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RNA synthesis in the liver of rats treated with 5-fluorouracil
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
RNA Synthesis
501403 (1967)
11%
in the Liver of Rats Treated
with 5-Fluorouracil ROGER Department
WILLEN of Pathology,
UIKNE STENRAM
AND
University
Received
of Uppscda, Sweden
June 29, 1966
The incorporation of cytidine-3H into rat liver RNA was completely or almost completely inhibited by high doses of 5-fluorouracil. Low doses gave high incorporation into heavy RNA molecules. Less activity was observed in the ribosomal RNA peaks. The results indicate a block in the ribosomal RNA synthesis and support the hypothesis of an accumulation of ribosomal RNA precursors in the nucleoli.
Rats treated with high doses of the cancertherapeutic agent FU1 have enlarged liver cell nucleoli with characteristic ultrastructural alterations, and, as shown by radioautography, a heavily depressed RNA synthesis of the liver cell (1). In the present paper the RNA synthesis was also studied by biochemical methods and in variant doses. The results indicated a block in the ribosomal RTU’A synthesis with an accumulation of precursors of ribosomal RNA in the nucleoli. MATERIALS
AND
METHODS
Twenty-foiir male whit,e rats (Sprague-Dawley strain), weighing about 45 gm, were starved for one day, fed on a protein-free diet (2) for 4 days, and then starved overnight. On the following day at 8:0%9:00 XVI, at which they weighed about 36 gm, they were given a subcutaneous injection of 50, 20, 5, 0.75, or 0.10 mg FU (generously supplied by Hoffman-La Roche, Switzerland) or 1 ml of 0.9% NaCl. Exactly one hour later they were given an intraperitoneal injection of 10 PC cytidine-3H per gram body weight (Schwara BioResearch, U.S.A.), and killed 20 minutes or 3 hours later. Small pieces of the livers were taken for radioautographic examination as described by Stenram and Willen (3). The remainder was frozen on dry ice for RNA preparation and stored at -30”. Liver tissue from two aniRNA preparation. mals treated in the same way, in all about 0.8 gm, 1 Abbreviations used: FU, 5-fluorouracil; RNA, ribonucleic acid; SDS, sodium dodecyl sulfate.
was homogenized in a microhomogenizer (Type Nelco. Measuring and Scientific Equipment Ltd., London, England) at 0” for 2 minutes in 5 ml 0.01 M acetate buffer, pH 5.6, containing low3 M MgCl,, lye bentonite [prepared according to FraenkelConrat et ~2. (4)] and 0.015 M naphtalene-1,5disulfonate. To the homogenate was added an equal volume of 90% (w/v) redistilled, watersaturated phenol containing 0.5% SDS and 0.1% 3hydroxyquinoline. The mixture was vigorously shaken for 20 minutes in a water-bath at 40”. After centrifugation at 30009 for 20 minutes, as much as possible of the aqueous and phenol layers was carefully withdrawn. The remainder was brought to the original volume by the addition of the abovementioned phenol solution, and the above-mentioned buffer system, supplemented with 0.5% SDS and 0.1% %hydroxyquinoline, and again shaken for 20 minutes at 40”, and centrifuged. A third extraction was performed at 60” for 15 minutes. The three aqueous layers were combined and three parts of 96yG alcohol were added. Having been kept overnight (about 15 hours) at -15”, the precipitate was dissolved in 2 ml of 0.01 M acetate buffer, pH 5.0, with 0.1 M NaCI, 10m3M MgCb, and 0.1% bentonite to a concentration of about 20 OD units/ml at 2.600& and dialyzed during magnetic stirring against 100 ml of the same buffer-salt system for 24 hours at 4” with four changes of the buffer. Ultracentrifugation and analysis. Ribonucleic acid solutions of about 0.8 ml were layered onto 24 ml of 5-207& (w/v) sucrose density gradients, prepared according to a modification of the methods of Bock and Nan-Sing (5) and Britten and Roberts (6)) and containing 0.0251 potassium phos501
502
WILLaN
AND
phate buffer, pH 7.0. After centrifugation in an SW 25 swinging bucket rotor, Spinco model L centrifuge, for 12 hours at 24,000 rpm (83,OOOg), the liquid was removed by a peristaltic pump from the bottom of the tube through a hypodermic needle carefuloy inserted from above. The absorption at 2537 A was recorded continuously by an LKB UVICORD 4701A, and the liquid was automatically fractioned into 32-drop samples (about 1 ml). The radioactivity of the samples was determined in duplicate in a dioxan-PPO-PoPoPnaphthalene system in a tritium scintillator counter, type 6012A (Isotope Developments Ltd., Reading, Berkshire, England); the mean of the two values is given in the figures. RESULTS
AND
DISCUSSION
The ultraviolet absorption curves were similar for all groups, and only one was therefore recorded in each figure. The peak in the 4-65 region was as a rule more evident in the FU-treated rats. A small shoulder was regularly found in t,he treated animals in the heavy region (Fig. 1). The RNA synthesis was completely or
STENRAM
almost completely inhibited by 50, 20, and 5 mg FU. There might, however, have been a slight labelling after cytidine-3H administration in the heavy and, at 3 hours, also in the 4-65 regions. Low doses of FU (0.75 and 0.10 mg) gave high activity in these regions. Less activity was observed in the ribosomal RKA peaks. As the ribosomal RNA precursors, elaborated in the nucleoli, are found at 35s and 455 (7-9), the results might indicate a block in their transformation to ribosomal RNA. It should be pointed out, however, that labelling in the heavy region can also be due to non-nucleolar, nuclear RNA of messenger type, but this labelling is more polydisperse than that obtained here. In view of the previously demonstrated enlargement of liver nucleoli with preservation of their intense basophilia during treatment with high doses of FU (I), the present results argue for an accumulation of ribosomal RNA precursors in the nucleoli during this condition.
0.6 00
18s
a FIG. 1. Sedimentation patterns of rat liver RNA. (a) Rats labelled for 20 minutes; (b) rats labelled for 3 hours with cytidine-3H. Faster moving components are at the left. Twenty and 5 mg FU gave the same results as 50 mg and are not reported in the figure.
5FLUOROURACIL
AND RAT LIVER
The radioautographs of the livers from the rats given 50, 20, or 5 mg FU showed low labelling, after cytidine-3H administration, over the whole cell. The rats given 0.75 mg FU showed heavy labelling over the nuclei, especially the nucleoli, and, at 3 hours, slight labelling over the cytoplasm. The rats given 0.10 mg FU showed slightly more, and the controls showed considerably more cytoplasmic labelling at 3 hours. The radioautographic results thus substantiate the view that at a suitable dose of FU, 0.75 mg, the nuclear, probably nucleolar precursors of ribosomal RNA are labelled but not transformed to cytoplasmic ribosomal RKA. ACKNOWLEDGMENTS The investigation was supported by grants from the Swedish Medical Research Council (12X-
RNB
50X
623-02), the Swedish Cancer Societ,y (65:85), and the Medical Faculty, Uppsala. REFERENCES 1.
STENRIM,
2. STENRIM,
3.
U., 2. Zellforsch. li.,
U., 7G5 (1966).
STENRAM,
dcta
Anat.
AND WILL-AN,
71, 207 (19G6). 26, 350 (195F). It., Cancer Res. 26,
H., SINGER, B., AND TSUGIT.~, A., Virology 14, 54 (1961). 5. BOCK, It. M., AND %X-SING, L., dnal. Chem. 10, 1543 (1954). 6. BRITTEN, H. J., AND ROBERTS, R. B., Science 131, 32 (1960). 7. Pssm~, S., J. Mol. ljiol. 17, 117 (1966). 8. PERRY, It. P., Monographs iTall. Cancer Inst. 23, (1966) (in press). 9. MUKAMA~TSU, M., IIODSSTT, J., AND BUSCH, H., J. Riol. Chew. 241, 1544 (1966).
4. FRLENKEL-COSR.\T,
OF
BIOCHEMISTRY
AND
BIOPHYSICS
RNA Synthesis
501403 (1967)
11%
in the Liver of Rats Treated
with 5-Fluorouracil ROGER Department
WILLEN of Pathology,
UIKNE STENRAM
AND
University
Received
of Uppscda, Sweden
June 29, 1966
The incorporation of cytidine-3H into rat liver RNA was completely or almost completely inhibited by high doses of 5-fluorouracil. Low doses gave high incorporation into heavy RNA molecules. Less activity was observed in the ribosomal RNA peaks. The results indicate a block in the ribosomal RNA synthesis and support the hypothesis of an accumulation of ribosomal RNA precursors in the nucleoli.
Rats treated with high doses of the cancertherapeutic agent FU1 have enlarged liver cell nucleoli with characteristic ultrastructural alterations, and, as shown by radioautography, a heavily depressed RNA synthesis of the liver cell (1). In the present paper the RNA synthesis was also studied by biochemical methods and in variant doses. The results indicated a block in the ribosomal RTU’A synthesis with an accumulation of precursors of ribosomal RNA in the nucleoli. MATERIALS
AND
METHODS
Twenty-foiir male whit,e rats (Sprague-Dawley strain), weighing about 45 gm, were starved for one day, fed on a protein-free diet (2) for 4 days, and then starved overnight. On the following day at 8:0%9:00 XVI, at which they weighed about 36 gm, they were given a subcutaneous injection of 50, 20, 5, 0.75, or 0.10 mg FU (generously supplied by Hoffman-La Roche, Switzerland) or 1 ml of 0.9% NaCl. Exactly one hour later they were given an intraperitoneal injection of 10 PC cytidine-3H per gram body weight (Schwara BioResearch, U.S.A.), and killed 20 minutes or 3 hours later. Small pieces of the livers were taken for radioautographic examination as described by Stenram and Willen (3). The remainder was frozen on dry ice for RNA preparation and stored at -30”. Liver tissue from two aniRNA preparation. mals treated in the same way, in all about 0.8 gm, 1 Abbreviations used: FU, 5-fluorouracil; RNA, ribonucleic acid; SDS, sodium dodecyl sulfate.
was homogenized in a microhomogenizer (Type Nelco. Measuring and Scientific Equipment Ltd., London, England) at 0” for 2 minutes in 5 ml 0.01 M acetate buffer, pH 5.6, containing low3 M MgCl,, lye bentonite [prepared according to FraenkelConrat et ~2. (4)] and 0.015 M naphtalene-1,5disulfonate. To the homogenate was added an equal volume of 90% (w/v) redistilled, watersaturated phenol containing 0.5% SDS and 0.1% 3hydroxyquinoline. The mixture was vigorously shaken for 20 minutes in a water-bath at 40”. After centrifugation at 30009 for 20 minutes, as much as possible of the aqueous and phenol layers was carefully withdrawn. The remainder was brought to the original volume by the addition of the abovementioned phenol solution, and the above-mentioned buffer system, supplemented with 0.5% SDS and 0.1% %hydroxyquinoline, and again shaken for 20 minutes at 40”, and centrifuged. A third extraction was performed at 60” for 15 minutes. The three aqueous layers were combined and three parts of 96yG alcohol were added. Having been kept overnight (about 15 hours) at -15”, the precipitate was dissolved in 2 ml of 0.01 M acetate buffer, pH 5.0, with 0.1 M NaCI, 10m3M MgCb, and 0.1% bentonite to a concentration of about 20 OD units/ml at 2.600& and dialyzed during magnetic stirring against 100 ml of the same buffer-salt system for 24 hours at 4” with four changes of the buffer. Ultracentrifugation and analysis. Ribonucleic acid solutions of about 0.8 ml were layered onto 24 ml of 5-207& (w/v) sucrose density gradients, prepared according to a modification of the methods of Bock and Nan-Sing (5) and Britten and Roberts (6)) and containing 0.0251 potassium phos501
502
WILLaN
AND
phate buffer, pH 7.0. After centrifugation in an SW 25 swinging bucket rotor, Spinco model L centrifuge, for 12 hours at 24,000 rpm (83,OOOg), the liquid was removed by a peristaltic pump from the bottom of the tube through a hypodermic needle carefuloy inserted from above. The absorption at 2537 A was recorded continuously by an LKB UVICORD 4701A, and the liquid was automatically fractioned into 32-drop samples (about 1 ml). The radioactivity of the samples was determined in duplicate in a dioxan-PPO-PoPoPnaphthalene system in a tritium scintillator counter, type 6012A (Isotope Developments Ltd., Reading, Berkshire, England); the mean of the two values is given in the figures. RESULTS
AND
DISCUSSION
The ultraviolet absorption curves were similar for all groups, and only one was therefore recorded in each figure. The peak in the 4-65 region was as a rule more evident in the FU-treated rats. A small shoulder was regularly found in t,he treated animals in the heavy region (Fig. 1). The RNA synthesis was completely or
STENRAM
almost completely inhibited by 50, 20, and 5 mg FU. There might, however, have been a slight labelling after cytidine-3H administration in the heavy and, at 3 hours, also in the 4-65 regions. Low doses of FU (0.75 and 0.10 mg) gave high activity in these regions. Less activity was observed in the ribosomal RKA peaks. As the ribosomal RNA precursors, elaborated in the nucleoli, are found at 35s and 455 (7-9), the results might indicate a block in their transformation to ribosomal RNA. It should be pointed out, however, that labelling in the heavy region can also be due to non-nucleolar, nuclear RNA of messenger type, but this labelling is more polydisperse than that obtained here. In view of the previously demonstrated enlargement of liver nucleoli with preservation of their intense basophilia during treatment with high doses of FU (I), the present results argue for an accumulation of ribosomal RNA precursors in the nucleoli during this condition.
0.6 00
18s
a FIG. 1. Sedimentation patterns of rat liver RNA. (a) Rats labelled for 20 minutes; (b) rats labelled for 3 hours with cytidine-3H. Faster moving components are at the left. Twenty and 5 mg FU gave the same results as 50 mg and are not reported in the figure.
5FLUOROURACIL
AND RAT LIVER
The radioautographs of the livers from the rats given 50, 20, or 5 mg FU showed low labelling, after cytidine-3H administration, over the whole cell. The rats given 0.75 mg FU showed heavy labelling over the nuclei, especially the nucleoli, and, at 3 hours, slight labelling over the cytoplasm. The rats given 0.10 mg FU showed slightly more, and the controls showed considerably more cytoplasmic labelling at 3 hours. The radioautographic results thus substantiate the view that at a suitable dose of FU, 0.75 mg, the nuclear, probably nucleolar precursors of ribosomal RNA are labelled but not transformed to cytoplasmic ribosomal RKA. ACKNOWLEDGMENTS The investigation was supported by grants from the Swedish Medical Research Council (12X-
RNB
50X
623-02), the Swedish Cancer Societ,y (65:85), and the Medical Faculty, Uppsala. REFERENCES 1.
STENRIM,
2. STENRIM,
3.
U., 2. Zellforsch. li.,
U., 7G5 (1966).
STENRAM,
dcta
Anat.
AND WILL-AN,
71, 207 (19G6). 26, 350 (195F). It., Cancer Res. 26,
H., SINGER, B., AND TSUGIT.~, A., Virology 14, 54 (1961). 5. BOCK, It. M., AND %X-SING, L., dnal. Chem. 10, 1543 (1954). 6. BRITTEN, H. J., AND ROBERTS, R. B., Science 131, 32 (1960). 7. Pssm~, S., J. Mol. ljiol. 17, 117 (1966). 8. PERRY, It. P., Monographs iTall. Cancer Inst. 23, (1966) (in press). 9. MUKAMA~TSU, M., IIODSSTT, J., AND BUSCH, H., J. Riol. Chew. 241, 1544 (1966).
4. FRLENKEL-COSR.\T,