Nonhistone Protein Changes during the

[CANCER RESEARCH 41, 1187-1192, March 1981] 0008-5472/81/0041-0000$02.00

Nonhistone Protein Changes during the Diethylnitrosamine-induced Carcinogenesis of Rat Liver Vicenta Martinez-Sales,

Maria Gabaldon, and Jose Baguena

Centro de InvestigaciÃ³n, Ciudad Sanitaria La Fe. Avenida Campanai. 21. Valencia. 9. Spain

ABSTRACT Diethylnitrosamine (DENA) was administered with drinking water (40 mg/liter) to male Wistar rats for 4, 6, 8, and 10 weeks. The protein:DNA ratio and the ultraviolet light spectral properties of liver chromatin were not modified by DENA treat ment. Nonhistone proteins were separated by sodium dodecyl sulfate:polyacrylamide electrophoresis on slab gels and ana lyzed by densitometry with a scanning microphotometer con nected on line to a computer. There were no qualitative changes in the pattern of nonhistone proteins during the treat ment with DENA. The quantitative changes statistically signifi cant at p < 0.005 were detected only in the 4th and 10th week, increases in fractions with molecular weights of 41,000 to 47,000 and 51,000 to 64,000 and decreases in fractions with molecular weights of 27,000 to 32,000 and 47,000 to 51,000 having been found. The proteinase activity of liver chromatin was assayed in incubation mixtures with 0.2 and 2 M NaCI and the measure ment of the cleavage products was performed with ninhydrin. Proteolytic activity was found only in 0.2 M NaCI and was higher in rats treated for 8 weeks with DENA than in controls. Autolysis of chromatin for 24 hr at 37Â° showed a severe breakdown of the nonhistone protein, being greater in the highmolecular-weight fractions than in the low-molecular-weight fractions. INTRODUCTION Components

of the NHP1 are involved in the regulation of

gene expression, and this justifies the growing interest which has been focused on the alterations of chromosomal proteins in malignant tissues and in tissues undergoing carcinogenesis (1, 26). An increase of the protein:DNA ratio and variations in the NHP content have been found in chromatins isolated from spontaneous primary hepatomas (21) and from slow- as well as fast-growing transplantable Morris hepatomas (2, 10, 25). There are few data concerning the changes in the compositon of proteins in liver chromatin during the chemically induced hepatocarcinogenesis. Sporn and Dingman (24) found varia tions in the protein:DNA ratio of liver chromatin during the feeding of A/-hydroxy-2-acetylaminofluorene and 3'-methyl-4dimethylaminoazobenzene. Gronow and Thackrah described variations in the thiol components of the NHP (15) and changes in the composition of liver chromatin fractions (14) in DENAtreated rats. Tsanev and Hadjiolov (30) made a study of the chromosomal proteins of rat liver during the process of nitrosomorpholine-induced hepatocarcinogenesis. In an attempt to know to what extent the NHP are altered in ' The abbreviations used are: NHP, nonhistone proteins; DENA, diethylnitrosamine; SDS, sodium dodecyl sulfate; PMSF, phenylmethanesulfonyl fluoride. Received July 10, 1980; accepted November 11,1980.

malignant transformation, we have performed a comparative study of the NHP of rat liver chromatin at different steps of the process of DENA-induced carcinogenesis. We have studied the relative amount of several NHP fractions which were sep arated by SDS:polyacrylamide electrophoresis on slab gels and analyzed by a densitometric method in which accuracy was improved by using a computer-controlled recording sys tem. Tsanev and Hadjiolov (30) observed in the course of hepa tocarcinogenesis induced by nitrosomorpholine that the NHP pattern from chromatins isolated in the presence or absence of PMSF was quite different. This phenomenon was found only in carcinogen-fed rats, indicating that the proteolytic activity of chromatin was due to treatment. In order to obtain more data about the effect of the hepatocarcinogens on the proteolytic activity of liver chromatin, we have isolated chromatin from DENA-treated rats in the absence as well as the presence of PMSF and have evaluated the proteinase activity in incubation mixtures with NaCI at both 0.2 and 2 M. We have also per formed autolysis assays of chromatin without any addition of salt in order to compare the results obtained from the electrophoretic profiles with those obtained by chemical procedures. MATERIALS

AND METHODS

Animals. Male Wistar rats were used. Animals that weighed 380 g served as controls. DENA solution was prepared weekly and administered with drinking water (40 mg/liter) for 4, 6, 8, and 10 weeks. Treatment was started when the rats weighed approximately 340 g. The total doses of DENA ingested after 4, 6, 8, and 10 weeks of treatment was 32, 49, 64, and 84 mg, respectively. Under these conditions, the first hepatomas ap peared in about 4 months. Rats were killed by cervical dislo cation. An aliquot of each liver was fixed with formalin for histological examination, and livers were considered only when absence of nodular hyperplasia or differentiated hepatoma was observed. Chemicals. DENA, SDS, PMSF, and acrylamide were pro vided by Merck (Darmstadt, West Germany). DNA type I, bovine serum albumin, Coomassie Brilliant Blue G, ninhydrin, histone type II-AS, A/.N'-methylenebisacrylamide, phosphorylase a, ovalbumin type VI, a-chymotrypsinogen A type II, and cytochrome c type VI were provided by Sigma Chemical Co. (St. Louis, Mo.). Analytical Techniques. Protein was determined by the method of Lowry et al. (18) with bovine serum albumin as standard. DNA was determined by the method of Burton (5) with calf thymus DNA as standard. The UV absorption spectra were performed by the dilution of chromatin in 0.5 mM Tris. Absorbances at 240, 260, and 280 nm were corrected for any minimal light scattering calculated, as described by Bonner ef al. (4).

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Isolation of Chromatin. All procedures were carried out at 2-4Â°. Liver was dropped into 0.25 M sucrose. Liver (2.5 g) was minced and suspended in 25 ml of 1.5% citric acid. The tissue was homogenized in a Potter-Elvehjem glass:Teflon homogenizer (clearance, 0.10 to 0.15 mm) with 10 up-and-down strokes at 500 rpm and 10 up-and-down strokes at 1,500 rpm. The homogenate was filtered through 4 layers of surgical gauze and was next centrifuged at 700 x g for 10 min. The pellet was resuspended in 4 ml of 0.25 M sucrose:1.5% citric acid, then layered over 9 ml of 0.88 M sucrose: 1.5% citric acid, and centrifuged at 1000 x g for 10 min. The pellet was resus pended in 4 ml of 0.25 M sucrose:1% Triton X-100 (v/v):3 row CaCI?, then layered over 9 ml of 0.88 M sucrose:1% Triton X100 (v/v):3 rriM CaCI2, and centrifuged at 1000 x g for 10 min. The morphological purity of the nuclear preparation at this step was checked by phase and electron microscopy. From this point, standard centrifugation conditions of 700 x g for 5 min were used to pellet nuclei during the washing procedures. The pellet nuclei were washed 3 times in 7.5 ml of 0.075 M NaCI:0.024 M EDTA, pH 8, and washed twice in 7.5 ml of 0.1 5 M NaCI:0.1 M Tris, pH 8.2. The resulting sediment was resus pended in 4 ml of 0.5 mw Tris and stirred in ice for 18 hr in order to permit the swelling of the chromatin. Chromatin was sheared gently by forcing the solution through a syringe with a 40/7 needle 5 times. The solution was centrifuged at 1000 x g for 10 min to remove unbroken nuclei and any large aggregates of chromatin, and the transparent supernatant was handled as pure chromatin. Chromatin was also obtained in the presence of PMSF. In this case, PMSF (50 mvi stock solution in isopropyl alcohol) was added at a final concentration of 1 mM to all solutions used in the isolation procedure except those in which citric acid was present. Electrophoresis of Chromosomal Proteins. Chromosomal proteins were analyzed by SDS:polyacrylamide gel electrophoresis using the method of Laemmli (17). Electrophoresis was performed on slab gels on a very simple apparatus designed by Reid and Bielesky (20) and modified by Studier (28). Sep arating gels containing 13.7% acrylamide were prepared from a stock solution of 30% acrylamide and 0.8% A/.W-methylenebisacrylamide. The stacking gel usually contained 6.3% acryl amide and was prepared from the same stock solution. The final concentration of SDS was 0.1% in both gels. The electrode buffer was 0.05 M Tris:0.38 M glycine:0.1% SDS; pH was approximately 8.3. Samples were prepared for electrophoresis by mixing 7 volumes of chromatin with 3 volumes of a solution containing 0.25 M Tris-HCI, pH 8; 5.8% SDS; 14.7% /2-mercaptoethanol (v/v); and 28.6% glycerol (v/v). Proteins were completely dissociated by immersing the samples for 4 min in boiling water. Samples of 20 to 30 mg of proteins were applied to each well. Electrophoresis was carried out at 18 ma/slab gel for 6 hr at 4Â°. At the end of the electrophoresis, the gels were stained overnight with 0.05% Coomassie Brilliant Blue G in 25% isopropyl alcohol:10% acetic acid and destained accord ing to the method of Fairbanks et al. (13). Molecular weight markers used were phosphorylase a (M.W. 94,000), bovine serum albumin (M.W. 68,000), ovalbumin (M.W. 43,500), a-chymotrypsinogen (M.W. 25,700), and cytochrome c (M.W. 11,700). The apparent molecular weights were roughly estimated by assuming a linear relationship be 1188

tween log molecular weight and mobility (23). Densitometric Recording. Gel densitometry was performed on color slides (Kodak Ektachrome) obtained under standard photographic conditions. Slides were measured in the direction of the electrophoretic migration with a scanning microphotometer (Leitz) connected on line to a microcomputer (HP/ 2100) programmed for this purpose. The program was de signed to record the density value of each scanning point and later to reproduce the densitometric profile on the computer display. To facilitate the comparative study, the densitometric profile was divided into 13 groups, always comprising the same peaks, by drawing vertical lines on the computer display. The densitometric value of each group was calculated by the com puter by integration and was expressed as percentage of the densitometric value of the complete profile. The control pattern was obtained from 9 rats, and the pattern of each period of DENA treatment was obtained from 5 rats. For comparison of the means, Student's f test was used, and significances were considered at p < 0.005. Reproducibility of the Gels. In order to know the interval in which the absorption of the bands was proportional to the amount of protein and thus to work in the zone where the relative densitometric values were independent of the protein load in the well, we have run in the same slab gel 6 samples of a given chromatin with protein ranging from 18 to 36 ,ug, and this assay was performed 6 times. The range of protein, in which the densitometry of the stained bands was within the linear portion of the absorbance measurements was 20 to 30 M9To determine the reproducibility of the electrophoretic sys tem, a sample of chromatin with 20 to 30 jug of protein was run on 5 different gels. We found that the reproducibility within the same gel was quite good, and the relative amounts of every fraction showed a S.D. of less than 11%. The reproducibility between different gels was also good, and the S.D. found was less than 13%. Assay of Proteolytic Activity. A modification of the tech nique described by Chong ef al. (11) has been used. The composition of the incubation mixture was as follows: NaCI, 0.2 M; Tris, 10 rnw; histone, 1 mg; chromatin protein, 400 /Â¿g; pH, 8; and final volume, 1 ml. Care must be taken that bacterial contamination does not affect the assay and, when it does, incubations must be done under conditions that prevent bac terial growth. Assays were done in triplicate. After incubation in screw-capped test tubes for 46 hr at 37Â°,2.5 ml of ninhydrin reagent were added to each assay sample. Ninhydrin reagent was: 1.60 g of ninhydrin; 4.96 g of CdCI2-2.5H2O; 80 ml of 95% ethanol; 10 ml of acetic acid; and 20 ml of water. Similar assay mixtures kept at â€”20Â°served as controls. The assay tubes were placed In a boiling-water bath for exactly 5 min, cooled quickly, and centrifuged. Absorbance of the supernatants was measured at 506 nm. One unit of proteinase activity was arbitrarily defined as the amount of the enzyme that causes a change in absorbance at 506 nm of 0.01 above control values after 46 hr of incubation using 10 mm light-path cells. For comparative purposes, we have also performed the assays under the same conditions as described by Chong ef al. (11). In order to check reproducibility of the method, absorbances were also referred to a calibration curve of alanine (0.1 to 0.5 HIM) prepared in 0.2 M NaCI:10 mM Tris; 2.5 ml of ninhydrin reagent were added to 1 ml of the alanine standard, and it was CANCER

RESEARCH

VOL. 41


NHP of Liver in DENA-fed followed as described previously. When changes in absorbance referred to the calibration curve, 1 unit is the amount of enzyme that produces the same absorbance as a 1 mw alanine solution under the specified conditions. Assays were also per formed in the same conditions as described before but with 2 M NaCI. In the inhibition studies, PMSF was applied dissolved either in 0.5 rtiM Tris, p-dioxane, or isopropyl alcohol. The final concentrations in the incubation mixtures were: PMSF, 1 HIM; p-dioxane, 10% (v/v) and isopropyl alcohol, 10% (v/v). Chromatin was preincubated for 30 min at 37Â°with PMSF or the organic solvents before addition of the histone. Chromatin Autolysis. One ml of freshly obtained chromatin (1.2 mg protein/ml, pH 8.1) was incubated without salt for 24 hr at 37Â°. At the end of the incubation, 2.5 ml of ninhydrin reagent were added, and the assay for proteolytic activity was carried out as described before. Another sample incubated under the same conditions was processed for electrophoretic assay as described before. RESULTS Chemical and Physical Properties of Chromatin. The protein:DNA ratio was 2 Â± 0.1 (S.D.); the A24o:A26oand the A28o:A26owere, respectively, 1.51 Â±0.02 and 1.63 Â±0.01. In ''any step of the treatment, DENA did not produce statistically significant changes either in the protein:DNA ratio or in the spectral properties of chromatin. The absorption spectrum of chromatin was in all cases similar to that described previously as a normal pattern (4), and the A32o:A26oratio was less than 0.05. Electrophoretic Pattern. The electrophoresis on SDS:polyacrylamide slab gels resolved the chromosomal proteins into at least 37 bands. The 4 bands of lowest molecular weight, between 12,000 and 20,000, were not considered because they belong partly to the histones. The histone H1 was abnor mally low because of its removal by the citric acid during the isolation of the chromatin (19). For convenience in the com parison of the different fractions, the 33 bands corresponding to the NHP were separated into 13 groups. Chart 1 shows the

Rats

densitometric profile of the NHP from liver chromatin of control rats. The same qualitative pattern as that of the control was obtained for liver chromatin of rats at every step of the treat ment with DENA. The densitometric profiles of NHP from chromatins obtained in the presence and in the absence of PMSF were similar in all the cases studied. Table 1 shows the apparent molecular weights and the relative amounts of the different groups of NHP from controls and from DENA-treated rats. After 4 weeks of DENA treatment, we have observed an increase in Groups 5 and 6 and a decrease in Groups 1 and 7. We have not found statistically significant differences in the relative amounts of the different fractions studied after 6 and 8 weeks of treatment. Changes newly appear by the tenth week and are reflected by increases in Groups 8 and 9 and decreases in Groups 2 and 7. The quantitative changes have been detected only at the beginning and at the end of the treatment with DENA, and in all cases the variations were discrete, comprising between 20 and 45% of the normal pattern. Proteolytic Activity of Chromatin. Table 2 shows the pro teolytic activity of chromatin obtained from livers of control rats and livers of rats treated for 8 weeks with DENA. These results refer to incubation mixtures with 0.2 M NaCI. DENA treatment produces an increase in the proteolytic activity that is statisti cally significant. No proteolytic activity of chromatin has been found either in controls or in DENA-treated rats when the incubation mixture was 2 M NaCI. This absence of activity was

I

2

3

I.

5

6

GROUP

7 8 9

10 II

12 13

NUMBER

Chart 1. Densitometric protiles of the NHP of liver chromatin from conirol rats. Electrophoresis on SDS:polyacrylamide slab gels. Vertical lines separate the 13 groups arbitrarily considered. The direction of migration was from right to left.

Table 1 Molecular weight and relative amounts of NHP in control and DENA-treated rats Liver chromatin obtained from rats receiving DENA with drinking water. NHP separated by SDS:polyacrylamide with a scanning microphotometer connected on line to a computer.

gel electrophoresis

and analyzed by densitometry

Relative amounts of NHP total)DENA (% of

treatmentGroup12345678910111213M.W.27,000-30,00030,000-32,00032,000-37,00037.000-41.00041,000-43,00043,000-47,00047,000-51,00051,000-56,00056,000-64,00064.000-68,00068,000-

(9)a16.20 1.83b5.10 Â± 0.984.01 Â± 0.813.74 Â± 0.533.00 Â± 0.483.09 Â± 0.548.39 Â± 0.814.23 Â± 0.765.23 Â± 0.959.41 Â± 0.759.06 Â± 0.906.61 Â± 0.7121.89 Â± Â±2.464

(5)12.52 wk 1.66C5.23 Â± 0.865.44 Â± 1.224.65 Â± 0.804.30 Â± 0.84C4.53 Â± 0.69C6.65 Â± 0.75Â°5.07 Â± 0.226.58 Â± .008.70 Â±1 0.778.95 Â± 0.847.24 Â± 0.7920.08 Â± Â±1.966

(5)13.35 wk

(5)14.91 wk

0.634.34 Â± 0.674.36 Â± 1.053.74 Â± 0.383.42 Â± 0.583.35 Â± 0.687.13 Â± 0.474.93 Â± 0.276.06 Â± 0.3410.02 Â± 0.349.82 Â± 0.317.34 Â± 0.5722.08 Â± Â±2.078

0.894.25 Â± 0.854.36 Â± 0.394.31 Â± 0.183.82 Â± 0.393.71 Â± 0.147.67 Â± 0.635.15 Â± 0.265.71 Â± 1.088.97 Â± Â±1.179.08 0.637.21 Â± 0.4020.80 Â± Â±2.681

(5)14.11 0 wk 1.503.07 Â± 0.63C3.73 Â± 0.504.38 Â± 0.633.77 Â± 0.793.01 Â± 0.296.32 Â± 0.64e5.74 Â± 0.33Â°7.16 Â± 0.64e9.15 Â± 0.459.17 Â± 0.357.73 Â± 0.5222.59 Â± Â±1.47

Numbers in parentheses, number of animals used for each group. 0 Mean Â±S.D. c p < 0.005 (Student's f test).

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confirmed in a concentration of protein chromatin ranging from 400 to 1200 jug/ml. We have found no proteolytic activity, using the conditions described by Chong ef al. (11), either in controls or in DENAtreated rats. This absence of activity was confirmed in concen trations of protein ranging from 50 to 800 fig/ml. In this respect, we must say that the ninhydrin reagent utilized by us has a sensitivity of more than twice that described by Chong ef al. (11). Complete inhibition of the proteolytic activity is obtained with isopropyl alcohol and p-dioxane at a final concentration of 10% (v/v). When PMSF is applied dissolved in 0.5 rriM Tris, the inhibition is only partial, the proteolytic activity being 45 to 60% that of the normal values. Chromatin Autolysis. Chart 2 shows the densitometric pro files of the NHP of native chromatin and chromatin incubated for 24 hr at 37Â°from control rats and rats treated for 10 weeks with DENA. In both cases, we observe a severe breakdown of the NHP starting from the M.W. 32,000 band, and this seems to be greater as the molecular weight increases. The autolytic process determines the appearance of several polypeptides with molecular weights lower than 10,000 which were evi denced by electrophoresis on 20% acrylamide gels. Compar ing the densitometric profiles of the NHP from control and DENA chromatin after autolysis, we observe in both cases a decrease from Group 2 to 13, being larger in the chromatin from rats treated with DENA. The proteolytic activity of the chromatin after 24 hr of autol ysis was of the same order in control as in DENA-treated rats, Table 2 Proteolytic activity of rat liver chromatin activityTreatmentControlDENA'Unitsa/mg Specific protein135.5

protein0.73

Â±34.0d (7)e

Â±0.17(7) 211.2 Â±36.1 (7)Units'Vmg 1.10 Â±0.18(7)P