Cell cycle analysis and drug inhibition studies of silver staining in synchronous HeLa cells

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Experimental

CELL

CYCLE ANALYSIS SILVER STAINING

HOWARD R. HUBBELL,’

Cell Research 129 (1980) 139-147

AND DRUG INHIBITION STUDIES IN SYNCHRONOUS HELA CELLS

YUN-FAI

OF

LAU,* RONALD L. BROWN and T. C. HSU

Department of Cell Biology, University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, TX 77030 and University of Texas Health Science Center, Graduate School of Biomedical Sciences, Houston, TX 77030, USA

SUMMARY Cytological staining with silver nitrate is specific for a protein associated with chromosomal nucleolus organizer regions and interphase nucleoli. At metaphase the amount of staining present is usually much less than that at interphase. During the transition from mitosis to Gl, as seen in synchronized HeLa cells, the amount of silver staining increases and, by late Gl, is located discretely and completely over the nucleolus. Such staining remains constant through G2. Towards late G2 a slight disorganization of the silver staining material is observed, possibly in preparation for the upcoming mitosis. Cells synchronized at mitosis and treated with either actinomycin D (AMD) or 2-mercapto-1-[2-(4-pyridyl)-ethyl]-benzimidazole (MPB), at concentrations which inhibit ribosomal RNA (rRNA) synthesis, show nucleolar fragmentation and little, if any, apparent increase in silver staining at early Gl. After removal of the MPB, the nucleolar fragments reform nucleoli and the staining increases to control levels. Treatment of mitotic cells with puromycin dihydrochloride does not effect nucleolar morphology or the increase in silver staining. These results directly demonstrate that silver staining is associated with rRNA synthesis.

Recent investigations have used silver nitrate to demonstrate the nucleoli and nucleolus organizer regions (NORs) of many species [lo!]. A direct correlation between the silver-stained NORs (AgNORs) and the sites of the ribosomal genes has been made by in situ hybridization of radioactive ribosomal RNAs (rRNA) [ 1, 51. Cytochemical studies have indicated that the silver binds specifically to protein material associated with the NOR [ 1,6-81. Recently, biochemical studies have demonstrated that a single nucleolar protein binds silver in the manner seen in cytological preparations [9]. In addition, previous studies employing the silver staining of mouse-human hybrids [ 10, 111, cricket oocytes [ 121,2-cell stage mouse embryos [13, 141, human tumor cells [15] and karyotypic variants [16, 171have suggested that the silver staining is related to active

ribosomal cistrons. Since nucleoli are more heavily silver stained than NORs under the same conditions [18], it is of interest to examine the silver staining patterns throughout the cell cycle and their relationship to both rRNA and protein synthesis. This report describes the behavior of silver staining throughout the cell cycle and its response to the inhibition of rRNA synthesis by actinomycin D (AMD) and 2mercapto- 1-[2-(4-pyridyl)-ethyl]-benzimidazole (MPB) and the inhibition of protein synthesis by puromycin dihydrochloride. Our results demonstrate the direct correlation between silver staining and rRNA syn’ Present address: Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, PA 19104, USA. ’ Present address: Departments of Medicine, Biochemistry and Biophysics, HSE1504, University of California, San Francisco, CA 94143, USA. Exp Cell Res 129 (1980)

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I. Sequential Hoechst 33258 fluorescence (fop) and silver staining (bottom) of synchronized HeLa cells throughout the cell cycle: (a) 30 min after plating mitotic cells, two cells having just divided and two cells which have progressed slightly further. The

Fig.

latter two cells do not show fluorescence in the cytoplasm, which is found 1 h after mitosis; (b) early Gl phase, 4 h post-plating; (c) late Gl, 121 h after plating; (d) early S, 15 h; (e) late S, 20 h and cf) G2, 24 h.

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2. Scintillation vial assay of the inhibition of HeLa cell total RNA synthesis by 0.04 pg/ml AMD. After 3 h of treatment, approx. 70% inhibition is found.

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thesis in both untreated and drug-inhibited synchronized HeLa cells.

MATERIALS

AND METHODS

Monolayer cultures of HeLa cells were mown on McCoy’s 5a medium supplemented with 20 % fetal calf serum (FCS) (Gibco). Metaphase cells were obtained by shaking off the mitotic cells from the flasks every 20 min for 3 h. After centrifueation in the cold. the mitotic cells were kept on i;e to prevent their progression through the cell cycle until the beginning of the experiment. In all experiments, the mitotic index was at least 85 %. Actinomycin D (AMD; Cosmegen@; Merck, Sharp & Dohme) was dissolved in medium at a stock concentration of 0.12 pg/ml. This stock was diluted with cells and medium to a final concentration of 0.04 pg/ml. At this concentration AMD preferentially inhibits rRNA svnthesis in HeLa cells 1191.The stock concentration of the MPB (Aldrich Chemical Co.) was 6 mg/ml, dissolved in dimethylsulfoxide (DMSO). This stock solution was diluted to 30 pg/ml with the appropriate amount of cells and medium. Puromycin dihydrochloride (Calbiochem), dissolved in medium, was diluted to a final concentration of 100pg/ml. Cells were released from the mitotic block by direct plating into chamber slides (Lab-Tek) or Petri dishes with microscope slides containing either growth medium or growth medium with a drug. After growth for a specified time interval, the cells were fixed in situ with 3: 1 methanol: acetic acid (Carnoy’s) or 5 : 1 methanol : acetic acid. The progression through the cell cycle and the effects of the drugs were monitored by autoradiography. For cell cycle analysis, cells were pulse-labeled with 2.0 &i/ml [3H]thymidine

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(spec. act. 6.7 Ci/mM; New England Nuclear) for the 1.5min before fixation. Drug effects on RNA or protein synthesis were monitored by similar 15 min pulses with 2.0 &i/ml r3H]uridine (spec. act. 25.9 Ci/mM; NEN) or. 2.0 &i/ml [3H]lysine (spec. act. 2.7 Ci/ mM: NEN), resuectivelv. The slides were coated with Kodak AR-IO fine gram autoradiographic stripping film and exposed for 7-30 days, depending upon the experiment. The film was developed in Kodak Dl9B and the slides were stained in 10% Giemsa. The extent of RNA or protein synthesis inhibition was quantitated by the following scintillation vial assay. Approx. 5x lo5 asynchronous HeLa cells were plated in- each scintillation vial and allowed to grow for 40 h. During the last 16 h, the cells were incubated in medium containing 0.01 @Zi/ml [‘4C]thymidine (spec. act. 54.5 Ci/mM; NEN). The 14C label allowed standardization of the results to a uniform number of cells per vial, and also gave an indication of the cell death rate due to drug treatment. The ‘*C medium was removed and medium containing one of the drugs, at the same concentration used for the cytological nrenarations, was added. The vials were then pulsed’ width 1.O &i/ml [3H]uridine or 1.O &ii ml [3H]lysine for the last 15 min before trichloroacetic acid (TCA) precipitation. The cells were given three 5-min washes with cold 5% TCA. The vials were rinsed well with ethanol and then toluene. ScintiVerse (Fisher) was added to the vials and they were counted in a Beckman LS-IOOC scintillation counter. All data points were corrected for the overlap of the 3H and YZ channels. All cytological preparations were stained with 50% aqueous AgN03 [20, 211. Some slides were counterstained with dilute Giemsa. Other slides were stained preferentially for RNA with the fluorochrome Hoechst 33258 at pH 2.0. The slides were treated with Carnay’s followed by 70% and 95 % ethanol. After airdrying, the slides- were stained with a 1.O pg/ml solution of Hoechst 33258 in phosphate-buffered saline (PBS), adjusted to pH 2.0 with HCI. The cells were rinsed with PBS, pH 2.0, and mounted with glycerol [22,23]. The slides were examined under UV ifiumination with an Olympus Vanox microscope.

RESULTS Metaphase HeLa cells were obtained by mitotic shake-off, replated in fresh medium and fixed at various stages of the cell cycle. The fixed preparations were stained preferentially for RNA with Hoechst 33258 followed by AgN03. Shortly after metaphase (fig. la), the two post-mitotic cells show a small amount of silver staining. The two other cells in fig. 1a are only slightly ahead of the post-mitotic cells, as indicated by the lack of cytoplasmic fluorescence due to Exp Cell Res 129 (1980)

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a Fig. 3. Silver staining of synchronous HeLa cells treated at their release from the mitotic block with 0.04 &ml AMD for (a) 1; (b) 2; and (c) 4 h. The amount of silver staining (arrows) pointed out in some

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of the nucleoli aooears to be about the same as that seen at mitosis. kil preparations were counterstained with Giemsa which causes the intense, non-silver, staining of some nucleoli.

(fig. 1b), show a large amount of silver staining with the beginnings of organization of this material over the nucleoli. Note that the fluorescence pattern demonstrates definite nucleoli and the presence of RNA in the cytoplasm. During late Gl (fig. lc), silver staining is found discretely over the nucleoli with no staining material scattered throughout the nucleus. A similar pattern is observed in early (fig. Id) and late (fig. 1e) S phase. G2 cells appear to have the same amount of staining material as in Gl and S phases, and may exhibit a slight disorganization of the silver grains, possibly in preparation for the upcoming mitosis. Continuous growth of asynchronous HeLa cells in 0.04 pg/ml AMD shows that after 3 h approx. 70 % of all RNA synthesis 1 2 3 4 5 6 7 a tiO”TS is inhibited (fig. 2). Comparison of the autoFig. 4. Scintillation vial assay of the inhibition of HeLa cell total RNA synthesis by 30 pg/ml MPB. radiographs of [3H]uridine pulses from conAfter 30 min of treatment, approx. 80% inhibition is trol cells and cells continuously treated for found. At 4 h (arrow) the MPB was removed by 1, 2 or 4 h demonstrates that no uridine is washing and the cells incubated in fresh medium. Within 1 h, a 4-fold increase in RNA synthesis occurs. incorporated into nucleoli in the presence of

RNA, which occurs within 1 h of plating. Apparently, the amount of silver staining increases shortly after cell division, at very early Gl. Early Gl cells, 4 h after plating

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5. Silver staining of synchronous HeLa cells treated at their release from the mitotic block with 30 pg/ml MPB for (a) 1; (6) 2; and (c) 4 h. The amount of silver staining (arrows) pointed out in some of the nucleoli appears to be about the same as that seen at mitosis. After 4 h of treatment the cells were

washed and then incubated in fresh medium for an additional (d) 1; (e) 2; and (f) 4 h. Note the increase in silver staining and reformation of nucleoli seen after 1 h in fresh medium. All preparations were counterstained with Giemsa which causes the intense, nonsilver, staining of some nucleoli.

AMD. These data indicate that the rRNA synthesis is inhibited by AMD treatment. Mitotic HeLa cells were directly plated into medium containing AMD and allowed to gl.ow for up to 4 h. After 1 h of treatment, the nucleoli appear as several small fragments and the amount of silver staining,

qualitatively, remains at levels seen only during metaphase (fig. 3a, arrows). Continued treatment for 2 or 4 h (fig. 3 b, c, respectively) shows nucleolar fragmentation with no increase in silver impregnation (arrows). Asynchronous cultures treated in the same manner show nucleolar segrega-

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Fig. 6. Scintillation vial assay of the inhibition of HeLa cell protein (0) and total RNA (A) synthesis by 100pg/ml puromycin dihydrochloride. After 15 min of treatment over 92% of protein synthesis was inhibited. A constant decrease in total RNA synthesis was observed and after 4 h, approx. 76% of RNA synthesis was inhibited.

tion with a decrease in silver staining from control levels to that apparently found in metaphase plates, within 1 h. Cells were also incubated in 30 pg/ml MPB, a reversible inhibitor of RNA synthesis [24]. Scintillation vial assays of RNA synthesis in the presence of MPB indicate that, within the first 30 min of treatment, over 78% of the synthesis is inhibited (fig. 4). After 4 h of continuous incubation, the drug was removed by washing the cells with Hanks’ balanced salt solution (without Ca2+ and Mg2+) and then medium. Within 1 h, a I-fold increase in the amount of RNA synthesis occurs. Autoradiography demonstrates that within 1 h no RNA synthesis is present in the nucleoli and that after the removal of the MPB, nucleolar RNA production is resumed. Mitotic cells, plated into MPB containing medium, show silver staining patterns similar to those seen after incubation in AMD. After 1, 2, and 4 h of treatment, the cells show typical nucleolar segregation and only Exp Cell Res 129 (1980)

Fig. 7. [“H]Uridine pulse-labeling of synchronous HeLa cells treated with 100 pg/ml puromycin dihydrochloride. All cells were nuked with 2.0 &i/ml [“HI uridine for the last 15’min before harvesting! (n) untreated control cells; (b) cells treated for 4 h. Note that in the treated cells there are grains over the nucleoli indicating that rRNA synthesis is still occurring. All control and drug-treated cells were pulselabeled, exposed and developed at the same time.

a small amount of silver staining, comparable to that found in metaphase (fig. 5 a-c, arrows). Removal of the MPB initiates the reformation of whole nucleoli and an increase in silver staining material (fig. S&f). Asynchronous cultures show similar reaction to MPB treatment. After incubation in the presence of MPB for up to 4 h, the nucleoli are typically dispersed and the amount of silver staining present is reduced to a level usually seen during mitosis. Upon washing out the MPB, normal nucleolar morphology and silver staining are restored. Cells were incubated in the presence of 100 pg/ml puromycin to explore the effect of protein synthesis inhibition on silver staining. This concentration of puromycin also inhibits rRNA synthesis [25]. At this concentration, over 92% of protein synthesis is halted within 15 min. RNA synthesis, however, is also inhibited. After 4 h continuous incubation in puromycin, approx. 76% of RNA synthesis is stopped (fig. 6). Autoradiography demonstrates that, even in the presence of puromycin,

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rRNA synthesis continues (fig. 7), although chromosomes [27, 281. These fragments apparently combine with the nucleolus orit is at a decreased level. Puromycin apparently has no effect on ganizer to form the interphase nucleoli [28]. silver staining. In synchronous cultures, in- At this point it is not clear whether the cubations of 1, 2 and 4 h (fig. 8a-c, re- silver staining protein material associated spectively) do not inhibit the increase in with these fragments undergoes a conforsilver staining material. These cells appear, mational change and increases its silver afboth in staining and nuclear morphology, finity, or whether the fusion of nucleolar to be quite similar to control cells (cf fig. particles increases the protein concentra1). Synchronous and asynchronous cultures tion in the area so that the staining betreated with puromycin for up to 10 h also comes visible. Regardless, the increase in show no change in silver staining as com- staining appears to coincide with the resumption of 4X3 rRNA synthesis and its pared with untreated controls. processing [29]. Previous studies of silver staining have DISCUSSION demonstrated its relationship with riboOur results demonstrate that the amount somal gene activity [IO-171. In all cases, of silver staining increases significantly the gene activity and staining were assayed towards the end of mitosis. The silver stain- separately and their relationship was deing material appears to be the same as the rived by temporal and spatial criteria. The pre-nucleolar bodies described by Tandler drug inhibition studies reported here di[26]. Similar nucleolar fragments have been rectly demonstrate the correlation between shown to reside scattered in the cytoplasm the synthesis of rRNA and silver staining or as a coat around the outside of the by controlling the rRNA synthesis in the Exp Cd

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cells being stained. Two recent reports [ 14, 301, using cycloheximide and AMD, gave a similar staining in asynchronous cells. The use of MPB is informative, not only because it is a reversible inhibitor of rRNA synthesis, but because it does not inhibit the reappearance of nucleolar components which fuse with the nucleolus organizers to reform the nucleoli at interphase [31]. Thus, the inhibition of silver staining in MPBtreated cells cannot be attributed to the lack of nucleolar components or the blockage of nucleolar reformation. Such blockage may not allow sufficient silver accumulation which would be visible at the light microscope level. Treatment of cells with 100 ,ug/ml puromycin dihydrochloride reduces, but does not eliminate, rRNA transcription. The reformation and normal staining of nucleoli in the presence of puromycin indicates that no new protein synthesis is necessary for nucleolar formation or silver binding. Incubation for up to 10 h (late Gl), demonstrates that the silver binding protein has a relatively long half-life. Our results correlate well with previous molecular reports of rRNA synthesis in synchronous HeLa cells in the presence of cycloheximide [29]. A recent investigation [25] has indicated the relationship between rRNA transcription and protein synthesis using either cycloheximide or puromycin. Both drugs appear to inhibit rRNA synthesis by inhibiting the production of a short-lived protein, or set of proteins, necessary for the binding of RNA polymerase I to rDNA. Thus, puromycin indirectly inhibits the production of 4% rRNA. Since the amount of silver staining seen in puromycin-treated cells is comparable to that seen in untreated cells, the silver staining protein is probably involved in the processing or packaging of rRNA and not in rRNA transcription. The fact Exp CellRes 129 (1980)

that some 45s rRNA is transcribed in the presence of puromycin probably explains the normal amount of staining present. Since silver staining has been unequivocably correlated with rRNA synthesis, a question arises about the staining of chromosomal NORs, in which no rRNA is synthesized [27, 291. One possibility is that during chromosome condensation, some of the silver staining protein is fortuitously trapped in the NOR. Alternatively, the structure with which the silver staining protein is associated remains in contact with the NOR so that the resumption of rRNA synthesis can be immediate and efficient. Silver staining of cytological preparations during chromosome condensation does show the decrease of staining as the cell progresses toward metaphase, with the remaining stain associated with the NOR [32]. In addition, earlier studies using AMD in a protocol similar to ours indicate that previously synthesized nucleolar RNA persists through mitosis [33]. Thus, it is possible that protein material associated with this RNA may be responsible for silver staining. Another possibility, however, is that the silver binding protein seen in the NORs differs from the protein which is involved in the increase in staining at early Gl which makes up the bulk of the staining material in the interphase nucleolus. Electron microscopy of NORs indicates that they are made up of an area of low electron density known as ‘fibrillar centres’ [28, 34-361. During late telophase the pars fibrosa and pars granulosa become reassociated with the ‘fibrillar centres’ [35] and rRNA is synthesized in the surrounding pars fibrosa [28]. Thus, the silver staining seen at metaphase, and that which remains during AMD or MPB treatment, may be related only to these ‘fibrillar centres.’ The staining there-

Cell cycle, drug inhibition and silver staining fore, may be indicative of two separate events and may even be due to silver binding of two different proteins or sets of proteins. Recently a silver binding protein, purified from isolated interphase nucleoli, has been identified in Novikoff hepatoma cells [9]. Further biochemical and immunochemical characterization of this protein should lead to an understanding of the relationship between interphase and metaphase silver staining. The authors would like to thank MS Cheryl Collie for technical assistance. This work was supported, in part, by research grants VC-21 from the ACS and ENV 7682241 from the NSF.

REFERENCES 1. Goodpasture, C & Bloom, S E, Chromosoma 53 (1975) 37. 2. Dev, V G, Tantravahi, R, Miller, D A & Miller, 0 J, Genetics 86 (1977) 389. 3. Schmid, M, Chromosoma 66 (1978) 361. 4. - Ibid 68 (1978) 131. 5. Hsu, T C, Spirito, S E & Pardue, M L, Chromosoma 53 (1975) 25. 6. Peters, A, Quart j microsc sci 96 (1955) 84. 7. Howell, W M, Denton, T E & Diamond, J R, Experientia 31 (1975) 260. 8. Schwarzacher, H G, Mikelsaar, A-V & Schnedl, W, Cytogenet cell genet 20 (1978) 24. 9. Hubbell, H R, Rothblum, L I & Hsu, T C, Cell biol int rep 3 (1979) 615. 10. Miller, D A, Dev, V G, Tantravahi, R & Miller, 0 J, Exp cell res 101 (1976) 235. 11. Miller, 0 J, Miller, D A, Dev, V G, Tantravahi, R & Croce, C M, Proc natl acad sci US 73 (1976) 4531.

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Sweden

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12. Howell, W M, Chromosoma 62 (1977) 361. 13. Engel, W, Zenzes, M T & Schmid, M, Human genet 38 (1977) 57. 14. Hansmann, I,’ Gebauer, J, Bihl, L & Grimm, T, EXD cell res 114 (1978) 263. 1.5. Hubbell, H R & Hsu, T C, Cytogenet cell genet 19 (1977) 185. 16. Miller, D A, Breg, W R, Warburton, D, Dev, V G & Miller, 0 J, Human genet 43 (1978) 289. 17. Lau, Y-F, Wertelecki, W, Pfeiffer, R A & Arrighi, F E, Human genet 46 (1979) 75. 18. Lau, Y-F & Arrighi, F E, Cytogenet cell genet 17 (1976) 51. 19. Perry, R P, Exp cell res 29 (1963) 400. 20. Bloom, S E & Goodpasture, C, Human genet 34 (1976) 199. 21. Lau, Y-F, Pfeiffer, R A, Arrighi, F E & Hsu, T C, Am j human genet 30 (1978) 76. 22. Hilwig, I & Gropp, A, Exp cell res 91 (1975) 457. 23. Lau, Y-F, Brown. R L & Arrighi, FE. EXD cell res 110 (1977) 57. 24. Bucknall, R A & Carter, S B, Nature 213 (1967) 1099. 25. Mishima. Y. Matsui, T & Muramatsu. M. J biochem 85 (1979) 807. 26. Tandler. C J. Exo cell res 17 (1959) 560. 27. Hsu, T C, Arrigh’i, F E, Klevecz, R R & Brinkley, B R, J cell biol26 (1965) 539. 28. Lepoint, A & Goessens, G, Exp cell res 117(1978) 89. 29. Fan, H & Penman, S, J mol biol59 (1971) 27. 30. Hofgartner, F J, Krone, W & Jain, K, Human genet 47 (1979) 329. 31. Lepoint, A, Thesis, University of Liege (1977). 32. Pelliccia, F, de Capoa, A, Belloni, G, Rocchi, A & Ferraro, M, Exp cell res 115 (1978) 439. 33. Phihips, S G, J cell biol53 (1972) 611. 34. Goessens, G, Compt rend acad sci 277 (1973) 325. 35. Goessens, G & Lepoint, A, Exp cell res 87 (1974) 63. 36. Mirre, C & Stahl, A, J ultrastruct res 56 (1976) 186. Keceived December 5, 1979 Revised version received March 27, 1980 Accepted March 31, 1980

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