Silver Staining of Nucleolar Granules in Tumor Cells'

[CANCERRESEARCH39,857-863,March1979] 0008-5472/79/0039-0000$02.00 Silver Staining of Nucleolar Granules in Tumor Cells'

Harris Busch, Yerach Daskal, Ferenc Gyorkey, and Karel Smetana

Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030

ABSTRACT concentration of silver nitrate in the staining solutions and developer. With this simplified method, large numbers and With the aid of a simple silver-staining procedure, large interesting formations of silver granules were detected in numbers and unusual arrays of nucleolar argyrophihic gran nucleoli of tumor cells including Novikoff hepatoma, HeLa, ules were found in Novikoff hepatoma, KB, and HeLa cells. and KB cells; different types of silver-staining structures Some of these arrays consisted of linearly arranged discrete were found in normal and regenerating liver nucleoli. Dun granules, and others were in two to three mows each ing these studies, analysis of the mitotic stages indicated containing three to five granules. Corresponding fomma that at metaphase the few granules present, usually in tions were not found in either the normal on regenerating doublets, were associated with NOR regions of chromo liver nucleoli which contained an argyrophilic network in somes (7-9, 11, 20). However, even before the completion which the dark granules were apparently associated with of telophase, a large number and unusual formations of the less dark argymophilic fibrils of a reticulum. The nucleo granules were present in the tumor cells. If these arrays of lam argyrophihic granules were readily identifiable in the granules reflect the increased nucleolar activity in the G1 separated daughter nuclei of the tumor cells in telophase, phase, the Novikoff hepatoma may be in the G1 phase even suggesting that the increased nucleolar activity of the G1 beforeterminationofmitosis. phase begins in these cells even before cell division has been completed. MATERIALSAND METHODS INTRODUCTION Tumors and Other Tissues. Novikoff hepatoma ascites In addition to their aberrations in size and shape, and cells were transplanted i.p. into adult male albino Holtzman unusual antigens (2, 3), tumor nucleoli have a high mateof rats (The Holtzman Co., Madison, Wis.) weighing 200 g. synthesis of pre-mRNA(1). In the search for approaches to Samples of these cells were taken 6 days after implantation light microscopic visualization of initiation and transcrip of the tumor. HeLa and KB cells were grown in tissue tion complexes of nucleolar pre-mRNAsynthesis, attention culture in modified Eagle's medium. The original samples was directed to the possible usefulness of silver staining of the KB cells were obtained from Flow Laboratories reported in the studies of Howell (9-i 1), Hubbell and Hsu (Rockville, Md.). Normal livers and regenerating livers were (12), Goodpasture and Bloom (8), Schwarzachem (20), and obtained from normal adult male albino Holtzman rats Giminez-Martin et a!. (7). weighing 200 g. Hepatectomy was done 6, 12, or 18 hr prior Silver staining of the nucleolus was developed first as an to sacrifice of the animals. Smears of cells, cell fractions, offshoot of the elegant methods for silver staining of tissues and tissues were spread on microscope slides and aimdried. (19) during the last century and then was used for the Silver Staining. The silver impregnation procedure (4, 8- demonstration of â€œnucleolonemasâ€•(5,13, 18), curled fibrils 12, 18, 20) was modified for tissues and cell smears to in the nucleolus. Its recent use has been the demonstration provide standard results in a relatively short period of time. of NOR2 in the specific â€˜â€˜nucleolam'â€˜chromosomes which Air-dried smears of the various types of cells were fixed in contain mDNA(4, 6-12). Preliminary cytochemical studies Camnoy's mixture (methanol:glacial acetic acid, 3:1) for 10 on these silver-staining NOR elements indicated that they mm and then washed with running water and dried. The were nonhistone proteins and not DNA, RNA, or histones smears were covered with concentrated A@NO:Csolution(1 (9, 10, 20). Particularly interesting questions from these g/ml)for5 to 7 mm, afterwhichthesolutionwasdrained results concern why the NOR regions of chromosomes off. The smears were next covered with a mixture prepared carry with them nonhistone protein(s) and the nature of by adding 37% formaldehyde containing 12% methanol thesenonhistoneproteins. (Fischer, Houston, Texas) to an equal volume of AgNO:@(1g/ Among the problems in the use of silver staining for ml)and thenwere incubatedfor3 to5 mm at40-50Â°ona biochemical studies on nucleolar proteins was the length of warm plate. The silver nitrate solution should not be kept time and complexity of the methods used for silver staining more than 2 days, and the formaldehyde:silvem nitrate solu and the degree of background (6, 8, ii , 13, 18). The present tion was prepared just prior to use. report indicates that silver staining could be simplified and To prevent the precipitation of silver salts, a thorough shortened with a high reproducibility by increasing the running-water wash was made. The sample was stained with May-Gmunwald (1.6 g of methylene blue eosinate pen liter of methyl alcohol) solution (Curtin Matheson Scientific , This research was supported in part by Cancer Program Grant CA 10893, awarded by the National Cancer Institute, Department of Health Instruments, Houston, Texas) and diluted with 1:1 distilled Education and Welfare; the Bristol-Myers Fund, the Pauline Sterne Wolff or tap water for 1 mm. After a thorough wash with the Memorial Fund; and a generous gift from Mrs. Jack Hutchins. 2 The abbreviation used is: NOR, nucleolus organizer region. running water, the smears were aim dried in the vertical Received September, 18, 1978; accepted December 1, 1978. position to prevent the formation of precipitates of silver

MARCH 1979 857

salts and then were examined on a light microscope under chromosomes in the NOR (7-9, 1i , 20) were present around oil immersion. the metaphase equatorial plates, and their total number was approximately 8 to 11. The size and density of these RESULTS granules were approximately the same as that of the nu cleolar granules, but it is not clean whether they contain the Silver-stained Granules in Tumor Cells. Fig. 1 is a same silver-staining proteins. composite showing various formations of silver granules in Duringanaphase,the number of granulesappearedto a number of tumor cells. The number of granules varied have increased but the granules were largely single; i.e., considerably from nucleolus to nucleolus and cell to cell in the doublets appear to have been separated (Fig. 4a). In these preparations. The mean number was 53 granules/ telophase (Fig. 4, a and b), when the cytoplasm of the nucleus and 21 granules/nucleolus in the populations of daughter cells had not separated, there was a marked Novikoff hepatoma ascites cells; 13 granules/nucleus and increase in the density and the number of the silver granules 20 granules/nucleolus in KB cells, or ii granules/nucleus in the newly formed nuclei. As noted in Fig. 4b (arrow), and 39 granules/nucleolus in HeLa cells (Table 1). Many there was a reappearance of the linear arrays of the sepa nucleolam granules appeared to be part of a reticulum in mated nucleolar granules. Apparently, even before the 2 these tumor cells as noted earlier by others (12, 17). daughter cells had fully formed, the nuclei contained the However, quite frequently, the nucleohar granules appeared large number of dense granules found in intemphase cells. to be isolated free of reticulum. Frequently, they were arrayed in one or more mowscontaining several granules DISCUSSION per row (Figs. 1, a to d, and 3a) in each of the types of tumors studied. The present studies represent an extension of attempts in Silver-staining Nucleolar Elements in Normal and Re this laboratory to investigate the elements of the nucleolus generating Liver. In normal liven (Fig. 2a) and in the 6-hr of the cancer cell, particularly with respect to the abemma (Fig. 2, b and C) and 18-hr regenerating liver (Fig. 2d), the tions in nucleolar size, shape, and hyperactivity. in human nucleolus contained the characteristic reticulum described neophasia (1). An important basis for such investigations as a â€˜â€˜nucleolonemaâ€•byEstable and Sotelo (5) and subse has been the development of rapid methods for staining quent workers (i, 7, 13, 18). The nucleolar granules were nucleoli and their substructures as an aid to biochemical thickenings of this network at a number of points; thus the analysis of nucleolam components. The present report of a structure resembles a reticulum. Unlike the tumors, the rapid, simple, and reproducible method for silver staining liver nucleoli did not contain individual granules on the will permit detailed studies on the constituents of the types of mowsnoted above. Both the density of the staining argymophihic nucleolar elements, their roles in nucleohar and the size of the nucleoli were markedly increased in the function, and their changes during the cell cycle. regenerating liver nucleoli (Fig. 2, C and d). An analysis of Stainingwithsilvernitraterevealedseveralinteresting the numbers of â€˜â€˜granulesâ€•presentper nucleolus indicated properties of nucleoli of tumor cells including a large they increased from approximately 4/nucleolus and 13/ number of nucheolar argymophihic granules and interesting nucleus in the normal liver to approximately 15/nucleolus formations of discrete granules in linear arrays, which were and 33/nucleus in the 18-hr regenerating liver. These values different from those observed in normal and regenerating correlate well with the increases reported in 45S RNA liver cells. There appears to be a relationship between the synthesis from 3 to 5 to 15 fg/min/nucleolus in the i8-hm number of granules and the rates of nucheolar synthesis of regenerating liver (i). prenibosomal 455 rRNA. In mature lymphocytes, where pne Nucleolar Granules in Cell Division of Novikoff Hepa rRNA synthesis is negligible (1), very few granules (0 to 4/ toma Cells. Fig. 3 shows that during the course of cell nucleus) were found in rat (Fig. la) or human (20) lympho division theme were marked changes in the number and cytes. In normal liven (1), where the rates of synthesis of appearance of the silver-staining nuclear granules. During 455 pme-mRNAarehow (3 to 5 fg/min/nucleolus), there were metaphase (Fig. 3), â€œdoubletsâ€•ofgranules associated with approximately 4 granules/nucleolus and 13 granules/nu cleus. In the advanced G, state of 18-hr regenerating liver, there were approximately 15 granules/nucleolus and 33 Table 1 granules/nucleus. In the Novikoff hepatoma, theme were The number of argyrophilic granules inucleoli of various cellsNo. approximately 21 granules/nucleolus and 53 granules/nu ofcellsGrains/nu cleus (1). Grains/nu ana These cytochemical findings must be extended and con CellscleohuscleuslyzedRat firmed in a number of systems before conclusions can be hepatocytes Â±0.3â€• Â±1.9 drawn either with respect to common features of these Regeneratingi5O@hepatocytesâ€• liven4.4 15.3 Â±0.913.1 33.4 Â±1 .7150b granules in tumors and their potential diagnostic or func tional significance. From the point of view of biology of 7.5150bcellsHeLaRat Novikoff hepatoma21 .0 Â±0.153.0 Â± cancer, it was interesting that even before termination of telophase, the nucleus and nucleoli contained large num 6.330KBcells1 1.2 Â±3.338.6 Â± cells13.5 Â±5.219.6 Â±4.825 bers of these granules in similar arrays to those found in a Mean Â±S.D. intemphasetumor cells. b From 3 animals. The nature of the argyrophilic elements in the nucleolus C Obtained 18 hr after partial hepatectomy. is not known. Preliminary studies have suggested that

858 CANCERRESEARCHVOL. 39

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1979 American Association for Cancer Research. Silver-staining Nucleolar Granules nucleolar phosphoproteins such as C23 and B23 (13-16) proteins associated with rRNA transcribed from oocyte chromosomes. Chromosoma (Berl.), 62: 361-368, 1977. stain positively in one-dimensional gels with the method 10. Howell,M. W., and Black,A. D. A rapid techniquefor producing silver used in this study. The relationship of these proteins to stained nucleolar organizer regions and trypsin-Giemsa bands on human those in the NOR and those that accumulate during telo chromosomes.Hum.Genet.,43: 53-56,1978. 11. Howell, M. W., Denton, T. E., and Diamond, J. R. Differential staining of -phase remains to be biochemically analyzed. The silver the satellite regions of human acrocentnicchromosomes.Expenientia staining techniques in smears and polyacrylamide gels will (Basal), 31: 260â€”262,1975. 12. Hubbell, H. R., and Hsu, T. C. Identification of nucleolus organizer be of value in such analyses. regions (NORs) in normal and neoplastic human cells by the silver staining technique. Cytogenet. Cell Genet., 19: 185â€”196,1977. 13. Lettre, R., Siebs, â€˜N.,andPaweletz, N. Morphological observations on REFERENCES the nucleolus of cells in tissue culture, with special regard to its composition. NatI. Cancer Inst. Monogr., 23: 107-123, 1966. 1. Busch, H., and Smetana,K. The Nucleolus.pp. 59-114,211-284,384- 14. Mamrack, M. D., Olson, M. 0. J. and Busch, H. Negatively charged 395. 448â€”471.New York: Academic Press, Inc., 1970. phosphopeptides of nucleolar nonhistone proteins from Novikoff hepa 2. Busch,R.K.,andBusch,H.Antigenicproteinsofnucleolarchromatin toma ascites cells. Biochem. Biophys. Res. Commun., 76: 150-157, of Novikoffhepatomaascitescells.Tumoni,63:847-387,1977. 1977. 3. Davis,F. M., Busch,R. K., Yeoman,L. C., and Busch,H.Differencesin 15. Mamrack, M. D., Olson, M. 0. J., and Busch, H. Acidic sequences nucleolar antigens of rat liver and Novikoff hepatoma ascites cells. containing phosphorylated sites in nucleolar nonhistone protein C23 of Cancer Res., 38: 1906-1915, 1978. Novikoff hepatoma ascites cells. Fed. Proc., 37: 1786, 1978. 4. Denton, T. E., Howell, M. W. , and Barrett, J. K. Human nucleolar 16. Olson, M. 0. J., Orrick, L. R., Jones, C., and Busch, H. Phosphorylation organizer chromosome: satellite associations. Chromosoma (Berl.), 55: of acid soluble nucleolar proteins of Novikoff hepatoma ascites cells in 81-84.1976. Vivo. J. Biol. Chem., 249: 2823-2827, 1974. 5. Estable,C., and Sotelo,J. R. Unanuevaestructuracelular;el nucleolo 17. Ornick, L. R., Olson, M. 0. J., and Busch, H. Comparison of nucleolar nema. PubI. Inst. Invest. Cien. Biol. (Montivideo), 1: 105-126, 1951. proteins of normal rat liver and Novikoft hepatoma ascites cells by two 6. Gimenez-Martin,G., de Ia Tome, C., Femandez-Gomez,M. E., and dimensional polyacrylamide gel electrophoresis. Proc. NatI. Acad. Sci. Gonzalez-Fernandez, A. Experimental analysis of nucleolar reorganiza U. S. A., 70: 1316-1320, 1973. tion.J.CellBiol.60:502-507, 1974. 18. Paweletz, N., Siebs, w., and Lettre, A. Untersuchungen zur Argentaff in 7. Gimenez-Martin,G.,de laTorre, C., Lopez-Saez,J.F., and Espona,P. reaktiondes Nukleolus.Z. Zellforsh.Mikrosk.Anat.,76:577-605,1967. Plant nucleolus: structure and physiology. Cytobiologie, 14: 421-462, 19. Ruzicka, V. Zur Geschichte und Kenntnis der Feineren Struktur der 1977. Nucleolen Centraler Nervenzellen. Anat. Anz., 16: 557-563, 1891. 8. Goodpasture,C., and Bloom,S. E. Visualizationof nucleolarorganizer 20. Schwarzacher, H. G., Mikelsaar, A. V., and Schnedl, W. The nature of regions in mammalian chromosomes using silver staining. Chromosoma the Ag-staining of nucleolus organizer regions. Cytogenet. Cell Genet., (Berl.),53: 37-50,1975. 9. Howell,M.W.Visualizationoftheribosomalgeneactivity:silverstains 20: 24â€”39,1978.

Fig. 1. Silver-staining granules in nucleoli of tumors cells. The diameter of the silver-staining granules range from 0.10 to 0.16 @i.m.a, 2 Novikoff hepatoma cells with large numbers of nucleolar granules, a number of which are in rows of 3 or more (arrowheads). Top nucleus, 64 granules; bottom nucleus, 46 granules, all of which are restricted in location to the nucleolus. A small adjacent WBC contains 4 granules (arrow). b, several Novikoff hepatoma cells containing silver granules. These granules differ in density, size, and distribution (arrowhead, arrow). c, HeLa cell nucleus showing granules similar to those in a . The nucleus on the left contains 43 granules in the larger nucleolus (arrowhead) and I 7 granules in the smaller. In the cell on the right, there is a total of 58 granules. In the nucleolus marked with an arrowhead, some granules are in a linear array. Others are in a reticular arrangement (arrow). d, silver-stained nucleus of a KB cell showing the linear arrangement (arrowheads) of separated silver granules similar to that noted in a . This cell contains 31 granules. x 1400. Fig. 2. a, silver stain of spread liver cell nuclei showing a silver-stained reticulum (arrowheads) connecting a few silver-stained granules (arrows). X 1250. b, 2 liver nuclei, 1 (arrowhead) with 1 granule. In the other, the denser silver-stained granules are associated with a less dense reticulum (arrow). x 1000. c, 6-hr regenerating liver showing increased silver-staining reticulum of the enlarged nucleolus. Arrows, granules. x 1000.d, 18-hr regenerating liver. Markedly enlargednucleoliarenotedin thesecells.Thesecellscontainlargernumbersofgranules(arrows)associatedwiththenucleolarsilver-stainedreticulum (pointers). x 1250. Fig. 3. a , Novikoff hepatoma cell in metaphase containing 4 pairs (arrowheads) and 2 single dense granules presumably at NOR regions of â€œnucleolar chromosomes.â€•Adjacent to this cell is an interphase cell containing 40 nucleolar granules in 3 nucleoli. The nucleoli contain rows of dense granules (arrows).b, a similar cell in metaphaseshowingthe presenceof 3 pairsof densegranules(arrowheads)and3 cellswith rowsof nucleolardensegranules (arrows).c, Novikoff hepatomacell in metaphasecontaining 4 pairs of dense granules (arrowheads).d, large Novikoff hepatomacell in metaphase containing several doublets of dense granules (arrowheads). Arrows, rows of nucleolar granules in interphase cells. x 1400. Fig. 4. a, 2 Novikoff hepatoma cells in mitosis showing anaphase and telophase states. Thin arrow, anaphase; thick arrow, telophase. In the anaphase cell, there are Ii dense granules in each nucleus. In the telophase cell, the nuceus has reformed, and many dense granules are visible in each nucleus. Some are arranged in rows (black arrowheads). Some unusually dense rectangular formations are also visible (white-edged arrowheads). Rows of granules are also visible in some interphase cells. b, Novikoff hepatoma cells in telophase (double arrowheads) showing multiple granules (arrows) in nuclei of the daughter cells. The number of granules in these cell pairs were 28 and 67. In adjacent interphase cells, rows of granules are noted. In a pair of cells apparently in very late telophase, similar rows of granules are visible x 1400.

MARCH 1979 859

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Harris Busch, Yerach Daskal, Ferenc Gyorkey, et al.

Cancer Res 1979;39:857-863.

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