puromycin was increased from 1 ,ug/ml (fig. lb) to 2 ,ug/ml (fig. 1c) and 3 pug/ml (fig. 1d) the distance that the epithelial cells migrated was obviously reduced progressively when compared with the control (fig. 1a). At 5 pg/ ml (fig. le) no migration was evident. A rough estimation of the extent of migration was obtained by counting the number of nuclei distal to a line drawn from the cut edge of the cornified layer and intersecting approximately at right angles the epithelialconnective tissue interface (black line on fig. 1a-d). As can be seenin table 1 at 1 ,ug/ml an obvious reduction (approx. 30%) had occurred. At higher concentration of the inhibitor the number of nuclei was progressively less until at 5 pg/ml no nuclei were counted. At 5 ,ug/ml most of the cells in the basal and suprabasal layers appeared morphologically intact but some were necrotic. At 7 pg/ml the number of necrotic cells had increased while at 10 pug/mlmost of the basal and suprabasal
Table 1. Estimate of the extent of epithelial migration in puromycin Treatment
No. of nuclei
Total
Mean
Control 1 &ml 2 ,&ml 3 Hz/~ 5 ks/ml 7 ,&ml 10 &ml
64, 36, 83, 59 43, 42, 17, 39 16, 20, 32 21, 16, 13 0, 0, 0, 3 Basal cell necrosis Total necrosis
242 205 68 50 3
60.3 41 22.6 16.6
Expt I Cell Res 80 (1973)
-
Table 2. Estimate of the extent of epithelial migration in cycloheximide Treatment
No. of nuclei
Total
Mean
Control 1 i4ml 5pglml 10 pg/mI 20 yg/ml
73, 16, 54, 69 4, 5, 8, 5, 10, 14, 10, 8 0 0 0
272 64
68 8 -
cells were dead. No migration was evident at 7 or 10 pg/ml. At concentrations of puromycin that prevented migration at 24 h the cells failed to recover if they were transferred to medium without inhibitor for a second 24 h. Cycloheximide. Exposure of explants to cycloheximide for 24 h also resulted in the inhibition of migration but the morphological picture was different from that with puromycin. At 1 pg/ml migration was still evident but much reduced in extent (table 2) when compared with the control. At 5 ,ug/ml no migration had occurred nor was it evident at 10 or 20 ,ug/ml. In contrast to the effect of puromycin however, cellular necrosis in the basal and suprabasal layers was not a feature with cycloheximide. The cells of the basal and suprabasal layers had a surprisingly normal appearance while the cells of the spinous layer above them had the ‘washed out’ appearance that was found in most explants in which migration had not occurred. When explants that had been exposed to inhibiting concentrations of cycloheximide were placed in recovery medium they failed to migrate but still appeared morphologically normal. Effects of puromycin and cycloheximide on amino acid uptake
Amino acid uptake into TCA-precipitable material was assessedby a dual label ratio technique that relies on the ratio of the dpm
Protein synthesis in migrating epithelium 285 When expressed as a percentage of the control, incorporation at 5 pg/ml of inhibitor for both cystine and glycine was nearly the same (70%): i.e. there was an apparent depression below the control of approx. 30~;. Migration was no longer evident (fig. 1e).
T
Cycloheximide. In the case of cycloheximide T
0
000 tI 21
.:
(
3
I
4
c-3 1 6I 5
0 I 7
I
8
I
9
0 ,;,
Fig. 2. Abscissa: puromycin concentration (pg/ml); ordinate: ratio of 14Cdpm to 3H dpm. Effect of puromycin on uptake of radioactive cystine into hot TCA-precipitable material. Mean i95 % confidence interval of the estimate of the mean. Relative to the control, a significant effect on amino acid uptake was not observed at concentrations of puromycin below 5 fig/ml. With increasing concentration, the depression of incorporation relative to the control increased. Signs within circles indicate presence or absence of epithelial migration as determined by morphological examination at the corresponding concentrations of inhibitor.
only one amino acid, glycine, was used. A significant depression in the incorporation ratio was found at all concentrations tested (fig. 4). Expressing the ratios obtained as a percentage of the control, at 1 pug/ml the incorporation ratio was depressed approx. 78 %. At 5 ,ug/ml the depression had increased to 93 % and did not increase significantly at 10 ,ug/ml when the degree of depression was 96%. In the presence of cycloheximide when amino acid incorporation was reduced 78 :‘,
04
obtained from one label in the absence of the inhibitor to the dpm at a second, energetically different label in the presence of the inhibitor.
Puromycin. The effect of puromycin was assessed in separate experiments with the amino acids cystine and glycine. The results of these experiments are llustrated graphically in figs 2 and 3. In both cases the ratio obtained in the presence of the inhibitor was not depressed below the control level at inhibitor concentrations of 1 and 2 pg/ml. In the case of glycine (fig. 3) the ratios were significantly greater at 1 and 2 pg/ml than in the control thus appearing to indicate an accelerated rather than depressed rate of incorporation. At 5 pg/ml the incorporation ratio was reduced significantly below the control in both cystine and glycine and, as the concentration of puromycin was increased, the degree of depression continued to increase.
03
O2 -1 o,
0
0
0
0 Y
0
1
2
5
10
Fig. 3. Abscissa: puromycin concentration (fig/ml): ordinate: ratio of r4C dpm to 3H dpm. Effect of puromycin on uptake of radioactive glytine into hot TCA-precipitable material. Mean + 95 % confidence interval. A similar effect to that illustrated in fig. 2 is apparent with a significant depression of uptake relative to the control at 5 pg/ml puromycin and a corresponding inhibition of migration. At low concentrations of puromycin (1 and 2 pg/ml) a significant increase in the incorporation ratio relative to the control is evident. This apparent increase results from the effect of puromycin which at low concentrations causes premature release of peptidyl-puromycin from polyribosomes. Signs within circles indicate presence or absence of epithelial migration. Exptl Cell Res 80 (1973)
286
J. R. Gibbins
Fig. 4. Abscissa: cycloheximide concentration &g/ml); ordinate: ratio of 14Cdpm to 3H dpm. Effect of cycloheximide on uptake of radioactive glycine into hot TCA-precipitable material. Mean t95 % confidence interval. A highly significant depression of amino acid uptake (approx. 78 % of control ratio) is evident at the lowest concentration tested (1 pg/ml). Signs in circles indicate presence or absence of epithelial migration.
Fig. 5. Abscissa: BUdR (5-bromodeoxyuridine) concentration @g/ml); ordinate: ratio of 14C dpm to 3H dpm. Effect of BUdR on uptake of radioactive leucine into hot TCA-precipitable material. BUdR did not produce a significant alteration in the uptake of radioactive amino acid at any of the concentrations used in these experiments. Epithelial migration occurred at all concentrations.
was still occurring though markedly reduced in amount (table 2).
Effect of BUdR on uptake of radioactive amino acid
migration
Effect of BUdR on epithelial migration When sections obtained from explants exposed to BUdR at concentrations of 1 x 1O-6 M, (0.3 ,ug/ml), 2.8 x 10~~ M (0.7 kg/ ml), 2.8 x 10~~ M (7.5 pug/ml), 1 x’ 1O-4 M (30 pug/ml) and 2.8 x 1O-4 M (75 ,ug/ml) were compared there was no morphological difference between the control and experimental incubations (table 3). The BUdR did not significantly reduce the extent of migration even at the highest concentration tested.
At each of the BUdR concentrations tested for its effect on epithelial migration the uptake of radioactively labelled leucine was determined by the dual label technique. As can be seen in fig. 5 the ratios of incorporation into hot TCA-precipitable material obtained at various concentrations of BUdR did not differ significantly from the control ratio during the incubation period. At the highest concentration of BUdR tested (2.8 x 10-4, 75 pg/ml) there was no significant difference from the control. These results indicate that
Table 3. Estimate of the extent of epithelial migration in BUdR Treatment
Number of nuclei
Total
Mean
S.E.
Control 0.7 pg/ml 7.5 pg/ml 75 ,alml
51, 39, 77, 67, 116, 0, 27, 21 34, 79, 44, 47 64, 56, 63, 19, 47, 41 22, 65, 35, 10, 35, 47, 51, 56, 62, 33
398 204 290 416
49.75 51 48.33 41.6
12.91 9.73 6.92 5.61
Exptl Cell Res 80 (1973)
Protein synthesis in migrating epithelium 281 DISCUSSION Analysis of effects of inhibitors
oi
75
Fig. 6. Abscissa: BUdR (5bromodeoxyuridine) concentration in pg/ml; ordinate: ratio of 14Cdpm to “H dpm. Effect of BUdR on the uptake of radioactive thymidine into the hot PCA-soluble fraction. Mean +95 % confidence interval. A significant denression of the incorporation ratio relative to the control is evident at concentrations of 1.5 and 15 pg/ml BUdR. At 0.1 ,ug/ml there is no significant difference from the control. Epithelial migration occurred at all concentrations of BUdR tested.
the BUdR is not having any effect on total protein synthesis in the organ culture system. Effect of BUdR on uptake of radioactive thymidine The uptake of labelled thymidine into acidextractable material was investigated using the dual label technique at BUdR concentrations of 2.8 x 1O-6(0.7 pg/ml), 2.8 x 1O-5(7.5 pg/ml) and 2.8 x 10u4M (75 ,ug/ml) (fig. 6). At 0.7 ,ug/ml the mean of the observations on the incorporation ratio was less than the mean of the control but the difference was not significant at the 5 % level. At 7.5 ,ug/ml the incorporation ratio was significantly less than the control and it was depressed still further at 75 pg/ml. At these last two concentrations BUdR was demonstrably competing with radioactive thymidine for incorporation into acid extractable material and undoubtedly being incorporated into cellular DNA.
In this study the effects on epithelial migration of three inhibitors was assessedby their effect firstly on cellular migration under the controlled conditions of a simple organ culture system and secondly on the uptake of specific metabolic precursors in same system. An attempt was made to ensure that observations on the two effects of each inhibitor could be correlated as far as possible. This correlation of the effects of the inhibitors in the organ culture systemwas madepossible by the use of a radioactive dual-label technique. Earlier attempts with a single radioactive tracer did not provide reproducible results mainly because there was no sound basis of comparison within or between experiments. Despite care in their preparation there is variation between explants in the amount of connective tissue, heterogeneity in the cellular composition of the connective tissue and variation in the relative numbers of epithelial and connective tissue cells (fig. 1). Clearly if the weight of tissue or the amount of cellular DNA were used as a basis for comparison between explants it would prove to be inadequate. The use of a dual label technique overcametheseproblems in large part. By using the ratio between the uptake of radioactive label in the absence and in the presence of the inhibitor these inherent variations were internally compensated in any one experiment. In addition to reducing systematic error, the dual label technique also allowed the relative effect of the inhibitor to be assessedover the prolonged period of exposure (24 h) that was necessary to correlate the biochemical and the morphological studies.
Exptl Cell Res 80 (1973)
288
J. R. Gibbins
Effects of puromycin and cycloheximide Puromycin and cycloheximide are both inhibitors of protein synthesis at the level of the ribosome but their modes of action at this site are quite different. Puromycin acts as an analogue of aminoacyl-t RNA for which it substitutes on the growing polypeptide chain. Peptidyl-puromycin is thus formed and incomplete peptide chains are released that are precipitable with TCA [8]. At low concentrations of puromycin this mode of action results in an accelerated release of peptidyl-puromycin and an apparent stimulation of protein synthesis as measured by amino acid incorporation into acid-precipitable material [9]. As the concentration of puromycin is increased inhibition of amino acid uptake becomes apparent and the peptides released are shorter and acid soluble. Cycloheximide on the other hand appears to exert its effect byinhibiting the’ formation” of the peptide chain probably by an action on transfer factors [lo] and by ‘freezing’ the growing peptide on the polysome and preventing peptide bond formation [l I]. Its action therefore is to stop the incorporation of amino acids into TCAprecipitable material and to inhibit the release of incomplete peptides from the polysomes. These two different effects on the incorporation of radioactive amino acids into TCAprecipitable material are evident in this study, especially in the case of glycine (figs 3, 4). With puromycin at 1 and 2 lug/ml there is an increase in incorporation relative to the control (fig. 3), whereas in the case of cycloheximide at 1 pg/ml there is a definite depression relative to the control (fig. 4). Despite this apparent stimulation of amino acid uptake that occurs at low puromycin concentration it is reasonable to assume that the peptidyl-puromycin is functionally defective. From the point of view of synthesis of functional protein the real depression produced by Exptl Cell Res 80 (1973)
puromycin at 5 pug/ml is almost certainly greater than the apparent depression of amino acid uptake measured in the experiment. The correlation of the results obtained from both morphology, and analysis of amino acid uptake indicate that a large disruption of protein synthesis is necessary before a cessation of epithelial migration is apparent. With cycloheximide, migration still occurs in the presence of a 78 “/o reduction of amino acid uptake. In both puromycin and cycloheximide the concentrations of inhibitor needed to prevent migration are also toxic to the cells because in both cases the cells failed to recover when the inhibitor was removed. Clearly these results indicate that protein synthesis is not a critical and sensitive step in the initiation of epithelial migration following injury. Effect of BUdR Since both puromycin and cycloheximide inhibit protein synthesis at the level of the ribosome, all the proteins being synthesized in the cell at the time of exposure to inhibitor will be affected. The investigation reported in this paper was directed mainly at the synthesis of ‘new’proteins that may be specificforepithelial migration and in order to study this question of specific protein synthesis more precisely, the effect of BUdR (5-bromodeoxyuridine) was investigated. BUdR is an analogue of thymidine that is incorporated into DNA during replication. It has been shown in a number of systems in vitro that BUdR exerts an effect on the synthesis of specific proteins characteristic of certain differentiated cells without affecting either the ability of these cells to synthesize other proteins required for their maintenance or their ability to divide. This effect of BUdR has now been demonstrated in a wide range of cells; chicken embryo myogenic cells [12, 131, chicken am-
Protein synthesis in migrating epithelium 289 nion cells [14, 151, mouse mammary glands [ 161, chicken embryo yolk sac erythropoietic cells [17] and mouse melanoma cells [18]. It appeared reasonable therefore to apply BUdR to the organ culture system used here to investigate the role of ‘new’ protein synthesis in epithelial migration. At all the concentrations tested BUdR was without effect on either the extent of migration, the morphology of the migrating cells or total protein synthesis over the 24 h period of the experiment. An objection may be raised, however, that over this period an insufficient number of cells will have passed through a mitotic cycle for the BUdR to be incorporated into DNA and to express its effect in the daughter cells. This objection can be met only in part but the following points lend support to the above interpretation. The depression of thymidine incorporation into acidsoluble material indicates that BUdR was being incorporated into the DNA replicated during the experimental period. Those cells that have undergone mitosis during this period would contain BUdR and one would expect that if these cells were affected by the BUdR then the number of cells migrating would have been reduced accordingly. However, when the extent of migration was estimated by counting the number of nuclei that moved beyond the incision point, no significant difference was detected between the control and the experimental incubations (table 3) thus indicating that the BUdR was not affecting epithelial migration.
Role of protein synthesis in epithelial migration The combination of all the results of these experiments appears to indicate that the induction and synthesis of a ‘new’ protein is not an essential requirement for epithelial migration after injury. The facts that epithelial cells can continue to migrate in the face
of a significant reduction in amino acid uptake into protein, and are unaffected by BUdR over a wide range of concentrations during the period in which the stimulus to migrate is being expressed, require that the original hypothesis be rejected and alternatives proposed. The most obvious alternative hypothesis is that the epithelial cells that migrate already possess a metabolic machinery for motility and that the injury merely gives this machinery an opportunity to move the cells in a preferred direction, that is, into the wound. Another hypothesis that would fit the observations is that the components of the migratory apparatus already exist in the cells in some other structural or functional form which may be disassembled into smaller intermediate units and then reassembled into the migratory apparatus following the stimulus of injury. A precedent for this type of disassembly and reassembly of structural and functional components of the cytoplasm in response to changes in the demands on a cell is to be found in the microtubular system [19]. Irrespective of what is the final mechanism of epithelial ‘migration, it is clear that these superficial cells that are so frequently exposed to traumatic insult are adapted to respond rapidly to such insults with minimal alteration to their metabolic machinery at the level of protein synthesis. Expert technical assistance has been provided by Mrs Jennifer Parcel]. The computer programmes used to calibrate the Liquid Scintillation Analyser and to process the data obtained from it were written in Algol in this laboratorv bv Mr E. Smvth. This work was supported by research grants from the National Health and Medical Research Council of Australia and the University of Sydney Cancer Research Fund.
REFERENCES 1. Gibbins, J R, Am j path01 53 (1968) 929. 2. -Pathology 1 (1969) 225. 3. Abercrombie, M, Wound healing (ed C Illingworth) p. 61. Churchill, London (1966). Exptl Cell Res 80 (1973)
290 J. R. Gibbins 4. Hoffman-Berling. H. Biochim bionhvs - . acta 19 (1956) 453. -’ 5. Shibata. N. Tatsumi. N. Tanaka. K. Okamura. Y & Senda, N, Biochim’biophys acta 256 (1972) 565. 6. Gibbins, J R, Exptl cell res 71 (1972) 329. 7. Patterson, M S & Greene, R C, Anal them 37 (1965) 854. 8. Nathans, D, Proc natl acad sci US 51 (1964) 585. 9. Stenesh, J & Shen, P Y, Biochem biophys res corn 37 (1969) 873. 10. Siegel, M R & Sisler, H D, Biochim biophys acta 87 (1964) 83. 11. Felicetti, L, Colombo, B & Baglioni, C, Biochim biophys acta 199 (1966) 120.
Exptl Cell Res 80 (1973)
12. Coleman, J R, Coleman, A W & Hartline, E J H, Dev biol 19 (1969) 537. 13. Bischoff, R & Holtzer, H, J cell bio144 (1970) 134. 14. - Exptl cell res 66 (1971) 224. 15. Mayne, R, Sanger, J W & Holtzer, H, Dev biol 25 (1971) 547. 16. Turkington, R W, Majumder, G C & Riddle, M, J biol them 246 (1971) 1814. 17. Miura, Y & Wilt, F H, J cell biol 48 (1971) 523. 18. Silagi, S & Bruce, S A, Proc natl acad sci US 66 (1970) 72. 19. Tilney, L G, Dev biol, suppl. 2 (1968) 63. Received December 5, 1972