Effects of Actinomycin D on Base Composition and NearestNeighbor Frequency of Nucleolar RNA of theWalker Tumor and Liver1
TAE SUK RO, K. SHANKAR NARAYAN, AND HARRIS BUSCH
Department of Pharmacology and the Tumor By-products Laboratory, Baylor University College of Medicine, Houston, Texas
Summary
The base compositions of nucleolar and extranucleolar nuclearIlNA's of the tumor and the liver were determined in controlsand rats treated with actinomycin D in a dose of 150 Mg/kg ofbody weight. A decrease in content of cytosine and an increase inA + U/G + C ratio in the nucleolar RNA of the liver werefound, but in the tumor the effects were not marked. UsingATP-U-32P2 or GTP-a-32P, the nearest neighbor frequencies ofnucleotides to AMP or GMP moieties of nucleolar RNA fromliver and Walker tumor of the control and actinomycin D-treatedrats were determined and found to be different in the liver andthe Walker tumor. The changes in nearest neighbor frequencyare consistent with the suppression of synthesis of high molecularweight, GC-rich RNA. As shown by autoradiographic studies,the greater suppressive effects of actinomycin D on nucleolarRNA synthesis than on extranucleolar nuclear RNA synthesisare apparently related to the localization of actinomycin D inthe extranucleolar chromatin in the form of a ring containing thenucleoli.
Introduction
A number of reports utilizing electron microscopic technicsand autoradiography indicated that nucleolar RNA synthesis wasmore markedly inhibited by actinomycin D than synthesis ofextranucleolar nuclear RNA (10-12, 20, 24) and changes innucleolar morphology occurred (4, 15). Studies on the uptake oflabeled precursors in vivo into nucleolar RNA of control andactinomycin D-treated animals supported earlier experiments onthe specificity of inhibition of biosynthesis of nucleolar RNA (7).In addition, in vitro studies have shown that DNA-primed RNAsynthesis was blocked in incubation systems containing isolated
1Supported by grants from the American Cancer Society, theJane Coffin Childs Fund, the National Science Foundation, andthe USPHS (CA 08182).
2The following abbreviations are used: AMP, adenylic acid;ATP, adenosine triphosphate; ATP-U-32P, ATP labeled uniformlywith 32P;A, adenine; C, cytosine; G, guanine; U, uracil; CTP,cytidine triphosphate; GMP, guanylic acid; GTP, guanosinetriphosphate; GTP-a-32P, GTP labeled with 32P in a-position;PEP, phospoenol pyruvate; PK, pyruvic kinase; TCA, trichloro-acetic acid; UMP, uridylic acid; UTP, uridine triphosphate;Tris, tris(hydroxymethyl)aminomethane.
Received for publication August 19, 1905.
nucleoli by actinomycin D (16, 18). Recently, it has been shownthat in vivoadministration of actinomycin D produced a markeddecrease in the amount of 35 S and 45 S RNA, which are maincomponents of the nucleolar RNA.3'4
In the present experiments, analyses have been made of thebase composition of nucleolar and extranucleolar nuclear RNAof the liver and Walker tumor of control and actinomycin D-treated animals. Marked changes were found in the base ratiosof the nucleolar RNA of the liver following injection of actinomycin D, but relatively little change was found in the baseratios of the extranucleolar RNA. Concomitant autoradiographicstudies have shown that actinomycin D-14C localizes in thenucleus of liver cells and to some extent in the perinucleolarchromatin. In the Walker tumor, autoradiographic studiesshowed little localization of actinomycin D and the changesfound were considerably less than those of the normal liver.
Materials and Methods
ANIMALS.Male rats weighing 180-250 gm obtained from theCheek Jones Company (Tombali, Texas) and fed ad libitum onPurina Laboratory Chow were used in these experiments. Thetumor studied was the Walker 256 carcinosarcoma, transplanted5-7 days prior to the experiment. Ten to 15 rats were used in eachexperiment.
PRETREATMENT.Some of the animals were injected i.v. withactinomycin D (150 Mg/kg body weight) 1 hr before they weresacrificed. Some rats were injected with actinomycin D-14C(approximately 1.5 X IO6cpm) 15 min before the animals were
sacrificed.ISOLATION OF NUCLEI AND SUBNUCLEAR FRACTIONS. The pro-
cedures for isolation of nuclei and nucleoli were essentially thesame as described previously (7, 8, 16, 18). After the nuclearsuspension was subjected to sonic oscillation in 0.25 Msucrose, 20ml of the sonicate were layered over 25 ml of 0.88 Msucrose andthe sample was centrifuged at 2000 X g for 20 and 10 min for theliver and the tumor, respectively. The layer containing 0.25 Msucrose and the layer containing 0.88 M sucrose were collected
3Muramatsu, M., Steele, W. J., and Busch, H., to be published.4Recently, Honig and Rabinovitz (1) reported that with very
high doses of actinomycin D, protein synthesis was inhibited inSarcoma 37 cells. Lower doses were employed in this study sinceonly ISO/jg/kg body weight were administered as compared to 1.5mg/liter of culture medium used by Honig and Rabinovitz.
Effects of Actinomycin D on Walker Tumor and Liver
separately. The pellet contained the purified nucleoli. On electron microscopic examination of nucleolar preparations, lessthan 10% of the particles were nonnucleolar contaminants. Lightmicroscopic studies of these preparations showed that less than0.1% of the particles were nuclei. An equal volume of 20% TCAwas added to each of the sucrose layers. The precipitates fromthe sucrose layers and the nucleolar pellet were washed successively with 5% TCA and 95% ethanol, containing 2% potassiumacetate, and twice with absolute ethanol (22, 23).
DETERMINATIONOF BASECOMPOSITIONS.The washed precipitates were hydrolyzed with 0.3 N KOH for 16-18 hr at 37°C(19).
The hydrolysate was chilled and acidified with 0.5 N perchloricacid to precipitate DNA and protein. After centrifugation at3000 X g for 10 min the supernate was transferred to anothertest tube and neutralized with 0.5 N KOH. The neutralizedhydrolysate of RNA was chromatographed on a Dowex-1-for-mate column using a linear gradient of formic acid (2). Sampleswere collected and dried in a desiccator. The dried samples weredissolved in 0.1 N HC1. The optical density was measured at thewavelength of maximum absorbancy for the individual nucleo-tide and the ratio of Amólesof individual nucleotides to totalnucleotides was calculated. The procedure employed (2) provided results for the determination of 2'- and 3'-nucleotides;
minor contaminants were not determined. Standard mixtures ofnucleotides were analyzed to provide comparisons for location ofnucleotides in the chromatographs.
EXPERIMENTSWITH ACTINOMYCIND-"C. Slices of liver and
Walker tumor excised from these rats were fixed in 10% neutralformalin. The samples were also stained with hematoxylin(Harris) before autoradiographs were prepared by dipping sections 5 n in thickness in Kodak emulsion NTB-3. After drying,the slides were stored for 2-8 weeks at 4°Cover silica gel. Grain
counts were made over 20 random fields using a pair of 10 Xcompensation oculars and a 100X phase contrast, oil immersionobjective. The grain counts were made after 5 weeks in view ofthe fact that at this time maximum grain counts were obtained;exposure of the film for longer periods did not produce any increase in the number of grains.
INCUBATIONOF ISOLATEDNUCLEOLIIN VITRO.The incubationmixture was essentially the same as previously reported (15, 18);
in a final volume of 2 ml were 50 jug pyruvic kinase, 20 jumólesphosphoenolpyruvate, 4 ¿miólesATP, 1 jumóleeach of CTP andITTP, 10 jumólesMgCl2, 5 jumólesmercaptoethanol, 100 jumólesTris-Hel, pH 8.2, 5-10 »cof GTP-a-^P (obtained from Inter
national Chemical and Nuclear Corporation, 13332 E. AmarRoad, City of Industry, California) and nucleoli obtained from10 gm of rat liver or 3-5 gm of Walker tumor. In the experimentswith ATP-U-^P (obtained from Schwarz BioResearch Labora
tories), unlabeled ATP was omitted and unlabeled GTP wasadded in the place of GTP-32P. The incubation was carried outfor 20 min at 37°C.After incubation, samples were chilled in ice
and 0.1 ml of 8.5% phosphoric acid was added to each tube. Twoml of 20 % TCA were added to each tube and the precipitate waswashed 3 times with 5% TCA, once with 90% ethanol containing2% potassium acetate (22), and twice with absolute ethanol.
DETERMINATION OF RADIOACTIVITY IN STUDIES ON NEAREST
NEIGHBORFREQUENCIES.Contamination of the samples by ortho-phosphate-32? was eliminated by addition of 0.1 ml of phosphoric
acid immediately after the incubation mixture was cooled. Analiquot from each nucleotide fraction was plated on a stainlesssteel planchet and dried; the radioactivity was determined in agas flow counter (Nuclear Chicago) with a Micromil window.The total counts in each peak were calculated and compared tothe total counts in the AMP peak; the radioactivity in individualnucleotides was determined as the percentage of total radioactivity in the 4 nucleotides (3).
Results
INTEACELLULAR LOCALIZATION OF ACTINOMYCIN D-14C. Fig. 1
shows the patterns of labeling found in liver and tumor cellsfollowing administration of actinomycin D-"C. Although livercells showed some specific labeling over the nucleus, none wasapparent in the tumor. In the liver, about 15% of all cells examined had more than 5 grains over the nucleus. The actinomycinD in liver nuclei appeared to be localized in the extranucleolarregions of the nucleus, in the form of a ring.
EFFECTSOF ACTINOMYCIND ON NUCLEOLARRNA. The basecompositions of nucleolar RNA in the tumor and the liver (Table1) were quite similar (5) although the A + U/G + C ratio was
TABLE 1EFFECTSOF ACTINOMYCIND ONBASE COMPOSITIONSOF NUCLEOLAKRNA FROM
WALKERTUMORANDTHE LIVERThe purified nucleoli were hydrolyzed with 0.3 NKOH for 16-18hr. Base ratios were determined using
TABLE 2EFFECTOF ACTINOMYCIND (150 jug/kg) ON BASE COMPOSITIONSOF LIVER NUCLEAR
RNA'sAfter sonication of the nuclear suspension (see "Materials and Methods"), 20 ml of the sonicate was
layered over 25 ml of 0.88 Msucrose and centrifugad at 2000X g for 20 min. The precipitate was purifiednucleoli. The 0.88 Msucrose layer was designated as the middle layer and the 0.25 Msucrose layer wasdesignated as the supernate. Base ratios of each fraction were determined using a Dowex-1 columnafter alkaline hydrolysis.
IN NUCLEOLARRNAThe complete system contained in 2 ml: 50 ¿igpyruvic kinase (PK), 20 Amólesphosphoenol pyruvate
(PEP), 1 /imole each of GTP, CTP, and UTP, 10 MmolesMgCU, 5 Amólesmercaptoethanol, 100/miólesTris-HCl, pH 8.2,5-10 /ucof ATP-U-32P,and nucleoli obtained from 10gm of rat liver or 3-5 gm of Walkertumor. The incubation was carried out for 20 min at 37°C.The values are % of total 32Pin nucleo-tides, and the numbers in parentheses are the ratios of 32Pin the nucleotide to 32Pin adenylic acid.
slightly greater in the liver than in the tumor. The effects ofactinomycin D in vivo on base composition of whole nucleolarRNA of the Walker tumor and the liver are shown in Table 1. Amarked decrease was found in the content of cyto.sine along withan increase in A + U/G + C ratio in liver nucleolar RNA following injection of 150 MgAg of actinomycin D. The decrease incytosine content after actinomycin D treatment might be expected from the reported specific binding of the actinomycin Dto the guanine moiety of the DNA-double helix (13).
The effects of the same amount of actinomycin D on thenucleolar RNA of the Walker tumor were not marked. Thechanges found in the nucleolar RNA of the Walker tumor wereof a similar order of magnitude as those found in the liver following injection of 30 jug/kg of actinomycin D (Table 1).
EFFECTS OF ACTINOMYCIN D ON THE BASE RATIO OF MIDDLELAYERANDSUPERNATERNA's OFNUCLEI.Earlier reports showed
that the biosynthesis of extranucleolar nuclear RNA is less sensitive to actinomycin D than that of nucleolar RNA (4, 7, 10-12,
15). These results were supported by studies on the base ratios ofextranucleolar nuclear RNA's after actinomycin D treatment
(Table 2). The increase in the A + U/G + C ratio in the extranucleolar nuclear RNA was 5-10% compared to 30-35% in thenucleolar RNA.
EFFECTS OF ACTINOMYCIN D ON THE NEAREST NEIGHBOR FRE
QUENCIES TO THE AMP AND THE GMP OF WHOLE NUCLEOLAR
RXA. The nearest neighbor frequencies of other nucleotides toAMP (Table 3) and to GMP (Table 4) were different in the RNAof tumor nucleoli than in RNA of liver nucleoli (16). The nearestneighbor frequency of GMP to GMP (G to G) in the tumornucleolar RNA was somewhat less than in the liver. The frequencies of C to A and U to A of the tumor nucleolar RNA werealso somewhat greater than those of the liver (Tables 3 and 4).
After treatment with actinomycin D, a marked increase wasfound in the frequencies of U to A and U to G in the liver nucleolar RNA. In the tumor, a decrease was found in the frequencyof G to A but the remainder of the changes were not marked.
The marked inhibition of biosynthesis of nucleolar RNA byactinomycin D has been ascribed to the block of DNA-dependentRNA synthesis by the specific binding of actinomycin D to thedeoxyguanosine residues of the DNA-double helix (13, 14).From the base composition of the nucleolar RNA of the liver itcan be estimated that guanine comprises 26 % of the total basesin the DNA that codes for nucleolar RNA synthesis. Similarly, inthe DNA that codes for synthesis of extranucleolar nuclear RNAof the liver, guanine comprises 23.5-26.5% of the total bases.
However, no significant change was found in the cytidylic acidcontent of the RNA of extranucleolar fractions of the livernuclei following administration of actinomycin D, despite thefact that marked decreases were found in the cytidylic acid content of the nucleolar RNA following administration of actinomycin D. Studies on the nearest neighbor frequency of nuclearRNA indicate that RNA with a higher than normal U to A andU to G content is synthesized. However, this RNA has a C to Aand C to G content that is only slightly below control values.
The discrepancy in inhibition of nucleolar and extranucleolarRNA synthesis is apparently unrelated to the mechanism ofaction of actinomycin D. From the autoradiographic studies itappears that actinomycin D specifically localizes in the extranucleolar chromatin. The mechanism of localization of actinomycin D to the extranucleolar chromatin is not yet clear, but itseems possible that actinomycin D may diffuse or be transportedalong the nuclear ribonucleoprotein network (21). Evidence hasbeen obtained that the nucleolus is the primary site of biosynthesis of high molecular weight RNA, including 35 S, 45 S, and 55 SRNA fractions, as defined by sucrose density gradient sedimentation patterns (6). Binding of a relatively small number of molecules of actinomycin D to DNA priming for synthesis of the longRNA molecules would be sufficient to inhibit their biosynthesis.However, synthesis of lower molecular weight RNA would beblocked only if a greater number of molecules of actinomycin Dwere bound to DNA strands.
The present study supports the previous results that have indicated a significant difference in the nearest neighbor frequencyof nucleolar RNA of the Walker tumor and the liver (16). Thesedifferences, along with the other evidence of differences in basecomposition and percentage of total nuclear synthesis of nucleolar RNA of the Walker tumor and liver, support the concept that
different genes are functional in these 2 different types of tissues(5, 9, 16). The lesser effects of actinomycin D on nucleolar basecomposition in the tumor appear to result from a lower tissueconcentration of the inhibitor (17).
Acknowledgments
The actinomycin D was generously provided by Dr. GeorgeBoxer of Merck, Sharp and Dohmc Co., Rahway, N. J., and theactinomycin D-14Cwas generously provided by Dr. Edward Reich,of the Rockefeller Institute of New York.
References
1. Honig, G. R., and Rabinovitz, M. Actinomycin D: Inhibitionof Protein Synthesis Unrelated to Effect on Template RNASynthesis. Science, 149: 1504-5, 1965.
2. Huribert, R. B., Schmitz, H., Brumm, A. F., and Potter, Y. R.Nucleotide Metabolism: Chromatographie Separation of AcidSoluble Nucleotides. J. Biol. Chem., 209: 23-39, 1954.
3. Josse, J., Kaiser, A. D., and Kornberg, A. Enzymatic Synthesis of Deoxyribonucleic Acid. VIII. Frequencies of NearestNeighbor Base Sequences in Deoxyribonucleic Acid. Ibid.,236: 864-75, 1961.
4. Journey, L. J., and Goldstein, M. N. Electron MicroscopicStudies on HeLa Cell Lines Sensitive and Resistant to Actinomycin D. Cancer Res., el: 929-32, 1961.
5. Muramatsu, M., and Busch, H. Studies on Nucleolar RNA ofthe Walker 256 Carcinosarcoma and the Liver of the Rat.Ibid., 24: 1028-34, 1964.
6. . Studies on Nucleolar and Nucleolar Ribonucleie Acidof Regenerating Rat Liver. J. Biol. Chem., 240: 3960-66, 1965.
7. Muramatsu, M., Hodnett, J. L., and Busch, H. Studies on the"Independence" of Nucleolar Ribonucleie Acid Synthesis.Biochim. Biophys. Acta, 91: 592-97, 1964.
8. Muramatsu, M., Smetana, K., and Busch, H. Quantitativeaspects of Isolation of Nucleoli of the Walker Carcinosarcomaand Liver of the Rat. Cancer Res., 23: 510-18, 1963.
9. Okamura, N., and Busch, H. Base Composition of HighMolecular Weight Nuclear RNA of Walker Tumor and Liverof the Rat. Ibid., 85: 693-97, 1965.
10. Perry, R. P. The Cellular Sites of Synthesis of Ribosomal and4S RNA. Proc. Nati. Acad. Sci., 48: 2179-86, 1962.
11. . Selective Effects of Actinomycin D on the IntracellularDistribution of RNA Synthesis in Tissue Culture Cells. Exptl.Cell Res., 29: 400-8, 1963.
12. . The Role of the Nucleolus in RNA Metabolism andOther Cellular Processes. Nati. Cancer Inst. MonographNo. 14, pp. 73-83, 1964.
13. Reich, E. Actinomycin Correlation of Structure and Functionof Its Complexes with Purines and DNA. Science, 143: 684-89,
1964.14. Reich, E., Franklin, R. M., Shatkin, A. J., and Tatus, E. L.
Action of Actinomycin D on Animal Cells and Viruses. Proc.Nati. Acad. Sci., 48: 1238-45, 1962.
15. Reynolds, R. C., Montgomery, P. O., and Hughes, B. Nu-clcolar "Caps" Produced by Actinomycin D. Cancer Res., 24.:
1269-77, 1964.
16. Ro, T. S., and Busch, H. In Vitro Labeling of RNA in IsolatedNucleoli of the Walker Tumor and Liver. Ibid., 24: 1630-33,
1964.17. . Concentration of Actinomycin D-14C in Various Tis
sues Following Intravenous Injection. Biochim. Biophys.Acta, 108: 317-18, 1965.
18. Ro, T. S., Muramatsu, M., and Busch, H. Labeling of RNA
of Isolated Nucleoli with UTP-14C. Biochem. Biophys. Res.Commun., 14: 149-55, 1964.
19. Schmidt, G., and Thannhauser, S. J. Method for Determination of Deoxyribonucleic Acid, Ribonucleic Acid and Phos-phoproteins in Animal Tissues. J. Biol. Chem., 161: 82-89,1945.
20. Sirlin, J. L., Tandler, C. J., and Jacob, J. The RelationshipBetween the Nucleolar Organizer and Nucleolar RNA. Exptl.Cell Res., 31: 611-13, 1963.
21. Smetana, K., Steele, W. J., and Busch, H. A Nuclear Ribo-nucleoprotein Network. Exptl. Cell Res., 31: 198-201, 1963.
22. Steele, W. J., Okamura, N., and Busch, H. Prevention of Lossof RNA-DNA and Protein into Lipid Solvents. Biochem.Biophys. Acta, 87: 490-92, 1964.
23. . Effects of Thioacetamide on the Composition and Biosynthesis of Nucleolar and Nuclear Ribonucleic Acid in RatLiver. J. Biol. Chem., êJfl:1742^9, 1965.
24. Stenram, U. Radioautographic RNA and Protein Labeling andthe Nucleolar Volume in Rats Following Administration ofModerate Doses of Actinomycin D. Exptl. Cell Res., 36:242-55, 1964.
Effects of Actinomycin D on Walker Tumor and Liver
" *
.'•*
FIGS. 1-6. Autoradiographs of tissues of tumor-bearing rats injected with actinomycin D-14C 15 min before they were sacrificed.
FIG. 1. Autoradiograph of a section of the Walker tumor. The distribution of the grains is random. X 1200.FIG. 2. Autoradiograph of a section of liver of a tumor-bearing rat. The arrow points to a nucleus containing an intranuclear ring of
grains. Other such nuclei are visible in the same field. X1200.FIG. 3. Autoradiograph of another section of liver. The long arrow points to a cell with grains in the cytoplasm and in close proximity
to the nucleus. The short arrow points to a nucleus containing an intranuclear ring of grains. X 1200.FIG. 4. A phase contrast micrograph showing extranucleolar localization of actinomycin D-14C in nuclei of a liver of a tumor-bearing
rat. X 1200.FIGS. 5, 6. Autoradiographs of areas of liver of a tumor-bearing rat containing a number of dense intranuclear rings of grains. As in
1966;26:780-785. Cancer Res Tae Suk Ro, K. Shankar Narayan and Harris Busch LiverNeighbor Frequency of Nucleolar RNA of the Walker Tumor and Effects of Actinomycin D on Base Composition and Nearest
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