Segregation of the Nucleolus Produced by Anthramycin1

[CANCER RESEARCH 28, 81-90, January 1968] Segregation of the Nucleolus Produced by Anthramycin1

Curtis Harris, Harold Grady, and Donald Svoboda Department oj Pathology, University oj Kansas Medical Center, Kansas City, Kansas 66103

SUMMARY mycin does not produce toxicity in the bone marrow and gastrointestinal tract (49). It possesses certain structural fea Anthramycin, an antibiotic possessing antitumor properties, tures in common with actinomycin D and the postulated biosyn- was administered to rats and mice in a single intraperitoneal thetic intermediates of the actinomycin^, 3-hydroxy-4-methyI- close ranging from 1 to 10 mg/kg body weight. Light micro anthraniloyl peptides (3,47). Like kanamycin, neomycin, and scopy showed that, at the highest dose, anthramycin produced other antibiotics (48), anthramycin permanently bleaches necrosis in the pancreas of rats but not in mice. By electron Euglena gracilis (11). microscopy, nucleolar changes were observed in the spleen and This report demonstrates that anthramycin is another agent kidney of both species by 1 hour. Acinar cells of the pancreas which causes nucleolar changes in pancreatic cells, reticulo showed nucleolar caps and segregation of nucleolar constituents endothelial cells of liver and spleen, and in proximal tubular in both species at 3 to 4 hours. By 10 hours segregation of the epithelium in the kidney. nucleolus and focal cytoplasmic degradation were prominent in pancreatic cells of rats given higher doses but were not MATERIALS AND METHODS observed in hepatocytes of either species. Anthramycin produced nucleolar alterations in Kupffer cells, Thirty-six inbred Fisher-344 male rats weighing between 95 endocrine and exocrine pancreatic cells, proximal tubular cells and 240 gm and fifteen C57 black mice weighing between 20 of the kidney, and in the reticuloendothelial cells of the spleen and 30 gm were used. Anthramycin (anthramycin methyl in rats and mice. The mechanism by which anthramycin acts ether) dissolved in 100% ethanol was administered by a single is unknown, but its ultrastructural effects, as well as its struc intraperitoneal injection. The dosages used were 1.0, 1.7, 5, and tural similarity to actinomycin D, suggest that it may inhibit 10 mg/kg body weight. At least two rats at each dose were RNA synthesis at the DNA level. The functional consequences sacrificed at 1, 4, 10, and 24 hr. Mice were sacrificed at 1, 3, of segregation of the nucleolus have not been established. 10, 12, and 24 hr. Microscopic Studies. Small blocks of liver, pancreas, kidney, INTRODUCTION and spleen were fixed in s-collidine-buffered 1% osmium tetroxide for electron microscopy. The blocks were dehydrated Recently, the effects of carcinogens and antitumor agents on in a graded series of alcohols and embedded in Epon 812 nucleolar morphology and function have been subjects of re containing 5% araldite (20). Thin sections were cut with an newed interest. Investigation of the acute ultrastructural and LKB microtome using glass knives, stained with lead hydroxide biochemical abnormalities following administration of pyrrolizi- and/or uranium acetate, and examined in an RCA 3B or 3G dine alkaloids (44), aflatoxin (42), ethionine (22), actinomycin electron microscope. Semi-thin sections (0.5 to 1.5 /j.) of Epon- D (13, 15, 26, 35, 36, 41), 4-nitroquinoline-A^-oxide (34), UV embedded tissue were stained with aqueous azure A in an radiation (24), proflavin (38), and mitomycin (17) has indi equal volume of 5% sodium bicarbonate. cated a variety of ultrastructural responses in the nucleus and For light microscopy, portions of pancreas, liver, spleen, has emphasized the critical role of the nucleus in cellular kidney, and duodenum were fixed in formalin, embedded in metabolism and inheritance. Particular emphasis has been paraffin, and stained with hematoxylin and eosin or by the given to the nucleolus because of its importance in ribosomal periodic acid-Schiff procedure. RNA (rRNA) synthesis and in the transfer of RNA from the nucleus to the cytoplasm (27-30). RESULTS Anthramycin, an antibiotic isolated from a thermophilic actinomycete (19), has in vitro antibacterial and in vivo anti- Light Microscopy tumor activity (49). At concentrations effective as an antitumor agent against transplantable tumors in mice, anthra- Loss of zymogen granules and necrosis were noted in the pancreatic acinar cells of rats but not in mice. Necrosis of i This study was supported in part by USPHS Grants CA-08055 duodenal crypts was present in both species. All pancreatic and CA-05680 and by the Kansas Division of The American Can alterations were most pronounced with the highest dose ( 10 cer Society. mg/kg) at 10 hr. No changes were observed in the liver, kid Received May 2, 1967; accepted September 8, 1967. ney, or spleen.

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Electron Microscopy the granular component into 2 types: one type resembles cytoplasmic ribosomes 100-150 A in diameter and the second Pancreas. At a dose of 5 mg/kg no changes were evident at consists of irregular aggregates 50-100 A in diameter. The 1 hr in the exocrine pancreas of rats. By 4 hr the endoplasmic reticulum of acinar cells showed marked distention and vesicu- nucleolus is surrounded, in part, by the nucleolus-associated chromatin which, like intranucleolar chromatin, contains DNA lation which was sustained at later intervals. The nucleoli of (2, 45). The morphology of normal nuclei show some degree acinar cells separated into granular and fibrillar components of organ and species specificity. Nucleoli of rat and mouse (Figs. 1, 2) with a denser granular component occasionally pancreatic cells are similar in their ultrastructure but nucleoli present at the periphery. It is uncertain whether these denser of hepatocytes of several species of mice characteristically granular components represent interchromatin granules, a newly-formed element, or a degradation of prÃ©existentele show slight separation of the fibrillar and granular components. ments. Nucleoli of islet cells were fragmented but no cyto- Organ Specificity, Mechanism of Action, and Biochemical Effects of Anthramycm. Nucleolar caps, produced by 4-nitro- plasmic changes were noted. quinoline-A^-oxide, was first described by Reynolds et al. (34). Nucleolar segregation was also apparent in many of the Like several other agents (39), anthramycin produces similar nuclei at the lower dose (1 mg/kg) at 10 hr, and all nucleoli changes in the nucleolus. In general, the initial separation of examined were altered at the highest dose (10 mg/kg). nucleolar constituents is followed by a diminution of the Granules similar in size to interchromatin granules were in granular component. creased in number and usually clustered in the center of the The various agents which cause nucleolar segregation demon nucleus (Figs. 3, 4). Occasionally, the entire nucleoplasm ap strate organ specificity. Actinomycin D produces this nucleolar peared to be distinctly partitioned into two different densities change in the exocrine pancreas (13), cell culture (15), neurons (Fig. 5). Focal cytoplasmic necrosis of exocrine cells was pres (16), and hepatocytes (26, 41). Anthramycin causes similar ent (Fig. 6). abnormalities in nucleoli of islet cells, the exocrine pancreas At 24 hr (1 mg/kg), most of the endocrine and exocrine cells (Fig. 1), reticuloendothelial cells of the spleen (Figs. 13, 14), of the rat pancreas contained normal nucleoli but a round dense and in Kupffer cells (Fig. 11), while hepatocytes show only nucleolar remnant consisting only of the fibrillar component slightly altered nucleoli (Fig. 10). Aflatoxin (42), tannic acid was present occasionally. Focal cytoplasmic necrosis persisted (31), and pyrrolizidine alkaloids (44) affect nucleoli of hepato in some exocrine cells. cytes and Kupffer cells but not those of the pancreas. Several The nucleolar response of mouse pancreas to anthramycin agents produce nucleolar segregation in cell culture ( 15, 17, 34, was similar to that in rats. At 3 hr amidst acinar cells with 35, 38). Two of the most unusual stimuli related to nucleolar normal nucleoli, there were cells with dispersed and disarranged segregation are Mycoplasma (14) and Herpes simplex virus nucleoli (Fig. 8). Nucleoli exhibited segregation at 4 and 12 (40), since these are the only known instances of nucleolar hr (Figs 7, 9). The only cytoplasmic change was dilation of segregation produced by microorganisms, though prominent the endoplasmic reticulum. nucleolar changes of a different type occur in liver cells in Liver. Segregation of the nucleolus was not observed in fected with Ectromelia (18). A separation of the entire nucleo either mouse or rat hepatocytes but granular aggregates meas uring 0.2-0.4 /j. and nucleolar plaques were present (Fig. 10). plasm into a lucent and a dense area was observed following anthramycin treatment (Fig. 7). Actinomycin D (13) and pro- In both species, Kupffer cells showed nucleolar alterations at flavin (38) produce similar partition of electron density of the all intervals (Fig. 11). No remarkable changes were observed nucleoplasm, and the same change has been observed follow in the cytoplasm except the accumulation of fat droplets in ing administration of cycloheximide (C. Harris, and D. J. Golgi vesicles of rat hepatocytes at 24 hr. Svoboda, unpublished results). Kidney and Spleen. Anthramycin produced a variety of The mechanism by which anthramycin acts is unknown. It nucleolar changes in the kidney of rats. These alterations has been found to inhibit RNA synthesis in Ehrlich tumor ranged from fragmentation of the nucleolus to complete separa cells (G. Zbinden, personal communication). It possesses struc tion of the nucleolar components (Fig. 12). In both species, tural features found in the actinomycins and their biosynthetic there were nucleolar alterations in reticuloendothelial cells of intermediates (3, 47). Leimgruber et al. (19) suggested that the spleen (Figs. 13, 14). "anthramycin could be transformed in vivo to 'actinomycin analogs', which actually may be responsible for the observed DISCUSSION anti-tumor activity." Ultrastructure of the Normal Nucleus. The variation in Although the majority of agents which cause nucleolar segre structure of normal nuclei of mammalian cells has been re gation bind DNA and/or RNA (39), some DNA-alkylating viewed by Davis (6), and attempts to unify the terminology agents such as dimethylnitrosamine (7), diethylnitrosamine of the nucleolus have been reported at a recent symposium (46). (23), and ethionine (22) do not cause the same nucleolar alter In general, the normal nucleolus is composed of (a) granules, ations. Work in progress in this laboratory indicates that, in 50-200 A in diameter; (o) fibrils, 50-100 A in diameter; and acute stages, dimethylnitrosamine, ethionine, and 3'-methyl- (c) "intranucleolar chromatin," consisting of microfibrillar ele dimethylaminoazobenzene, when given in high doses, produce ments (2). The first and second components are sensitive to a form of nucleolar segregation, suggesting that the nucleolar RNase digestion (21, 36) and the third component is partially effects of agents which alkylate nucleic acids are dependent digestible by trypsin. Marinozzi and Bernhard (21) divided upon dose and time.

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Although many of the nucleolar segregating agents are car of migration of labeled 18 S and 28 S rRNA into the cytoplasm cinogens, there is no evidence implicating the related alterations following actinomycin D treatment. in nucleolar ultrastructure to carcinogenesis (43). Indeed, the Ultrastructural evidence indicates that anthramycin is a raethylation or ethylation of nucleotide bases, as with the typical nucleolar segregating agent which produces separation nitrosamines, may be meaningless in terms of transmission of of nucleolar constituents followed by a diminution of the "granular component," 100-200 A in diameter. These nucleolar genetic information by DNA. A recent comparative study of antimetabolites by Simard granules and the 60 S ribosomal precursors observed in ultra- and Bernhard (39) suggests that nucleolar segregating agents centrifugation studies are most likely one and the same (25). ". . . bind to DNA and interfere with its template activity for In conclusion, nucleolar segregation is the ultrastructural the conduct of DNA-directed RNA synthesis by RNA poly- reflection of nucleolar dysfunction, that is, alterations in the merase." This hypothesis is compatible with the biochemical synthesis of ribosomal precursors by various agents which bind mechanism of aflatoxin (9) and the proposed mechanism for DNA and inhibit the synthesis of rRNA. Alterations in pre nucleolar caps induced by UV irradiation of the non-nucleolar existing nucleolar rRNA and protein synthesis may also be nucleoplasm (24). The recent finding in our laboratory that partly responsible for the ultrastructural changes observed. cycloheximide can produce nucleolar segregation indicates that an alteration in protein synthesis is an important facet of ACKNOWLEDGMENTS nucleolar segregation (C. Harris and D. J. Svoboda, unpub lished results). In this context, it is of interest that the primary We wish to thank Dr. Gerhard Zbinden, Research Division, action of cycloheximide appears to be in the cytoplasm and the Hoffmann-La Roche, Nutley, New Jersey, for providing the anthra drug interferes with protein synthesis at a step more distal in mycin used in these studies. The technical assistance of Claire the protein synthetic pathway than the site(s) of interference Nielson, Kay Balle, Faye Brady, and Lynne Schmutz is gratefully or inhibition by other agents which cause nucleolar segregation. acknowledged. The synthesis of rRNA seems to be particularly sensitive to low concentrations of DNA-binding agents. As noted by Chiga REFERENCES et al. (5), 80% of the nucleoli of hepatocytes undergo segrega tion after administration of concentrations of actinomycin D 1. Allfrey, V. G. Structural Modification of Histones and Their (13 /ig/100 gm body weight) which inhibit only a fractional Possible Role in the Regulation of Ribonucleic Acid Synthe amount of DNA-dependent RNA synthesis. At a slightly higher sis. Can. Cancer Conf., 6: 313-335, 1966. concentration of actinomycin D (20 ^g/100 gm body weight), 2. Bernhard, W., and Granboulan, N. The Fine Structure of the Jacob et al. (12) have indicated that the synthesis of the Cancer Cell Nucleus. Exptl. Cell Res., Suppl. 9, 19-53, 1963. initial rRNA species (45 S rRNA) is completely blocked within 3. Brockmann, H. Structural Differences of the Actinomycins and Their Derivatives. Ann. N.Y. Acad. Sci., 89: 323-335, 1960. 30 seconds. 4. Busch, H. Histones and Other Nuclear Proteins. New York: The protein matrix, of which 30% is histone, comprises ap Academic Press, 1965. proximately 70% of the nucleolar dry weight (4, 10). Histones 5. Chiga, M., Kume, F., and Millar, R. C. 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Cytol., 7: 393-396, 1960. that the inhibition of RNA synthesis by actinomycin D re sulted in a 50% decrease in synthesis of the lysine-rich histone 8. Freedman, M. L., Honig, G. R., and Rabinovitz, M. The Role of Newly Synthesized RNA on Nuclear Histone Synthesis by fraction. Chicken Immature Erythrocytes. Exptl. Cell Res., 44: 263- Anthramycin-induced changes parallel some of the morpho 272, 1966. logic effects of actinomycin D. Actinomycin D produces 9. Gelboin, H. V., Wortham, J. S., Wilson, R. C., Friedman, M., nucleolar segregation and inhibits DNA-dependent RNA syn and Wogan, G. N. Rapid and Marked Inhibition of Rat Liver thesis by binding the guanine residues of DNA (32, 33). As RNA Polymerase by Aflatoxin Br Science, 164: 1205-1206, suggested by Perry (29), actinomycin D would be expected to 1966. inhibit the synthesis of high molecular weight guanine + cyto- 10. Grogan, D. E., Desjardins, R., and Busch, H. 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13. JÃ©zÃ©quel,A.M., and Bernhard, W. Modifications Ultrastruc of Its Complexes with Purines on DNA. Science, 143: 648-689, turales du PancrÃ©asExocrine de Rat sous L'Effect de L'Actino- 1964. mycine D. J. Microscopie, 3: 279-296, 1964. 33. Reich, E., and Goldberg, I. H. Actinomycin and Nucleic Acid 14. JÃ©zÃ©quel,A.M., Shreeve, M. M., and Steiner, J. W. Segrega Function. In: J. N. Davidson and W. E. Cohn (eds.), Progress tion of Nucleolar Components in Mycoplasma-Infected Cells. in Nucleic Acid Research and Molecular Biology, Vol. 3, pp. Lab. Invest., 16: 287-304, 1967. 183-234. New York: Academic Press, Inc., 1964. 15. Journey, L., and Goldstein, M. Electron Microscope Studies 34. Reynolds, R. C., Montgomery, P. O'B., and Karney, D. H. on HeLa Cell Lines Sensitive and Resistant to Actinomycin D. Nucleolar "Caps"â€”a Morphologic Entity Produced by the Cancer Res., Zl: 929-932, 1961. Carcinogen 4-Nitroquinoline N-oxide. Cancer Res., 23: 535- 16. Koenig, H., and Jacobson, S. Nuclear Changes Induced in 538, 1963. Neurons by Actinomycin D. J. Cell Biol., 31: 61A, 1966. 35. Reynolds, R. C., Montgomery, P. O'B., and Hughes, B. 17. Lapis, K., and Bernhard, W. The Effect of Mitomycin-C on Nucleolar "Caps" Produced by Actinomycin D. Cancer Res., the Nucleolar Fine Structure of KB Cells in Cell Culture. 24: 1269-1277, 1964. Cancer Res., 25: 628-645, 1965. 36. Schoefl, G. The Effect of Actinomycin D on the Fine Struc 18. Leduc, E. H., and Bernhard, W. Electron Microscope Study of ture of the Nucleolus. J. Ultrastruct. Res., 10: 224-243, 1964. Mouse Liver Infected by Ectromelia Virus. J. Ultrastruct. Res., 37. Schwartz, H. S., and Garofalo, M. Degradation of RNA in 6: 46^488, 1962. Liver of Rats Treated with Actinomycin D. Mol. Pharmacol., 19. Leimgruber, W., Batcho, A., and Schenker, F. Isolation and S: 1-8, 1967. Characterization of Anthramycin, a New Antitumor Antibiotic. 38. Simard, R. Specific Nuclear and Nucleolar Ultrastructural Le J. Am. Chem. Soc., 87: 5791-5795, 1965. sions Induced by Proflavin and Similarly Acting Antimetabo- 20. Luft, J. H. Improvements in Epoxy Resin Embedding Meth lites in Tissue Culture. Cancer Res., 26: 2316-2328, 1966. ods. J. Biophys. Biochem. Cytol., 9: 409-414, 1961. 39. Simard, R., and Bernhard, W. Le PhÃ©nomÃ¨nede la SÃ©grÃ©ga 21. Marinozzi, V., and Bernhard, W. PrÃ©sencedans le NuclÃ©olede tion Nucleolaire : SpÃ©cificitÃ©D'Actionde Certains AntimÃ©tabo- Deux Types de RibonuclÃ©oprotÃ©inesMorphologiquement Dis lites. Intern. J. Cancer, /: 463-179, 1966. tinctes. Exptl. Cell Res., 32: 595-598, 1963. 40. Sirtori, C., and Bosisio-Bestetti, M. Nucleolar Changes in KB 22. Miyai, K., and Steiner, J. W. Fine Structure of Interphase Tumor Cells Infected with Herpes simplex Virus. Cancer Res., Liver Cell Nuclei in Subacute Ethionine Intoxication. Exptl. 27: 367-376, 1967. Mol. Pathol., 4: 525-566, 1965. 41. Smuckler, E. A., and Benditi, E. P. The Early Effects of 23. 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The Nucleolus and the Synthesis of Ribosomes. 48. Zahalsky, A. C., Hutner, S. H., Keane, M., and Burgess, R. M. Nati. Cancer Inst. Monograph, 18: 325-340, 1965. Bleaching Euglena gracilis with Antihistamines and Strepto 30. Perry, R. P. Ribosomal Biogenesis. Nati. Cancer Inst. Mono mycin-type Antibiotics. Arch. Mikrobiol., 42: 46-55, 1962. graph, 23: 527-546, 1966. 49. Zbinden, G. Fermentation Products Derived from a Thermo- 31. RÃ¡cela,A., Grady, H., and Svoboda, D. Ultrastructural Nuclear philic Streptomycete. In: P. A. Plattner (ed.), Chemotherapy Changes Due to Tannic Acid. Cancer Res., 27: 1658-1664, 1967. of Cancer, pp. 303-310. New York: Elsevier Publishing Com 32. Reich, E. Actinomycin Correlation of Structure and Function pany, 1964.

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Figs. 1-14. All electron micrographs are from sections stained with uranyl acetate and lead. Fig. 1. Rat pancreas, 4 hr after administration of anthramycin, 1 mg/kg body wt. The fibrillar component (/) and the granular com ponent (g) show complete segregation. Ill-defined granular densities (arrows) are adjacent to the segregated nucleolus. X 72,000. Fig. 2. Rat pancreas, 4 hr after administration of anthramycin, 1 mg/kg body wt. The granular component (g) is diminished and segregated from the two contrasted zones of the fibrillar component (jÂ¡and }t). A denser granular component (arrow) is present else where in the nucleoplasm. X 66,000. Fig. 3. Rat pancreas, 10 hr after administration of anthramycin, 5 mg/kg body wt. The nucleolus (nue) contains only the fibrillar component and a constellation of granules (bracket) is centrally located. X 16,000. Fig. 4. Rat pancreas, 10 hr after administration of anthramycin, 5 mg/kg body wt. The granular aggregate appears to be composed of at least two different sizes of granules (1 and 2). X 61,000. Fig. 5. Rat pancreas, 10 hr after administration of anthramycin, 5 mg/kg body wt. Distinct partition (marked by x) of the nucleo plasm into regions of contrasting density is present. X 5,800. Fig. 6. Rat pancreas 10 hr after administration of anthramycin, 5 mg/kg body wt. Foci of cytoplasmic necrosis containing mitochon dria (m) and endoplasmic reticulum (ER) are prominent in acinar cells after high doses of anthramycin. X 6,700. Fig. 7. Mouse pancreas, 4 hr after administration of anthramycin, 10 mg/kg body wt. The nucleolus is segregated into granular (g) and fibrillar (/) components. The denser granular component (arrow) adjacent to the nucleolus is similar to those found in Figs. 3 and 4. X 29,000. Fig. 8. Mouse pancreas, 3 hr after administration of anthramycin, 1.7 mg/kg body wt. The nucleolus shows fragmentation and partial segregation of the granular (g) and fibrillar (/) components. X 39,000. Fig. 9. Mouse pancreas, 12 hr after administration of anthramycin, 1 mg/kg body wt, Segregation of the fibrillar (/) and granular (g) components of the nucleolus is present in the majority of the cells observed at 12 hr. X 19,000. Fig. 10. Rat liver, 24 hr after administration of anthramycin, 1 mg/kg body wt. Hepatocytes show little response to anthramycin. Nucleolar plaques (p) and granular aggregates (arrows) are noted with increased frequency. X 19,000. Fig. 11. Rat liver, 24 hr after administration of anthramycin, 1 mg/kg body wt. Kupffer cell nucleoli demonstrate partial separation of components at all intervals tested. X 29,000. Fig. 12. Rat kidney, 12 hr after administration of anthramycin, 1 mg/kg body wt. Nucleolar alterations in the proximal tubule cells are evident at all intervals beginning at 1 hr. Large microbodies (mb) containing dense nucleoids are seen. X 7,900. Fig. 13. Rat spleen, 12 hr after administration of anthramycin, 1 mg/kg body wt. The fibrillar component (/) is partially encircled by a ring of dense granules (g). X 72,000.

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90 CANCER RESEARCH VOL. 28

Curtis Harris, Harold Grady and Donald Svoboda

Cancer Res 1968;28:81-90.

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