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Partial purification of a low molecular weight inhibitor of epidermal DNA synthesis

Partial purification of a low molecular weight inhibitor of epidermal DNA synthesis Departments of Biochemistry and *Dermatologv. The University, Newcastle upon Tyne, England (Received 19 August 1980; revision accepted 25 March 198 1) Abstract. An attempt has been made to purify factors present in aqueous extracts of pig epidermis which inhibit epidermal cell proliferation. A lipophilic factor of low molecular weight (less than lO,O00), has been shown to inhibit DNA synthesis as measured by the uptake of tritiated thymidine in mouse ear epidermis. Purification by alcohol precipitation, ethyl acetate extraction and silicic acid column chromatography produced a fifteen-fold increase in the specific activity of the inhibitory action. It seems likely that aggregation or absorption of this low molecular weight factor may explain the high molecular weight of epidermal cell proliferation inhibitors previously studied, as well as the difficulty in their characterization. Aqueous extracts of skin have an inhibitory effect on epidermal proliferation (Bullough & Laurence, 1964; Iversen el al., 1965; Hondius-Boldingh & Laurence, 1968: Marks, 1976). However, the factors responsible for this inhibition have not been purified or chemically characterized. Marks ( 1975) isolated an endogenous inhibitor of epidermal DNA synthesis which, when partially purified, acted upon the G, stage of the cell cycle and was thought to be a glycopeptide. Isaksson-Forsen et al. (1977) also partially purified an inhibitor from epidermal tissue; this inhibited the G , stage of the cell cycle but was not tested for G, inhibition, and was thought to be a glycoprotein. Neither of these inhibitors were diffusible on dialysis. Our previous attempts to fractionate the inhibitory activity present in crude aqueous extracts of epidermis, by techniques commonly used for the fractionation of macromolecules (e.g. gel filtration and cellulose ion-exchange chromatography) were unsuccessful because inhibitory activity was distributed throughout the fractions (M. C. Dahl & S. Shuster, G. unpublished data). It was therefore considered that the inhibitor might be a small molecule adventitiously attached to or associated with a number of larger molecules. In support of this proposal it was observed that, when crude aqueous extracts of epidermis were suspended in water at low concentrations (< 1 mg/ml), they yielded diffusible inhibitory material upon dialysis. Therefore, to test this hypothesis, we took the diffusible fraction as our starting point, and using this we have been able to extract and partly purify a low molecular weight inhibitor of DNA synthesis, as assessed by the inhibition of uptake of tritiated thymidine by mouse ear epidermis in vitro. t Present address: Department of Microbial Products, PHLS-CAMR, Porton, Salisbury, Wiltshire. Correspondence: Professor S. Shuster, Department of Dtrmatology, The University, Newcastle upon Tyne NE 1 7RU. 0008-8730/82/0100-003 lSO2.00 @ 1982 Blackwell Scientific Publications A . V. Quirk et al. MATERIALS AND METHODS Extraction The crude extracts used for fractionation were prepared from epidermis scraped from pig skin obtained from an abattoir. After rinsing briefly in cold distilled water, the scrapings were soaked in distilled water at 4OC for 1 hr with occasional stirring, and filtered through a Buchner funnel. The filtrate was centrifuged at 10OO g for 30 min at 4OC and the supernatant freeze dried and stored at , O -2OOC. The freeze dried extract amounted to 0.3% of the starting material. Purification All solvents used were of ANALAR reagent grade (BDH, Poole, Dorset, U.K.). Crude lyophilized extract (500 mg) was suspended in water (50 ml) and nine volumes of ethanol (4OC) were added, drop by drop, with stimng. The ethanol soluble fraction was obtained by centrifugation (l0,OOO g, 20 min, 4OC) and the volume reduced to 5 ml by rotary evaporation under vacuum (37OC). The volume of this solution was subsequently adjusted to 15 ml by the addition of distilled water, acidified to pH 2 with 6 M HCl and extracted (x3) with equal volumes of ice-cold ethyl acetate. The ethyl acetate soluble fraction was washed with one half volume of ice cold distilled water, dried by rotary evaporation at 37OC and redissolved in 1 ml chloroform. To separate neutral from polar lipids the chloroform soluble material was applied to a 7 x 1.5 cm column consisting of 5 g of heat activated silicic acid (BDH, Poole, Dorset, U.K.) equilibrated in chloroform (Carroll, 1976). The neutral lipid fraction was eluted by washing with chloroform (50 ml), recovered by rotary evaporation at 30°C and resuspended in 1 ml of hexane. To separate the major neutral lipid classes (Carroll, 1976), the hexane soluble material was applied to a 4 x 1-5 cm column consisting of 4 g of Florisil (Sigma, Poole, Dorset, U.K.) heat-activated for 16 hr at 105OC, and equilibrated in hexane. Fractions were eluted with the following solvent mixtures: hexane (25 ml), hexane-diethyl ether (47.5 :2-5 ml), hexanediethyl ether (25 :25 ml), diethyl ether-methanol (35 :0.75 ml) and diethyl ether-acetic acid (25 :O-2 ml). The fractionation of lipids was assessed using thin-layer chromatography. Aliquots of fractions, dissolved in ethanol, were applied to precoated plates of silica gel G (Camlab, Cambridge, Cambs., U.K.) and developed with petroleum ether (60-80)ethyl acetate-acetic acid (85 :15:1 by volume). Lipids were examined under ultraviolet light after the plates had been sprayed with 40% v/v sulphuric acid and heated for 5 min at 105OC. For preparative thin layer chromatography (TLC), after development, portions of silica gel were scraped off the plate and eluted twice with 2 ml of chloroform :methanol (2 :1 v/v) before resuspending in water. For assay of all fractions, the organic solvent was removed by rotary evaporation at 37OC and the residues were dissolved in 5 ml ethanol. To remove the ethanol, distilled water was added and the sample was reduced to a minimal volume of about 1 ml by rotary evaporation under vacuum at 37OC. More distilled water was added and the procedure repeated. Each fraction was finally suspended in 1 ml water. The refractive index of the suspension was measured before assay, enabling residual ethanol in excess of 2% (v/v) to be detected. In vitro assay This system used the in vifro assay of Dahl & Shuster (1972), as modified by Taylor (1977). The mice were adult male and female hairless (Hr/Hr); they were killed by cervical Epidermal DNA synthesis inhibitor dislocation and both ears were removed and placed in H n ’ balanced salt solution aks containing penicillin G 100 units/ml, streptomycin 100 pg/ml, Nystatin 100 unitdm1 and HEPES 40 mM,pH 7.3. The ears were placed on filter paper and a 8 x 6 mm rectangle was cut from the central portion of the pinna and cut into eight pieces each measuring 8 x 1 mm. Five randomly selected pieces were taken for each culture and placed in a screw-top bottle of 25 ml capacity in 0.9 ml Eagles minimum essential medium w t 2 mM glutamine, antibiotics ih and HEPES, pH 7.3. The test sample or distilled water was subsequently added in 0 - 1 ml volumes. The ear pieces were incubated in a shaking water bath at 35°C for 18 hr which is the peak time for DNA synthesis in this system. For the last hour of incubation tritiated thymidine (20 pCi, 0.1 ml) was added to produce a final concentration of M. The incubation was terminated by the addition of cold saline containing 1 mg/ml sodium azide. The pH of the supernatant was checked after incubation. The ear pieces were then dehydrated using acetic acid-methanol (1: 3 v/v) and two washes of methanol. The dried ear pieces were weighed separately and placed in individual vials for solubilization and liquid scintillation counting. Results were expressed as mean disintegration/min per mg dry weight, since we have shown that epidermal DNA corresponds to dry weight in the mouse ear (Taylor, 1977). The percentage inhibition was calculated by comparison with the control from the mean of five separate assays done in duplicate (reproducibility k 12%, SD) Cytotoxicity Inhibitory fractions were tested for cytotoxicity by incubation with secondary cell cultures of chick embryo fibroblasts, and using concentrations which produced a 50% inhibition of thymidine uptake in the mouse ear. After incubation at 37OC for 5 or 16 hr, cells were mixed with equal volumes of 0.2% w/v aqueous trypan blue (Gorer & O’Gorman, 1956) or incubated with tritiated thymidine (2 pCi) for 1 hr (37OC). The supernatant was removed and the cells washed the cold distilled water, solubilized and suspended in liquid scintillant for counting. RESULTS In initial experiments the crude aqueous extract was suspended in water and dialysed against distilled water at 4°C for 16 hr. Significant inhibitory activity was recovered in the diffusible fraction only if the original concentration of the aqueous extract dialysed was less than 1 mg/ml (Table 1); this lower molecular weight fraction (<lO,OOO) was therefore investigated further. Table 1. Significant inhibitory activity i low n molecular weight fraction (< l0,OOO) was consistently obtained only when the extract dialysed contained less than 1 mg/ml (although some was obtained at 5 mg/ml with this particular extract) Concentration of extract dialy sed (mdml) 13.0 10.0 5.0 1 .o Inhibitory activity in dialysate as % of control A . V. Quirk et al. After dialysis most of the inhibitory activity in the diffusible fraction was found to be soluble in ethanol (90% v/v). Thus, 220 pg/ml of ethanol soluble and insoluble material gave inhibitions of 5 1 and 15%, respectively. Subsequently, therefore, alcoholic precipitation was used as the first step in the purification scheme (Fig. 1). The specific activities and percentage yields of the various active fractions are shown in Table 2. The fraction which was insoluble i n ethyl acetate was readjusted to pH 7-0 with dilute sodium hydroxide (0.1 N)prior to assay. This ethyl acetate-insoluble fraction did not contain inhibitory activity. The chloroform Crude aqueous extract ( I mg/ml) 0I ALY S S I PREClPlTATl ON Ethanol Oiolysable ETHANOL PRECIPITATION . fraction - soluble ETHYL ACETATE EXTRACTION Ethanol -soluble fraction Ethyl ocetote soluble f roction SlLlClC ACID CH R OM ATOG R APHY +I froction pH 2 Chloroform -soluble froction Fig. 1. Purification procedures for the low molecular weight inhibitory material. Table 2. Summary of partial purification of the low molecular weight inhibitory material. The final concentration, giving a 50% inhibition of epidermal DNA synthesis, was obtained from dose-response curves for each extract, there being 6-8 independent extracts for each step. Note: 2a and 2b are alternative, not sequential steps (see Fig. 1) Final concentration (pg/ml) to produce a 50% inhibition 1 Crude extract 2a Dialysable fraction 2b Direct ethanol soluble fraction 3 Ethyl acetate soluble fraction 4 Chloroform soluble fraction % Recovery (w/w) 100 % Yield of total activity 1 .o Epidermal DNA synthesis inhibitor soluble fraction showed almost a fifteen-fold increase in specific activity compared to the crude extract. This represents a 15% yield of activity. On seven separate occasions the major neutral lipid classes were separated, as assessed by TLC, using the Florisil column. However, the inhibitory activity was not consistently confined to one fraction (Table 3). Representatives of the major neutral lipid classes, viz. cholesterol, oleic acid and glycerol trioleate were assayed for inhibitory activity, both individually and in a mixture. These materials when suspended in water were not inhibitory at final concentrations of 40, 500 and 200 ,ug/ml, respectively (Table 4a) which were well above the amounts of chloroform-soluble material required to produce a 50% inhibition of epidermal DNA synthesis. Hence, the inhibitory activity in the chloroform-soluble material resides in a minor neutral lipid or some other lipophilic component as yet uncharacterized. Table 3. Inhibitory activity associated with the major neutral lipid classes separated on a Horisil column. Results are given for the four independent separations in which all fractions were assayed. In three other separations only some of the fractions were assayed but, the results were similar % Inhibition of epidermal DNA synthesis Extract no. 'Florisil fraction F1 Florisil fraction F2 Florisil fraction F3 Florisil fraction F4 Florisil fraction F5 53 87 48 69 +43 Table 4. Data showing (a) lack of inhibition by major neutral lipids; (b) thermostability of inhibitory extracts; (c) absence of diffusion of partially purified inhibitory material from water. The results in (b) and (c) are for equal quantities of approximately 30 pglml (a) Lack of effect of major neutral lipids Neutral lipid Cholesterol Trioleate Oleic acid %Inhibition of DNA synthesis pglrnl (b) Thermostability of chloroform soluble material from ethyl acetate fraction Control % Inhibition of DNA synthesis ~ ~~ ~ ~ ~~ After l0OoC for 2 min 50% 42yo (c) Lack of diffusibility of chloroform soluble inhibitory material Before dialysis % Inhibition of DNA synthesis Dialysable Residue 78% 18% 75% A . V. Quirk el al. The inhibitory activity of the chloroform-soluble material, when suspended in water, was thermostable (lOO°C, 2 min) and did not diffuse upon dialysis (Table 4b,c). This last property indicates the formation of aggregates or micelles when the more purified material is suspended in water. Gel filtration could not be used for purification or to determine the approximate molecular weight of the inhibitory factor and Sephadex G10 or G15 equilibrated in distilled water or 0-1 M phosphate buffer (pH 7-3) adsorbed all the inhibitory activity present in the chloroform-soluble fraction. Inhibitory activity was not recovered after preparative TLC. This may have been due to denaturation of the inhibitory factor, failure to elute the factor from the silica gel or subsequent failure to resuspend the lipophilic factor in water for assay. The major problem of working with lipophilic materials was the transfer from organic solvents to water for assay. However, solvent residues were shown not to be responsible for the inhibitory activity. Solvent mixtures eluted from blank silicic acid and Florisil columns were dried and any residues resuspended in water via ethanol for assay: no activity was found. Residual ethanol was not responsible for inhibitory activity, although 0.1% (v/v) produced a 50% inhibition, since the inhibitory activity of the fractions was non-dialysable and resistant to the enzyme alcohol dehydrogenase (pH 7-3). The inhibitory activity of partially purified material was not due to a direct effect on the tritiated thymidine before it reached the cells; ear pieces were incubated, in the usual way, with inhibitor, but at 17 hr, instead of adding the label, the medium was changed and fresh inhibitor was added to half the samples and 0-1 ml water to the other half. Each sample was then pulsed with tritiated thymidine for 1 hr in the usual way. The results, whether or not the inhibitor was present, showed an equivalent uptake of tritiated thymidine. No evidence of cytotoxicity was indicated, as the uptake of trypan blue was not detected in fibroblast cell cultures incubated with the chloroform-soluble fraction. Similarly, there was no difference in the uptake of tritiated thymidine by fibroblast cells incubated in the presence or absence of active material for 5-22 hr (Table 5). Table 5. Lack of effect of chloroform extract on thymidine uptake by cultured fibroblasts Material Control Extract Control Extract Control Extract Time of incubation (hr) DPM DISCUSSION These experiments demonstrate the existence of a low molecular weight inhibitor of DNA synthesis in aqueous extracts of pig epidermis. Biological activity was assessed by an in uitro ear piece assay, which has the necessary ability to screen a large number of fractions in a Epidermal DNA synthesis inhibitor relatively short time ( 5 days). However, the measurement of DNA synthesis by the uptake of tritiated thymidine is open to certain criticisms (Lord, 1976) and independent confirmation of the findings are now required using biological assays which do not depend upon the uptake of this material. Likewise, further studies are required to confirm the present finding that impaired thymidine uptake was not due to cytotoxicity. The inhibitory effect of aqueous extracts of skin on epidermal proliferation have been known for some time (Bullough & Laurence, 1964) but the active components have not been purified or chemically characterized. A major reason is that when the extracts are subject to macromolecular separation techniques, there is a distribution of inhibitory activity throughout the fractions. One possible explanation is that a number of independent inhibitors of varying molecular weight are involved. This seems unlikely, since if it were the case, after the initial separation step, subsequent purification of each inhibitor should have been possible. Our findings support the alternative possibility that epidermal extracts contain a small molecular weight inhibitor capable of aggregation and dissociation, depending upon its concentration or the conditions or of non-specific absorption to higher molecular weight material. This explains our finding that only when the aqueous extract is suspended in water at low concentrations will an inhibitor diffuse upon dialysis. The recent findings of Gradwohl (1978) also support this conclusion. The inhibitory factor may originate as a separate moiety or it could possibly be an active fragment released by enzymic hydrolysis from a higher molecular weight precursor. However, the non-diffusible or alcohol insoluble fractions did contain some inhibitory activity which indicates either i,ncomplete extraction of the low molecular weight inhibitor or the presence of additional higher molecular weight inhibitors. Loss of some of the total activity (Table 2) at the ethyl acetate extraction stage suggested that the active principle was generally labile or was particularly labile at pH 2. Alternatively the loss may be attributed to the problems of resuspending this fraction in water. The inhibitory activity was partially extracted into ethyl acetate at pH 4.5 but not at pH 7.0.This suggests that the active material may be weakly acidic. After ethyl acetate extraction and drying by rotary evaporation the inhibitory activity was soluble in chloroform, indicating the lipophilic nature of this fraction. The experimental evidence presented here demonstrates the presence of a low molecular weight inhibitor in aqueous epidermal extracts. This inhibitor has been partially purified and shown to be lipophilic and non-diffusible when suspended in the crude aqueous extract at concentrations above 1 mg/ml, when partially purified. These properties allow the proposal that this inhibitory factor may be the same as that previously studied by other workers, and that absorption of a low molecular weight inhibitor to higher molecular weight material and/or its aggregation explains the differences in molecular size previously found, as well as the problems of purification. Neutral lipids were detected in the chloroform-soluble fraction after TLC, but neutral lipids (e.g. oleic acid, cholesterol, etc.) were found not to be inhibitory when incorporated into the in vitro assay system. Although a minor neutral lipid may be the inhibitory factor it is more likely that a lipophilic peptide is the active principle in the chloroform soluble fraction. Thus we have found the chloroform extract to contain a material which reacted with ninhydrin or dansyl chloride only after total acid hydrolysis indicating the presence of a peptide or peptides with substituted N-termini in this fraction. Further purification of the low molecular weight active principal is currently being attempted and the relationship between the diffusible and the non-diffusible fractions may then be determined. A . V. Quirk et al. ACKNOWLEDGMENTS The work was supported by a grant from The Wellcome Trust. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell Proliferation Wiley

Partial purification of a low molecular weight inhibitor of epidermal DNA synthesis

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Publisher
Wiley
Copyright
1982 Blackwell Science Limited
ISSN
0960-7722
eISSN
1365-2184
DOI
10.1111/j.1365-2184.1982.tb01021.x
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Abstract

Departments of Biochemistry and *Dermatologv. The University, Newcastle upon Tyne, England (Received 19 August 1980; revision accepted 25 March 198 1) Abstract. An attempt has been made to purify factors present in aqueous extracts of pig epidermis which inhibit epidermal cell proliferation. A lipophilic factor of low molecular weight (less than lO,O00), has been shown to inhibit DNA synthesis as measured by the uptake of tritiated thymidine in mouse ear epidermis. Purification by alcohol precipitation, ethyl acetate extraction and silicic acid column chromatography produced a fifteen-fold increase in the specific activity of the inhibitory action. It seems likely that aggregation or absorption of this low molecular weight factor may explain the high molecular weight of epidermal cell proliferation inhibitors previously studied, as well as the difficulty in their characterization. Aqueous extracts of skin have an inhibitory effect on epidermal proliferation (Bullough & Laurence, 1964; Iversen el al., 1965; Hondius-Boldingh & Laurence, 1968: Marks, 1976). However, the factors responsible for this inhibition have not been purified or chemically characterized. Marks ( 1975) isolated an endogenous inhibitor of epidermal DNA synthesis which, when partially purified, acted upon the G, stage of the cell cycle and was thought to be a glycopeptide. Isaksson-Forsen et al. (1977) also partially purified an inhibitor from epidermal tissue; this inhibited the G , stage of the cell cycle but was not tested for G, inhibition, and was thought to be a glycoprotein. Neither of these inhibitors were diffusible on dialysis. Our previous attempts to fractionate the inhibitory activity present in crude aqueous extracts of epidermis, by techniques commonly used for the fractionation of macromolecules (e.g. gel filtration and cellulose ion-exchange chromatography) were unsuccessful because inhibitory activity was distributed throughout the fractions (M. C. Dahl & S. Shuster, G. unpublished data). It was therefore considered that the inhibitor might be a small molecule adventitiously attached to or associated with a number of larger molecules. In support of this proposal it was observed that, when crude aqueous extracts of epidermis were suspended in water at low concentrations (< 1 mg/ml), they yielded diffusible inhibitory material upon dialysis. Therefore, to test this hypothesis, we took the diffusible fraction as our starting point, and using this we have been able to extract and partly purify a low molecular weight inhibitor of DNA synthesis, as assessed by the inhibition of uptake of tritiated thymidine by mouse ear epidermis in vitro. t Present address: Department of Microbial Products, PHLS-CAMR, Porton, Salisbury, Wiltshire. Correspondence: Professor S. Shuster, Department of Dtrmatology, The University, Newcastle upon Tyne NE 1 7RU. 0008-8730/82/0100-003 lSO2.00 @ 1982 Blackwell Scientific Publications A . V. Quirk et al. MATERIALS AND METHODS Extraction The crude extracts used for fractionation were prepared from epidermis scraped from pig skin obtained from an abattoir. After rinsing briefly in cold distilled water, the scrapings were soaked in distilled water at 4OC for 1 hr with occasional stirring, and filtered through a Buchner funnel. The filtrate was centrifuged at 10OO g for 30 min at 4OC and the supernatant freeze dried and stored at , O -2OOC. The freeze dried extract amounted to 0.3% of the starting material. Purification All solvents used were of ANALAR reagent grade (BDH, Poole, Dorset, U.K.). Crude lyophilized extract (500 mg) was suspended in water (50 ml) and nine volumes of ethanol (4OC) were added, drop by drop, with stimng. The ethanol soluble fraction was obtained by centrifugation (l0,OOO g, 20 min, 4OC) and the volume reduced to 5 ml by rotary evaporation under vacuum (37OC). The volume of this solution was subsequently adjusted to 15 ml by the addition of distilled water, acidified to pH 2 with 6 M HCl and extracted (x3) with equal volumes of ice-cold ethyl acetate. The ethyl acetate soluble fraction was washed with one half volume of ice cold distilled water, dried by rotary evaporation at 37OC and redissolved in 1 ml chloroform. To separate neutral from polar lipids the chloroform soluble material was applied to a 7 x 1.5 cm column consisting of 5 g of heat activated silicic acid (BDH, Poole, Dorset, U.K.) equilibrated in chloroform (Carroll, 1976). The neutral lipid fraction was eluted by washing with chloroform (50 ml), recovered by rotary evaporation at 30°C and resuspended in 1 ml of hexane. To separate the major neutral lipid classes (Carroll, 1976), the hexane soluble material was applied to a 4 x 1-5 cm column consisting of 4 g of Florisil (Sigma, Poole, Dorset, U.K.) heat-activated for 16 hr at 105OC, and equilibrated in hexane. Fractions were eluted with the following solvent mixtures: hexane (25 ml), hexane-diethyl ether (47.5 :2-5 ml), hexanediethyl ether (25 :25 ml), diethyl ether-methanol (35 :0.75 ml) and diethyl ether-acetic acid (25 :O-2 ml). The fractionation of lipids was assessed using thin-layer chromatography. Aliquots of fractions, dissolved in ethanol, were applied to precoated plates of silica gel G (Camlab, Cambridge, Cambs., U.K.) and developed with petroleum ether (60-80)ethyl acetate-acetic acid (85 :15:1 by volume). Lipids were examined under ultraviolet light after the plates had been sprayed with 40% v/v sulphuric acid and heated for 5 min at 105OC. For preparative thin layer chromatography (TLC), after development, portions of silica gel were scraped off the plate and eluted twice with 2 ml of chloroform :methanol (2 :1 v/v) before resuspending in water. For assay of all fractions, the organic solvent was removed by rotary evaporation at 37OC and the residues were dissolved in 5 ml ethanol. To remove the ethanol, distilled water was added and the sample was reduced to a minimal volume of about 1 ml by rotary evaporation under vacuum at 37OC. More distilled water was added and the procedure repeated. Each fraction was finally suspended in 1 ml water. The refractive index of the suspension was measured before assay, enabling residual ethanol in excess of 2% (v/v) to be detected. In vitro assay This system used the in vifro assay of Dahl & Shuster (1972), as modified by Taylor (1977). The mice were adult male and female hairless (Hr/Hr); they were killed by cervical Epidermal DNA synthesis inhibitor dislocation and both ears were removed and placed in H n ’ balanced salt solution aks containing penicillin G 100 units/ml, streptomycin 100 pg/ml, Nystatin 100 unitdm1 and HEPES 40 mM,pH 7.3. The ears were placed on filter paper and a 8 x 6 mm rectangle was cut from the central portion of the pinna and cut into eight pieces each measuring 8 x 1 mm. Five randomly selected pieces were taken for each culture and placed in a screw-top bottle of 25 ml capacity in 0.9 ml Eagles minimum essential medium w t 2 mM glutamine, antibiotics ih and HEPES, pH 7.3. The test sample or distilled water was subsequently added in 0 - 1 ml volumes. The ear pieces were incubated in a shaking water bath at 35°C for 18 hr which is the peak time for DNA synthesis in this system. For the last hour of incubation tritiated thymidine (20 pCi, 0.1 ml) was added to produce a final concentration of M. The incubation was terminated by the addition of cold saline containing 1 mg/ml sodium azide. The pH of the supernatant was checked after incubation. The ear pieces were then dehydrated using acetic acid-methanol (1: 3 v/v) and two washes of methanol. The dried ear pieces were weighed separately and placed in individual vials for solubilization and liquid scintillation counting. Results were expressed as mean disintegration/min per mg dry weight, since we have shown that epidermal DNA corresponds to dry weight in the mouse ear (Taylor, 1977). The percentage inhibition was calculated by comparison with the control from the mean of five separate assays done in duplicate (reproducibility k 12%, SD) Cytotoxicity Inhibitory fractions were tested for cytotoxicity by incubation with secondary cell cultures of chick embryo fibroblasts, and using concentrations which produced a 50% inhibition of thymidine uptake in the mouse ear. After incubation at 37OC for 5 or 16 hr, cells were mixed with equal volumes of 0.2% w/v aqueous trypan blue (Gorer & O’Gorman, 1956) or incubated with tritiated thymidine (2 pCi) for 1 hr (37OC). The supernatant was removed and the cells washed the cold distilled water, solubilized and suspended in liquid scintillant for counting. RESULTS In initial experiments the crude aqueous extract was suspended in water and dialysed against distilled water at 4°C for 16 hr. Significant inhibitory activity was recovered in the diffusible fraction only if the original concentration of the aqueous extract dialysed was less than 1 mg/ml (Table 1); this lower molecular weight fraction (<lO,OOO) was therefore investigated further. Table 1. Significant inhibitory activity i low n molecular weight fraction (< l0,OOO) was consistently obtained only when the extract dialysed contained less than 1 mg/ml (although some was obtained at 5 mg/ml with this particular extract) Concentration of extract dialy sed (mdml) 13.0 10.0 5.0 1 .o Inhibitory activity in dialysate as % of control A . V. Quirk et al. After dialysis most of the inhibitory activity in the diffusible fraction was found to be soluble in ethanol (90% v/v). Thus, 220 pg/ml of ethanol soluble and insoluble material gave inhibitions of 5 1 and 15%, respectively. Subsequently, therefore, alcoholic precipitation was used as the first step in the purification scheme (Fig. 1). The specific activities and percentage yields of the various active fractions are shown in Table 2. The fraction which was insoluble i n ethyl acetate was readjusted to pH 7-0 with dilute sodium hydroxide (0.1 N)prior to assay. This ethyl acetate-insoluble fraction did not contain inhibitory activity. The chloroform Crude aqueous extract ( I mg/ml) 0I ALY S S I PREClPlTATl ON Ethanol Oiolysable ETHANOL PRECIPITATION . fraction - soluble ETHYL ACETATE EXTRACTION Ethanol -soluble fraction Ethyl ocetote soluble f roction SlLlClC ACID CH R OM ATOG R APHY +I froction pH 2 Chloroform -soluble froction Fig. 1. Purification procedures for the low molecular weight inhibitory material. Table 2. Summary of partial purification of the low molecular weight inhibitory material. The final concentration, giving a 50% inhibition of epidermal DNA synthesis, was obtained from dose-response curves for each extract, there being 6-8 independent extracts for each step. Note: 2a and 2b are alternative, not sequential steps (see Fig. 1) Final concentration (pg/ml) to produce a 50% inhibition 1 Crude extract 2a Dialysable fraction 2b Direct ethanol soluble fraction 3 Ethyl acetate soluble fraction 4 Chloroform soluble fraction % Recovery (w/w) 100 % Yield of total activity 1 .o Epidermal DNA synthesis inhibitor soluble fraction showed almost a fifteen-fold increase in specific activity compared to the crude extract. This represents a 15% yield of activity. On seven separate occasions the major neutral lipid classes were separated, as assessed by TLC, using the Florisil column. However, the inhibitory activity was not consistently confined to one fraction (Table 3). Representatives of the major neutral lipid classes, viz. cholesterol, oleic acid and glycerol trioleate were assayed for inhibitory activity, both individually and in a mixture. These materials when suspended in water were not inhibitory at final concentrations of 40, 500 and 200 ,ug/ml, respectively (Table 4a) which were well above the amounts of chloroform-soluble material required to produce a 50% inhibition of epidermal DNA synthesis. Hence, the inhibitory activity in the chloroform-soluble material resides in a minor neutral lipid or some other lipophilic component as yet uncharacterized. Table 3. Inhibitory activity associated with the major neutral lipid classes separated on a Horisil column. Results are given for the four independent separations in which all fractions were assayed. In three other separations only some of the fractions were assayed but, the results were similar % Inhibition of epidermal DNA synthesis Extract no. 'Florisil fraction F1 Florisil fraction F2 Florisil fraction F3 Florisil fraction F4 Florisil fraction F5 53 87 48 69 +43 Table 4. Data showing (a) lack of inhibition by major neutral lipids; (b) thermostability of inhibitory extracts; (c) absence of diffusion of partially purified inhibitory material from water. The results in (b) and (c) are for equal quantities of approximately 30 pglml (a) Lack of effect of major neutral lipids Neutral lipid Cholesterol Trioleate Oleic acid %Inhibition of DNA synthesis pglrnl (b) Thermostability of chloroform soluble material from ethyl acetate fraction Control % Inhibition of DNA synthesis ~ ~~ ~ ~ ~~ After l0OoC for 2 min 50% 42yo (c) Lack of diffusibility of chloroform soluble inhibitory material Before dialysis % Inhibition of DNA synthesis Dialysable Residue 78% 18% 75% A . V. Quirk el al. The inhibitory activity of the chloroform-soluble material, when suspended in water, was thermostable (lOO°C, 2 min) and did not diffuse upon dialysis (Table 4b,c). This last property indicates the formation of aggregates or micelles when the more purified material is suspended in water. Gel filtration could not be used for purification or to determine the approximate molecular weight of the inhibitory factor and Sephadex G10 or G15 equilibrated in distilled water or 0-1 M phosphate buffer (pH 7-3) adsorbed all the inhibitory activity present in the chloroform-soluble fraction. Inhibitory activity was not recovered after preparative TLC. This may have been due to denaturation of the inhibitory factor, failure to elute the factor from the silica gel or subsequent failure to resuspend the lipophilic factor in water for assay. The major problem of working with lipophilic materials was the transfer from organic solvents to water for assay. However, solvent residues were shown not to be responsible for the inhibitory activity. Solvent mixtures eluted from blank silicic acid and Florisil columns were dried and any residues resuspended in water via ethanol for assay: no activity was found. Residual ethanol was not responsible for inhibitory activity, although 0.1% (v/v) produced a 50% inhibition, since the inhibitory activity of the fractions was non-dialysable and resistant to the enzyme alcohol dehydrogenase (pH 7-3). The inhibitory activity of partially purified material was not due to a direct effect on the tritiated thymidine before it reached the cells; ear pieces were incubated, in the usual way, with inhibitor, but at 17 hr, instead of adding the label, the medium was changed and fresh inhibitor was added to half the samples and 0-1 ml water to the other half. Each sample was then pulsed with tritiated thymidine for 1 hr in the usual way. The results, whether or not the inhibitor was present, showed an equivalent uptake of tritiated thymidine. No evidence of cytotoxicity was indicated, as the uptake of trypan blue was not detected in fibroblast cell cultures incubated with the chloroform-soluble fraction. Similarly, there was no difference in the uptake of tritiated thymidine by fibroblast cells incubated in the presence or absence of active material for 5-22 hr (Table 5). Table 5. Lack of effect of chloroform extract on thymidine uptake by cultured fibroblasts Material Control Extract Control Extract Control Extract Time of incubation (hr) DPM DISCUSSION These experiments demonstrate the existence of a low molecular weight inhibitor of DNA synthesis in aqueous extracts of pig epidermis. Biological activity was assessed by an in uitro ear piece assay, which has the necessary ability to screen a large number of fractions in a Epidermal DNA synthesis inhibitor relatively short time ( 5 days). However, the measurement of DNA synthesis by the uptake of tritiated thymidine is open to certain criticisms (Lord, 1976) and independent confirmation of the findings are now required using biological assays which do not depend upon the uptake of this material. Likewise, further studies are required to confirm the present finding that impaired thymidine uptake was not due to cytotoxicity. The inhibitory effect of aqueous extracts of skin on epidermal proliferation have been known for some time (Bullough & Laurence, 1964) but the active components have not been purified or chemically characterized. A major reason is that when the extracts are subject to macromolecular separation techniques, there is a distribution of inhibitory activity throughout the fractions. One possible explanation is that a number of independent inhibitors of varying molecular weight are involved. This seems unlikely, since if it were the case, after the initial separation step, subsequent purification of each inhibitor should have been possible. Our findings support the alternative possibility that epidermal extracts contain a small molecular weight inhibitor capable of aggregation and dissociation, depending upon its concentration or the conditions or of non-specific absorption to higher molecular weight material. This explains our finding that only when the aqueous extract is suspended in water at low concentrations will an inhibitor diffuse upon dialysis. The recent findings of Gradwohl (1978) also support this conclusion. The inhibitory factor may originate as a separate moiety or it could possibly be an active fragment released by enzymic hydrolysis from a higher molecular weight precursor. However, the non-diffusible or alcohol insoluble fractions did contain some inhibitory activity which indicates either i,ncomplete extraction of the low molecular weight inhibitor or the presence of additional higher molecular weight inhibitors. Loss of some of the total activity (Table 2) at the ethyl acetate extraction stage suggested that the active principle was generally labile or was particularly labile at pH 2. Alternatively the loss may be attributed to the problems of resuspending this fraction in water. The inhibitory activity was partially extracted into ethyl acetate at pH 4.5 but not at pH 7.0.This suggests that the active material may be weakly acidic. After ethyl acetate extraction and drying by rotary evaporation the inhibitory activity was soluble in chloroform, indicating the lipophilic nature of this fraction. The experimental evidence presented here demonstrates the presence of a low molecular weight inhibitor in aqueous epidermal extracts. This inhibitor has been partially purified and shown to be lipophilic and non-diffusible when suspended in the crude aqueous extract at concentrations above 1 mg/ml, when partially purified. These properties allow the proposal that this inhibitory factor may be the same as that previously studied by other workers, and that absorption of a low molecular weight inhibitor to higher molecular weight material and/or its aggregation explains the differences in molecular size previously found, as well as the problems of purification. Neutral lipids were detected in the chloroform-soluble fraction after TLC, but neutral lipids (e.g. oleic acid, cholesterol, etc.) were found not to be inhibitory when incorporated into the in vitro assay system. Although a minor neutral lipid may be the inhibitory factor it is more likely that a lipophilic peptide is the active principle in the chloroform soluble fraction. Thus we have found the chloroform extract to contain a material which reacted with ninhydrin or dansyl chloride only after total acid hydrolysis indicating the presence of a peptide or peptides with substituted N-termini in this fraction. Further purification of the low molecular weight active principal is currently being attempted and the relationship between the diffusible and the non-diffusible fractions may then be determined. A . V. Quirk et al. ACKNOWLEDGMENTS The work was supported by a grant from The Wellcome Trust.

Journal

Cell Proliferation – Wiley

Published: Jan 1, 1982

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