Biol Cell (1992) 74, 281-286

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© Elsevier, Paris

Original article

Effects of camptothecin, an inhibitor of D N A topoisomerase I on ribosomal gene structure and function in TG cells Fulvia Farabegoli, Marzia Govoni, Francesco Novello * Dipartimento di Patologia Sperimentale, sezione di Patologia Generale, via S Giacomo 14, Universitfl di Bologna, Italy (Received 13 September 1991; accepted 11 February 1992)

Summary - The effects of camptothecin treatment and topoisomerase I inhibition on ribosomal gene structure and function were

investigated in TG cells, a human tumour cell line. 90- and 180-min treatments with 25/aM camptothecin resulted in an increased DNA fragmentation and decreased activity of topoisomerase I in cell extracts. After 180-min treatment, the incorporation of labelled uridine into total cell RNA was reduced to 39% and the ribosomal RNA synthesis to 10%, as compared to values of control cells. At the ultrastructural level, the nucleolar components appeared to be segregated; after selective DNA staining, with osmium-amine complex, a part of the nucleolar chromatin of treated cells showed the presence of thin, extended DNA filaments, superimposable to those present in control cells. ribosomal genes / rRNA synthesis / electron microscopy / topoisomerase ! / camptothecin

Introduction

In interphasic eukaryotic cells, r R N A synthesis and processing occur in the nucleolus, where highly repeated copies of ribosomal genes are clustered and transcribed by R N A polymerase I molecules [12]. Using Ag-nucleolus organizer region (NOR) staining, a technique which contrasts a group of acidic proteins associated to ribosomal genes; the latter have been located in two nucleolar components, the fibrillar centres (FC) and the surrounding dense fibrillar component (DF) [18, 25]. The fine structure of ribosomal genes in situ has been widely investigated in mammalian cells using a Feulgen-like reaction which selectively contrasts D N A in thin sections using the osmium-amine complex [5, 12]. After Feulgen-like staining, the nucleolar D N A appeared to be organized either in nucleosomal-like fibres, 11 - 30 nm thick, or in roundish, loose agglomerates of extended fibres, 2 - 3 nm thick, which did not show a nucleosomal-like organization [9, 10] and which corresponded to the Ag-NOR stained nucleolar components [8]. The fully extended structure of ribosomal genes associated to Ag-NOR proteins was also visible when r R N A synthesis was arrested, in metaphasic chromosomes [19] and after actinomycin D treatment [20], suggesting .that structural modifications of this part of ribosomal genes were not involved in transcriptional control [11]. Recently, it has been suggested that a new enzyme, topoisomerase I, might be involved in ribosomal gene transcription; this enzyme is closely associated with actively transcribed ribosomal genes and with RNA polymerase I molecules [3, 15, 16, 23, 28, 29]. Using a immunocytochemical technique, the enzyme has been detected

in the peripheral zone of the fibrillar centres and in the surrounding dense fibrillar component of the nucleolus [26], the same nucleolar components where Ag-NOR proteins and non-nucleosomal ribosomal D N A are located. Topoisomerase I can induce conformational and topological modifications in the D N A molecule, by introducing and resealing transient DNA breaks in the phosphodiester backbone of one D N A strand [33] ; the resealing step can be specifically inhibited by camptothecin [21]. We have investigated the effects of topoisomerase I inhibition by camptothecin treatment on T G cells, a human tumour cell line which actively synthesizes rRNA. The aim of this investigation was to verify if this treatmer~t affected r R N A synthesis and the fine structure of that part of ribosomal genes which have an extended structure.

Materials a n d m e t h o d s

TG cells were grown in plastic flasks in RPMI medium, supplemented with non-essential amino acids, 100 U/ml penicillin, 100/zl/ml streptomycin and 10% of foetal calf serum. Camptothecin (NSC 94600) was dissolved in dimethylsulfoxide immediately before use [14] and was added to the culture medium at 25 tzM final concentration for 90 min or 180 min. This concentration was shown to inhibit topoisomerase I activity [21] without affecting cells viability in our experimental condition.

Fluorometric analysis of DNA unwinding (FADU) Fluorometric analysis of DNA unwinding was performed to evaluate the DNA strand breaks induced by camptothecin treatment, as described by Birnboim and Jevcak [2].

Electron microscopy * Correspondence and reprints

TG cells were f'Lxedin flasks with 4°70p-formaldehyde, in 0.I M S6rensen buffer pH "].4, for I h at room temperature, scraped

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and centrifuged. The pellet was fragmented and rinsed with S6rensen buffer. Some fragments were post-fixed in 107o OsO 4, dissolved in S6rensen buffer 0.1 M pH 7.4, dehydrated in ethyl alcohol and embedded in Epon. The remaining fragments, fixed only in p-formaldehyde, were dehydrated in dimethylformamide and embedded in Lowicryl K4M [1]. Thin sections of Epon embedded samples were stained with uranyl acetate and lead citrate. The Feulgen-like reaction using the osmium-amine complex was performed on thin sections of Lowicryl K4M embedded samples as previously described [ 8 - 1 I, 19, 20]. R N A synthesis

[3H]-uridine (15/~Ci/~ml) was added to control and treated TG cells for the last 15 min of culture, thereafter 0.1 mM unlabelled uridine in culture medium was added and the cells harvested by centrifugation. After two washings in PBS, samples were used to measure the radioactivity of the acid soluble fraction [1 I]. Two nuclease RNA fractions were separated from isolated and purified nuclei by a hypertonic sucrose procedure [7] and by stepwise extraction with phenol at 4 and 50°C [17]. Radioactivity was evaluated as previously described [11] and RNA was measured in nuclear and nucleolar fractions according to Munro and Fleck [24]. DNA was estimated according to Burton [41.

Results Biochemical findings

In T G cells treated with 2 5 / z M c a m p t o t h e c i n the activity o f topoisomerase I in total cell extract decreased to 66 and 43% o f control values (table I). At the same experimental times measured in F A D U assay, the a m o u n t o f double stranded D N A remaining after partial alkali treatment was reduced to 3 and 13% respectively, while control value showed 47% double stranded D N A ; both these results suggest that camptothecin affected the activity o f topoisomerase I, resulting in an increased n u m b e r o f strand breaks in D N A treated cells. T o p o i s o m e r a s e I activity p r o b a b l y involves regulation o f R N A synthesis [34] ; therefore we studied (table II) the synthesis o f total nuclear R N A and o f ribosomal R N A , represented by the 50°C fraction, by means o f [3H]uridine incorporation in control and c a m p t o t h e c i n treated cells. After 180 min incubation with the drug, the incorporation o f [3H]-uridine in total nuclear R N A decreased to less than 40% o f control values; this inhibition reached 90% in the 50°C R N A fraction which c o r r e s p o n d e d to nucleolar R N A .

Topoisomerase I assay

Electron m i c r o s c o p y

The extract containing topoisomerase I activity was prepared according to the procedure described by Tricoli et al [32] : control and treated TG cells were PBS-washed and resuspended in 10 mM Tris-HCl, pH 7.8, 0.5 mM EDTA, 0.005°7o Triton X-100 and 1 M KCl and frozen at 20°C. The topoisomerase I activity was determined by the method of Kowalski [22], modified by Tricoli and Kowalski [31], measuring the ATP-independent relaxation of supercoiled pM2-DNA.

Uranium a n d lead staining In figure 1 a control T G cell is s h o w n ; in the nucleolus three small fibrillar centres (FC) are clearly visible, surr o u n d e d by a very electron-opaque layer, c o r r e s p o n d i n g to the dense fibrillar component (DF). The nucleolus shows a reticulated aspect due to the thread-like organization o f fibrils and granules (G).

Table I. Topoisomerase I activity and percentage of double stranded (ds) DNA of TG cell after 25 ~M camptothecin treatment. Topoisomerase I activity a A U/ng protein %

Control Camptothecin Camptothecin

2.58 1.70 I. 10

90 min 180 rain

% ds b

-66 43

47 3 13

P

a Preparation of cell extracts and determination of enzyme activity have been performed according to Tricoli et al [32]. Results are expressed in arbitrary units per ng of protein. b Percent of double stranded DNA remaining 45 rain after partial alkali treatment in fluorometric analysis of DNA unwinding (FADU) according to Birnboim and Jevcak [2].

Table II. RNA s~'nthesis of TG cells after 25 tzM camptothecin treatment. Nuclear R N A [SH]-urid ine incorporation

Control Camptothecin Camptothecin

90 min 180 min

0.13 0.09 0.05

070 Control

-70 39

50 °C RATA fraction a [3H]-uridine % incorporation

0.29 0.15 0.03

-52 10

aNuclear RNA was fractionated with phenol at different temperatures. The RNA fraction extracted at 50°C corresponded to nucleolar RNA [17]. Results are expressed in CPM//zg DNA/acid soluble fraction (CMP/~g DNA).

Effects of camptothecin on ribosomal genes

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Fig 1. Nucleolar aspect of a control TG cell. Three fibrillar centres (FC) surrounded by a thick rim of dense fibrillar component (DF) and granules (G) are visible; p-formaldehyde and OsO 4 fixation, uranium and lead staining. Bar l tzm ( x 28000). Fig 2. TG cell after 3 h, 25/~M camptothecin treatment showing segregation of nucleolar components (FC, DF, G). Arrows indicate perichromatin granules at the nucleolar border; p-formaldehyde and OsO4 fixation, uranium and lead staining. Bar 1 tzm ( x 28000).

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Fig 3. Control TG cell after selective DNA staining, using osmium-amine complex. Nucleolus appears as an electron-translucent area, where intranucleolar chromatin clumps and fibres (arrows) are in contact with lighter DNA agglomerates (small arrows). Bar 0.5 tzm ( x 33 000). a. At higher magnification agglomerates appear to be composed of thin DNA filaments (small arrows). Bar 0.2 tzm ( x 75 000). Fig 4. TG cell after 3 h, 25/zM camptothecin treatment. Selective DNA staining using osmium-amine complex. A large agglomerate of lightly contrasted DNA fibres (small arrows) is present at the periphery of the nucleolus, where a few clumps of more contrasted intranucleolar DNA are also visible (arrows). Bar 0.5/zm ( x 33000). a. higher magnification of figure 4. Very thin DNA filaments of the large agglomerate are shown (small arrows). Bar 0.5/zm ( x 75000).

Effects of camptothecin on ribosomal genes After 180 rain camptothecin treatment (fig 2) the threadlike organization of the nucleolar components is no longer visible : dense fibrillar (DF) and granular (G) components are distributed in two separate compact agglomerates. The material derived from fibrillar centres (FC) is visible as an unique, large agglomerate. Perichromatin granules are present at the periphery of the nucleolus (arrows). The fine structure of ribosomal genes in situ was investigated using the Feulgen-like osmium-amine reaction which selectively stains DNA. In control T G cells (fig 3) the nucleolus appeared as an extremely electron-translucent area, where only intranucleolar chromatin is contrasted ; this chromatin is organized into highly contrasted clumps and fibres (arrows) or in slightly electron-opaque agglomerates (small arrows) which are composed, at high magnification, by very thin filaments of DNA (fig 3a). After 180 min camptothecin treatment, redistribution of intranucleolar chromatin occurred (fig 4) : a few clumps of condensed intranucleolar chromatin (arrows) are present at the periphery of the nucleolar area. This chromatin appears to be in contact with a large DNA agglomerate (small arrows) which is composed of thin extended filaments (fig 4a).

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which maintained an extended structure, superimposable to that present in control cells (figs 3a, 4a). Our findings support previously reported ultrastructural data that a part of ribosomal genes is maintained in a highly dispersed state, independent of the transcriptional activity [11, 19, 20]. The presence of a permanent, nonnucleosomal structure of part of ribosomal genes has also been demonstrated in Friend cells by means of a psoralen photo cross-linking technique [6]. At the moment we are still unable to establish whether this part of chromatin corresponds to the inactive, potentially active or active ribosomal genes. Comprehension of its functional role may contribute to understanding the fine regulation of ribosomal gene transcription.

Acknowledgments The authors are grateful to Dr Christine Betts-Eusebi for technical assistance. This work was supported by grants from Ministero Pubblica Istruzione, Pallotti's legacy for Cancer Research and Progetto Finalizzato Oncologia 88/781.

References Discussion Eukaryotic ribosomal genes represent a good model for investigating the relationship between structure and function of chromatin in situ at the ultrastructural level. In mammalian cells, after selective DNA staining by osmiumamine complex, ribosomal genes, which are associated with acidic proteins, appeared to be organized in roundish agglomerates composed of thin filaments 2 - 3 nm thick. This structure is completely different from the rest of nucleolar and nuclear chromatin which clearly showed a nucleosomal and supranucleosomal organization [ 8 - l 1, 13, 19, 20]. The extended structure of ribosomal chromatin also persisted into metaphase [19] and after actinomycin D treatment [20]; for these reasons it has been suggested that structural modification was not involved in the transcriptional control of this part of ribosomal genes. In the present report we have investigated the effects of topoisomerase I inhibition on ribosomal gene structure and function after 25 tzM camptothecin treatment. This enzyme which modifies DNA conformation [33], has been presumed to be involved in ribosomal gene transcription [3, 15, 16, 28]. Our results have shown that after camptothecin treatment extensive DNA fragmentation occurred and topoisomerase I was inhibited (table I). The enzyme inhibition affected the [3H]-uridine incorporation in treated T G cells (table II) : this effect was particularly marked in the nucleolar fraction, thus suggesting an inhibition of rRNA symhesis. An inhibition of ribosomal RNA synthesis was also supported by ultrastructural findings which showed segregation of the nucleolar components (fig 2). This nucleolar aspect resembled the results obtained using actinomycin D in the same cells [20] and it is in agreement with previous ultrastructural data about effects of camptothecin on rat liver and ME-180 cell culture nucleoli [13, 27]. We also investigated the effects of topoisomerase I inhibition on the fine structure of ribosomal genes in T G cells after selective DNA staining. Camptothecin treatment caused changes in the distribution of intranucleolar chromatin but did not seem to modify the extended configuration of a part of putative ribosomal genes,

1 Altman LG, Schneider BG, Papermaster DS (1984) Rapid embedding of tissues in Lowicryl K4M for immuno-electron microscopy. J Histochem Cytochem 32, 1217-1223 2 Birnboim HC, Jevcak JJ (1981) Fluorometric method for rapid detection of DNA strand breaks in human white blood cells produced by low doses of radiation. Cancer Res 41, 1889-1892 3 BrillSJ, Di Nardo S, Voelkel-Meiman K, Sternglanz R (1987) Need for DNA topoisomerase activity as a swivel for DNA replication and for transcription of ribosomal DNA. Nature 326, 414-416 4 Burton K (1956) A study of the condition and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic-acid. Biochem J 62, 315-323 5 Cogliati R, Gautier A (1973) Mise en 6vidence de I'ADN et des polysaccharides/~ I'aide d'un nouveau r6actif ttde type Schiff~. CR Acad Sci D Paris 276, 3041-3044 6 Conconi A, Widmer R, Koller T, Sogo JM (1989) Two different chromatin structures coexist in ribosomal RNA genes throughout the cell cycle. Cell 57, 753-761 7 Dabeva MD, Dubov KP, Hadjiolov AA, Stoykova AA (1978) Quantitative analysis of rat liver nucleolar and nucleoplasmic ribosomal ribonucleic acids. Biochem J 171, 367-374 8 Derenzini M, Hernandez-Verdun D, Bouteille M (1981) Relative distribution of DNA and NOR-proteins in nucleoli visualized by simultaneous Feulgen-like and Ag-NOR staining procedures. Biol Cell 40, 147-150 9 Derenzini M, Hernandez-Verdun D, Bouteille M (1982) Visualization in situ of extended DNA filaments in nucleolar chromatin of rat hepatocytes. Exp Cell Res 141,463-469 10 Derenzini M, Hernandez-Verdun D, Bouteille M (1983) Subunit configuration of rat hepatocyte chromatin fixed in situ as visualized in thin sections selectively stained for DNA. Biol Cell 41, 161-163 11 Derenzini M, Pession A, Betts-Eusebi CM, Novello F (1983) Relationship between the extended non-nucleosomai intranucleolar chromatin in situ and ribosomal RNA synthesis. Exp Cell Res 145, 127-143 12 Derenzini M, Thiry M, Goessens G (1990) Ultrastructurai cytochemistry of the mammalian cell nucleolus. J Histochem Cytochem 38, 1237-1256 13 Gajkowska B, Puvion E, Bernhard W (1977) Unusual perinucleolar accumulation of ribonucleoprotein granules induced by camptothecin in isolated liver cells. J Ultrastruc Res 60, 335-347

286

F Farabegoli et al

14 Gallo RC, Whang-Peng J, Adamson RH (1971) Studies on the antitumor activity, mechanism of action and the cell cycle effects of camptothecin. J N a t l Cancer Inst 46, 789-795 15 Garg LC, Di Angelo S, Jacob ST (1987) Role of DNA topoisomerase .I in the transcription of supercoiled rRNA gene. Proc Natl Acad Sci USA 84, 3185-3188 16 Gilmour DS, Pflugfelder G, Wang JC, Lis JT (1986) Topoisomerase I interacts with transcribed regions in Drosophila cells. Cell 44, 401-407 17 Hadjiolov AA, Dabeva MD, Mackedonski VV (1974) The action of a-amanitin in vivo on the synthesis and maturation of mouse liver ribonucleic acid. Biochem J 138, 321-334 18 Hernandez-Verdun D, Hubert J, Bourgeois C, Bouteille M (1978) Identification ultrastructurale de l'organisateur nucl6olaire par la technique ~ l'argent. CR Acad Sci Sdr D Paris 287, 1421-1423 19 Hernandez-Verdun D, Derenzini M (1983) Non-nucleosomal configuration of chromatin in nucleolar organizer regions of metaphase chromosomes in situ. Eur J Cell Biol 31, 360-365 20 Hernandez-Verdun D, Derenzini M, Bouteille M (1984) Relationship between the Ag-NOR proteins and ribosomal chromatin in situ during drug-induced RNA synthesis inhibition. J UItrastruct Res 88, 55-65 21 Hsiang YH, Hertzberg R, Hecht S, Liu LF (1985) Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem 260, 14873-14878 22 Kowalski D (1979) A procedure for the quantitation of relaxed closed circular DNA in the presence of superhelical DNA: an improved fluorometric assay for nicking-closing enzyme. Anal Biochem 93, 346-354 23 Muller MT, Pfund WP, Mehta VB, Trask DK (1985) Eukaryotic type I topoisomerase is enriched in the nucleolus and catalytically active on ribosomal DNA. EMBO J 4, 1237-1243 24 Munro HN, Fleck A (1966) Recent developments in the

measurements of nucleic acids in biological materials. Analyst 91, 78-88

25 Ploton D, Bobichon H, Adnet J (1982) Ultrastructural localization of NOR in nucleoli of human breast cancer tissues using a one-step Ag-NOR staining method. Biol Cell 43, 229-232 26 Raska I, Reimer G, Jarnik KM, Kostrouch Z, Raska K (1989) Does the synthesis of ribosomal RNA take place within nucleolar fibriUar centers or dense fibrillar component ? Biol Cell 65, 79-82 27 Recher L, Chan H, Briggs L, Parry N (1972) Ultrastructural changes inducible with the plant alkaloid Camptothecin. Cancer Res 32, 2495-2501 28 Rose KM, Szopa J, Han FS, Cheng YC, Richter A, Scheer U (1988) Association of DNA topoisomerase I and RNA polymerase I: a possible role for topoisomerase I in ribosomal gene transcription. Chromosoma 96, 411-416 29 Scheer U, Benavente R (1990) Functional and dynamic aspects of the mammalian nucleolus. BioEssays 12, 14-21 30 Trask DK, Muller MT (1988) Stabilization of type I topoisomerase-DNA covalent complexes by actinomycin D. Proc Natl Acad Sci USA 85, 1417-1421 31 Tricoli JV, Kowalski D (1983) Topoisomerase I from chicken erythrocytes : purification, characterization and detection by a deoxyribonucleic acid binding assay. Biochem 22, 2025- 2031 32 Tricoli JV, Sahai BM, McCormick P J, Jarlinski SJ, Bertram JS, Kowalski D (1985) DNA topoisomerase I and II activities during cell proliferation and the cell cycle in cultured mouse embryo fibroblast (C3HIOT1/2) cells. Exp Cell Res 158, 1-14 33 Vosberg HP (1985) Topoisomerases: enzymes that control DNA conformation. Curr Top Microbiol Immunol 114, 19-102

34 Zhang H, Wang JC, Liu LF (1988) Involvement of DNA topoisomerase I in transcription of human ribosomal RNA genes. Proc Natl Acad Sci USA 85, 1060-1064