CANCER RESEARCH VOLUME 23 MARCH 1963 NUMBER 3 The Nucleolus of the Cancer Cell : A Reviews HARRIS BUSCH, PAUL BYVOET,t AND KAREL SMETAN4 (Department of Pharmacology, Baylor University College of Medicine, Houaton@ Texas) Introduction 314 Identification and Morphology of the Nucleolus 314 Isolation of Nucleoli from Cells 314 Staining of nucleoli for biochemical procedures Methods for disruption of the nucleus Purification of the nucleoli Composition and Internal Structure of the Nucleolus 316 DNA RNA Quantity of Nucleolar RNA Protein Shape, Size, and Number of Nucleoli 3@O Hypertrophy of the nucleolus in tumor cells Other nucleolar changes in tumor cells Modification of Nucleolar Size, Shape, and Number in Nontumor Cells 3@1 Enlargement of the nucleolus Diminution in size of nucleoli External influences on nucleolar RNA Origin of Nucleoli Functions of the Nucleolus 3@4 Synthesis of RNA in the nucleus Role of the nucleolus in biosynthesis of RNA Biochemical evidence for rapid RNA synthesis in the nucleolus Role of DNA in biosynthesis of cytoplasmic RNA Role of the nucleolus in the biosynthesis of ribonucleoproteins Role of the nucleolus in the biosynthesis of protein Nucleolus as a storage depot Relationship of Nucleolar RNA to RNA of the Remainder of the Cell 3@6 “Messenger―-RNA s-RNA Ribosomes Discussion 3@8 * The original studies reported in this manuscript have been t Research Associate of the American Cancer Society. supported in part by grants from the U.S. Public Health Serv- Present address: Department of Pharmacology, University of ice, the American Cancer Society, and the Jane Coffin Childs Florida, Gainesville, Florida. @ Fund. Postdoctoral Trainee in Pharmacology, on leave from the Received for publication September 20, 1962. National Academy of Science, Prague. 313 Thia One @1@II@II@\I\\\UII\IIIIU@ POB6-BTA-D1L4 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1963 American Association for Cancer Research. 314 Cancer Re8earch Vol. 923, March 1963 INTRODUCTION species (179, 9249).The technics employed included Several factors have contributed to the in light microscopy of fixed and stained specimens, creasing interest in the role of the nucleolus in the as well as fresh, supravitally stained material, cancer cell. Among these are included the accumu ultraviolet-absorption microscopy of fresh or fixed lating evidence that the nucleoli of mammalian material (9245—48), phase contrast microscopy, in celLs (a) occupy a central role in the production or terference microscopy, and electron microscopy. distribution of RNA, (b) have a significant role in Although fixation of tissues produces many arti the production of components of the cytoplasmic facts (130, 314, 4092),cytochemical strains are gen ribosomes, and (c) can now be isolated as purified erally made with fixed material. The solutions entities from both neoplastic and other mam used vary from aqueous solutions, such as formal malian cells. The development of important new dehyde, 1 per cent chromic acid, Zenker's fluid, technics for the isolation of RNA and fractionation Cajal's fluid, or formol sublimate to partly or of nuclear proteins has further increased interest largely organic solutions, such as Bouin's fluid, in both the mechanisms and kinetics of biochemi Zenker-formol, Carnoy's fluid, formaldehyde-ab cal events occurring in the nucleolus. These tech solute alcohol-propionic acid, 95 per cent ethanol, flies are added to previously existing procedures or methanol (68, 158, 196, 9213, 92924—928,255,9257, which have served to bring attention to the impor 307, 3928, 3929, 3492, 4092). Fixation by freeze-drying tance of the nucleolus by showing that the nude or air-drying has also been employed. Similarly, a olus had a high concentration of RNA and by large variety of stains is available for studies on permitting determination of its size in specific the nucleolus. Phase contrast microscopy, and cells as well as the effects of changes in cell func especially supravital staining of fresh or living tion on nucleolar size. Staining technics and ultra material, have proved to be the most successful violet-absorption measurements also showed that in making the nucleolus clearly visible. Stains rec the nucleoli of neoplastic cells were increased in ommended for the nucleolus include methylene size by comparison with a variety of other types blue or its homolog Azure C (Fig. 1), Giemsa's of cells. The primary objective of this review is to stain, brilliant cresyl blue and dilute toluidine blue correlate some of the extensive earlier studies with (9231—33,9259,9260,9288,301, 3928,374—76,386, 387). the biochemical information now rapidly increas Recently, Seman (3927) reported that the borate ing with regard to the nucleolar components, their salt of methylene blue was useful for visualization turnover and functional role in the cell. For more of the nucleolus. More classical examples of this extensive reviews of the morphological aspects of class of stains are hematoxylin or hematoxylin the subject, the interested reader is referred to a iron, and impregnation technics with silver or number of previous excellent reviews (392,33, 158, osmium tetroxide. Tanaka (3792) has successfully 335). used acetocarmine and hematoxylin solution so as to stain both nucleoli and chromosomes. IDENTIFICATION AND MORPHOLOGY OF THE NUCLEOLUS ISOLATION OF NUCLEOLI Nucleoli of mammalian cells are readily visual FROM CELLS ized by a variety of optical procedures, of which Until recently, satisfactory methods for the the simplest is phase microscopy. In unstained isolation of highly purified nucleolar preparations mammalian cells or cell nuclei one can readily were unavailable. The recent reports (50)1 that visualize, within the nuclei, one or more bodies nucleoli can be isolated in reasonable yields from with a different refractive index from that of the liver and neoplastic cells in a reproducible manner remainder of the nucleus. Depending upon the suggest the possibility that direct analysis of the concentration of divalent ions in the medium, the components and functional activity of the nucleo nucleoli may be stippled either lightly or heavily. lus will soon be accomplished. Fundamentally, Some questions have arisen whether the bodies there are three basic problems in the isolation of seen by phase microscopy are identical with those nucleoli—namely, rapid recognition of nucleoli, which are stained by commonly employed meth release of the nucleoli from the nucleus, and puri ods (9217); but there is very little doubt that this fication of the nucleoli away from the attached is the case, since the particles isolated by proce nuclear debris and other nuclear components. dures to be discussed later not only look like those Staining of nucleolifor bücheirticalprocedures.— in the nuclei, but also are stained with “nucleolar―One of the problems in studies on the isolation of stains. nucleoli was the need for a method for rapid stain The morphology of the nucleolus has been ing of preparations. Although a variety of stains studied in a variety of cells of tissues of different 1 R. Maggio, P. Siekevitz, and G. Palade, in manuscript. Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1963 American Association for Cancer Research. BUSCH et al.—Nucleolus of the Cancer Cell: Review 315 would seem to be potentially useful for this pur which differ considerably in density. One of the pose (9217, 3927), the method which proved most simplest procedures for purification of the nucleoli satisfactory was the simple procedure of mixing a was that of Monty et al. (9250), who permitted the drop of 0.1 per cent Azure C in distilled water or nucleoli and other components of the nuclei to 0.925M sucrose with the tissue preparation (Fig. 1). settle slowly in tragacanth solutions. There are a Within a few seconds the nucleoli were stained a number of problems involved, particularly the deep purple, and the cytoplasmic ribonucleopro long time required (366). Another procedure is teins were stained to a lesser extent (Fig. 1). This differential centrifugation of the nucleoli with the procedure also permitted quantitation of the num use of sucrose gradients or several steps in centri ber of nucleoli and determination of recovery in fugation so as to provide separations of the heavier various steps of the purification procedure.2 nucleolar masses from the lighter nuclear con Methods for disruption of the nucleus.—Methods taminants. Recent reports have suggested that for isolation of nuclei from tumor cells and liver similar methods have been effective in the prepa cells have been improved (51, 67), but the condi ration of nucleoli from plants (928, 311). A purely tions required for isolation of nuclei are different chemical method for obtaining a “nucleolo-chro from those required for isolation of nucleoli. The mosomal apparatus― was reported by Georgiev et nuclei of tumor cells can be freed from cytoplasmic al. (1926—929),whoextracted nuclei with dilute salt tags when isolated in media which do not contain solutions and then subjected the extracts and the divalent ions, but the presence of latter is abso residues to differential centrifugation. They re lutely essential for isolation of nucleoli. According ported that this method permitted the separation ly, in preparation of the nucleoli of tumor cells, it of nuclear components into a number of types of has been necessary to use a less satisfactory prepa particles such as microsomes, ultramicrosomes,