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Chromosome Research

, Volume 27, Issue 1–2, pp 95–108 | Cite as

Cytogenetic instability of chromosomal nucleolar organizer regions (NORs) in cloned mouse L929 fibroblasts

  • Olga V. ZatsepinaEmail author
Original Article
  • 181 Downloads

Abstract

Ribosomal DNA (rDNA) gene codes for 18S, 5.8S, and 28S rRNA form tandem repetitive clusters, which occupy distinct chromosomal loci called nucleolar organizer regions (NORs). The number and position of NORs on chromosomes are genetic characteristics of the species although within a cell, the NOR sizes can significantly vary due to loss or multiplication of rDNA copies. In the current study, we used mouse L929 fibroblasts, the aneuploid cells which differ in the FISH- and Ag-NOR numbers, to examine whether the parental NOR variability is inherited in clones. By statistical analysis, we showed that the cloned fibroblasts were able to restore the NOR numerical characteristics of the parental cells after long-term culturing. These results support the idea that mammalian cells may have mechanisms which control the number and activity of NORs at the population level. In L929 fibroblasts, we also regularly observed laterally asymmetry of FISH-NORs that evidenced in an unequal distribution of the mother rDNA copies between the daughter cells in mitosis.

Keywords

mouse L929 fibroblasts clones nucleolar organizer regions rDNA-FISH Ag-NOR staining variability 

Abbreviations

NOR

Nucleolar organizer region

rDNA

Ribosomal DNA

rRNA

Ribosomal RNA

5′ETS

5′ External transcribed spacer

ITS1 and ITS2

The first and second internal transcribed spacers

FISH

Fluorescence in situ hybridization

FISH-NOR

NOR hybridized with rDNA-FISH probes

Ag-NOR

Silver-positive NOR

Nucleolar organizer region (NOR) is the term to designate particular chromosomal sites organizing the nucleolus, the largest multifunctional nuclear domain which plays main roles in ribosome biogenesis, participates in lifespan regulation, and is a guardian of cellular homeostasis and genome integrity (Boisvert et al. 2007; Grummt 2013; Lam and Trinkle-Mulcahy 2015; Tsekrekou et al. 2017; Tiku and Antebi 2018). The NOR sites are present only on certain chromosomes of a karyotype (referred to as secondary constrictions on opposite to primary constrictions, or centromeres, present in all chromosomes), and their location is an important cytogenetic characteristic of the species and various cell lines (McStay 2016).

In mammals, NORs consist of tandemly repeated sequences of ribosomal DNA (rDNA) genes encoding 18S, 5.8S, and 28S ribosomal RNA (rRNAs) which are separated by intergenic nontranscribed spacers. Under normal conditions, RNA polymerase I transcribes rRNA genes to produce a single 47S precursor for the three rRNAs that undergoes chemical modification and processing cleavage reactions to remove the external (ETS) and internal (ITS) transcribed spacers to yield mature 18S, 5.8S, and 28S rRNAs (Mullineux and Lafontaine 2012). These processes require a participation of numerous appropriate proteins and auxiliary factors and are strictly coordinated with cell proliferation. Genes encoding the forth rRNA type, 5S rRNA, are physically unlinked to NORs (Gibbons et al. 2015).

In mitosis, when rDNA transcription drops down, nucleoli disintegrate and the majority of their proteins and rRNAs intermingle with the cytoplasm content (Hernandez-Verdun 2011). However, the RNA polymerase I-related factors, which were involved in rDNA transcription, remain associated with chromosomal NORs. Among others, these proteins include transcription initiation factors UBF and SL1/TIF-1B (Zatsepina et al. 1993; Roussel et al. 1996), transcription termination factor TTF-1 (Sirri et al. 1999), and the UBF-interacted Treacle protein (Valdez et al. 2004). The presence of RNA polymerase I in mitotic NORs evidenced by immunocytochemistry (Roussel et al. 1996; Seither et al. 1997) was not confirmed by expression of the enzyme subunits in living cells (Leung et al. 2004) and therefore requires additional investigations.

A special group of NOR-associated proteins is called “Ag-NOR proteins” owing to their ability to reduce silver ions to metallic silver under appropriate conditions. The presence of these proteins ensures the silver-binding properties of NORs and permits their visualization on standard cytogenetic preparations (Howell and Black 1980). However, the identity of Ag-NOR proteins remains obscure. Two NOR-binding proteins, UBF and Treacle, which contain acidic domains required for the reduction of Ag+, are the most probable candidates to be involved (McStay 2016). It is noteworthy that the main silver-reducing proteins of interphase nucleoli such as the abundant NPM1/B23/nucleophosmin and nucleolin/C23 proteins (Sirri et al. 2000) are lacking from mitotic NORs (Leung et al. 2004). Thus, while the rDNA-fluorescence in situ hybridization (FISH) method provides an access to the rDNA arrays themselves, the Ag-NOR staining is a tool to discern active (or competent) NORs from silent ones, i.e., the NORs which do not contain rRNA genes transcribed during the preceding interphase.

In the mouse species (Mus musculus), NORs are highly polymorphic chromosomal loci which number and size vary among subspecies, laboratory strains, and individuals (Suzuki et al. 1990; Kurihara et al. 1994; Britton-Davidian et al. 2012). The standard house mouse karyotype (2n = 40) consists of only telocentric chromosomes, and well-defined NORs are carried by up to six autosomes (11, 12, 15, 16–18) which, however, can differ in frequency between subspecies and strains. In addition to the major NORs, a number of the minor NOR sites can also be present on other autosomes (Britton-Davidian et al. 2012), thereby evidencing in favor of the unique divergence of NORs in mice as compared to humans where NORs are carried by the constant chromosomes. On metaphase chromosomes, mouse NORs are usually located at centromeres and close to prominent constitutive heterochromatin that plays a role in the maintenance of chromosome stability and the silencing of neighboring genes (Nishibuchi and Déjardin 2017).

The mouse genome contains approximately 200 rDNA copies but, like in other species, only a half them is transcribed (Gibbons et al. 2015). Exploring in vitro activated spleenocytes of several mouse inbred strains showed that the number of Ag-NOR sites were in general agreement with the number of rDNA sites deduced from in situ hybridization, but about 12% rDNA clusters remained negative for Ag-NOR (Kurihara et al. 1994). The cytogenetic data on NORs in established mouse cell lines are rather poor, but it has been shown that L fibroblasts contain four Ag-NORs carried by the same number of chromosomes (Nielsén et al. 1979). We failed to find data on the NOR number in L929 fibroblasts.

Mouse strain L929 was established in 1948 by cloning L fibroblasts which in turn were derived from normal subcutaneous tissue of an adult C3H/An male. Strain L is one of the first cell strains established in continuous culture, and the clone 929 is the first cloned strain developed. L929 fibroblasts are known to be tumorigenic in immunosuppressed mice and have a very instable karyotype with the chromosome number ranges from 40 to above 100 (https://www.lgcstandards-atcc.org/products/all/CCL-1.aspx?geo_country=ru#characteristics; Kraemer et al. 1972; Andreeva et al. 1987; Sorokina et al. 1988) that complicates their karyological analysis, but they are capable for cloning.

In the current study, we took an advantage of this fact to determine the extent of the NOR stability in cloned fibroblasts as compared to parental L929 cells. The NOR phenotypes were explored using the FISH-NOR and Ag-NOR labeling techniques, and the results obtained were evaluated as statistically significant according to the Student’s t test. Our results showed that early clones were significantly more homogenous in the number and competence of NORs than the parental cells, but the clones restored the parental NOR heterogeneity after approximately 6 months of culturing or roughly after 150–200 cell generations. These results showed that cultured mammalian cells possess mechanisms to maintain the number and transcriptional competence of NORs at the population level.

Materials and methods

Cell culturing and cloning

Mouse L fibroblasts (clone 929, ESCC collection) were purchased from the Russian Cell Collection (Institute of Cytology, St. Petersburg, Russia) and grown as a monolayer in DMEM medium (PanEco Ltd., Russia) supplemented with 10% of fetal bovine serum (HyClone, USA), 50 IU/ml penicillin, and 50 μg/ml streptomycin. Cells were harvested with 0.25% trypsin-0.02% EDTA and re-seeded twice per week when they reached 80–90% confluence.

To clone L929 fibroblasts, they were yielded with trypsin-EDTA and resuspended in growth medium to the final concentration of one cell per 100 μl. Aliquots (100 μl) of the suspension were placed in F-base wells of 96-well plates which have the flatten bottom and perfect transparency capacities (TPP Techno Plastic Products AG, Switzerland). Three to 4 h later, which was sufficient for a cell attachment and spreading over the substrate, the plates were screened under an inverted Axiovert 200 microscope equipped with a phase contrast 10×/0.45 Plan-Apochromat objective (Carl Zeiss, Germany) to identify the wells which contained only one cell. Finally, the wells designated B4, D6, and E1 have been selected. Three days later, 100 μl of fresh growth medium was added to the selected wells, and then, it was renewed twice per week until the clones formed a monolayer. The clones B4, D6, and E1 were harvested as described above and then transferred subsequently to wells of 24-well plates, 6-well plates, and finally to 25-cm2 flasks for further culturing. The overall procedure lasted for 1.5–2 months and permitted to yield cloned fibroblasts in the amounts sufficient for statistical analysis of NORs. Two clones (B4 and D6) were cultured for another 4 months and their NORs were analyzed in parallel with those of parental fibroblasts which were continuously cultured during the same time period. In total, chromosomal spreads were obtained from the early clones (12th–15th passages), the late clones (47th–50th passages), and parental fibroblasts at the beginning (i.e., before cloning) and after 6 months of culturing.

Spreads of metaphase chromosomes

Spreads of metaphase chromosomes were prepared by the routine air-drying technique after incubation of exponentially growing cells with 50 ng/ml nocodazole (Merck, Germany) for 1–3 h. Cells were detached from the substrate as described above, pelleted by centrifugation (× 1000 g, 8 min), hypotonized with 0.56% KCl at 37 °C for 10 min, and fixed with an absolute methanol/glacial acetic acid mixture (3:1) at − 18 °C for 2.5–3 h. Cells were pippeted onto clean wet cold slides and air-dried, and the quality of chromosome spreading was controlled microscopically with the aim of a phase contrast 10×/0.45 Plan-Apochromat objective.

Ag-NOR staining

Active (competent) chromosomal NORs were revealed by a silver impregnation method according to Howell and Black (1980) with slight modifications. A 50% solution of AgNO3 (Merck) in bidistilled water was prepared immediately before use, while 2% solution of gelatin (Merck) in bidistilled water with addition of 1% formic acid (Merck) was stored at 4 °C until use. Chromosome spreads were incubated with a mixture of the AgNO3 and gelatin (2:1) solutions at 37 °C for 10–15 min, carefully washed in water, and mounted in Canada balsam mounting medium (Merck). Specimens were examined under an Axiovert 200 microscope using Plan-Apochromat 63/1.40 Oil Ph3 and Plan-Achromat 100×/1.3 Oil Ph3 objectives and photographed with a 12-bit digital camera CoolSnapcf (Roper Scientific, USA). For each experimental point, up to 50 metaphase plates were examined.

FISH of NORs

Dr. I. Grummt (German Cancer Research Center, Heidelberg, Germany) kindly provided the original plasmids containing different fragments of the mouse rDNA units. The rDNA fragments were subcloned into pBluescript II KSvector (Agilent Technologies, USA), purified with Wizard DNA Purification System (Promega, USA) and labeled with digoxigenin-UTP using Nick Translation Kit (Roche Holding AG, Switzerland) following recommendations of the manufacturers. Probe 1, a 11.35-kb EcoRI-EcoRI fragment, comprised parts of the nontranscribed spacer (NTS), the 5′ ETS, and the majority of the 18S sequence (residuals − 5715 to + 5635 relative to the transcription start site). Probe 2, a 6.6-kb EcoRI-EcoRI fragment, comprised a part of the 18S sequence, the ITS1 and ITS2, the 5.8S sequence, and the majority of the 28S sequence (residuals + 5635 to 12,235). A mixture of the probes was used to intensify FISH signals. Together, the probes recognize ~ 18 kb of the rDNA unit and the majority of an rDNA repeat. Adding to the hybridization mix a probe 3 to a minor part of the 28S rDNA, the 3′ ETS and a short NTS sequences did not affect localization and intensity of FISH signals.

Chromosomes were treated with 100 μg/ml RNase A (Roche) in 2 × SSC (0.3 M NaCl, 0.03 M sodium acetate, pH 7.3) at 37o C for 1 h, washed in 2 × SSC, and exposed to 0.01% pepsin (Merck) in 0.05 M sodium citrate (pH 2) for 2–4 min at room temperature. After thorough washing in water, spreads were dehydrated with ethanol of increasing concentrations and air-dried. Hybridization mix contained 10–25 μg rDNA probes, 500 ng/ml salmon carrier DNA (Merck), 500 ng/ml tRNA (Merck), 10% dextran sulfate, and 50% deionized formamide (Merck) in 2 × SSC. The target rDNA and rDNA probes were denatured simultaneously at 85 °C for 10 min. The hybridization reaction was executed at 37 °C for 14 h. Specimens were washed in 50% formamide in 4 × SSC/Tween 20 at 42 °C (3 × 10 min) and 2 × SSC at room temperature for 10 min and incubated with mouse anti-digoxigenin antibodies conjugated with rhodamine (Roche; diluted 1:20 in PBS containing 0.05% Tween and 5% nonfat dry milk) for 60 min at room temperature. Before mounting in the antifade/DAPI reagent (MetaSystems, Germany) chromosomes were stained with 0.1 μg/ml DAPI (4.6-diamidino-2-phenylindole, Merck) in water for 10 min. Specimens were examined with a Fluar 100×/1.3 Oil UV objective, photographed with a CoolSnapcf digital camera, and analyzed with Adobe Photoshop software, version CS6. NORs were considered as present on a chromosome if a FISH signal was detected regardless of its intensity and shape, and as absent when no signal was visible.

In some experiments, we tried to detect FISH-NORs and Ag-NORs simultaneously on the same metaphase spreads. For this aim, chromosomes were first exposed for rDNA-FISH as described above with two modifications. The pepsin treatment was omitted from the hybridization protocol, and chromosomes were additionally fixed with 2% paraformaldehyde in PBS (137 mM NaCl, 2.7 mM KCl, 6.7 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2–7.4) for 10 min at room temperature before Ag-NOR staining. Chromosomes were counterstained with DAPI (0.1 μg/ml, 10 min), mounted in the antifade reagent, and examined using a PlanAchromat100×/1.3 Oil Ph3 lens.

Serial 8-bit digital images of FISH-NORs were acquired with a confocal laser scanning LSM510 microscope (Carl Zeiss) equipped with a Plan-Apochromat 63×/1.40 Ph3 objective. The NOR sizes were measured using ImageJ software (http://imagej.nih.gov/ij/download.html).

Results are expressed as the mean ± standard error of the mean (SEM). Pairs of values were compared by the unpaired two sample t test assuming unequal variances. Differences are considered significant at a P < 0.05 and insignificant at a P > 0.05. Microsoft Excel 2007 software (Microsoft, USA) was used for statistical analysis of data.

Results

The number of NORs in the parental L929 fibroblasts

Before cloning, we examined the number of FISH- and Ag-NORs in parental cells. The results showed that L929 fibroblasts we used were heterogeneous in the number of both FISH- and Ag-NORs. The results of the NOR statistical analysis are summarized in Table 1. It shows that before cloning, L929 fibroblasts contained 7.40 ± 0.15 FISH-NORs on an average. The average number of Ag-NORs was lower, 7.08 ± 0.16, but the difference between two sets of data was insignificant (t test, P > 0.05). The latter indicates that a majority of FISH-positive NORs contained rDNA repeats which were transcribed in the preceding interphase. Figure 1a illustrates the frequency distribution of FISH-NORs in the parent cells before cloning. One can see that their number per cell varies from six to nine, while metaphases with seven FISH-NORs are most frequent. The number of Ag-NORs varied in a wider range, from five to nine (Fig. 2a), that corresponds to the statistical data which shows that in parental fibroblasts, the average number of Ag-NORs was lower than the average number of FISH-NORs (Table 1).
Table 1

Average numbers of chromosomes, FISH-NORs, and Ag-NORs in the parental and cloned L929 fibroblasts. Data are expressed as the mean ± SEM (standard error of the mean). n, the number of metaphases examined. See Supplementary data for an extended statistical analysis

Cells

Parental L929 fibroblasts

B4 clone

D6 clone

E1 clone

Parameters

Before cloning

After long-term culturing

Early (12–15 passages)

Late (47–50 passages)

Early (12–15 passages)

Late (47–50 passages)

Early (12–15 passages)

Chromosome number

53.1 ± 0.7, n = 45

53.2 ± 0.5, n = 50

52.9 ± 0.5, n = 32

53.0 ± 0.6, n = 50

53.1 ± 0.6, n = 26

53.2 ± 0.4, n = 50

52.3 ± 0.5, n = 27

FISH-NOR number

7.40 ± 0.15 (A), n = 35

7.35 ± 0.15 (A’), n = 40

6.65 ± 0.12 (a), n = 26

7.23 ± 0.15 (a’), n = 31

6.57 ± 0.08 (b), n = 37

7.30 ± 0.14 (b’), n = 46

6.89 ± 0.14 (c), n = 28

Ag-NOR number

7.08 ± 0.16 (B), n = 39

7.03 ± 0.18 (B′), n = 35

6.44 ± 0.13 (d), n = 25

6.95 ± 0.16 (d’), n = 40

6.52 ± 0.08 (e), n = 50

7.04 ± 0.14 (e’), n = 50

6.54 ± 0.13 (f), n = 26

The pairs of values which differ statistically significantly (t test, P < 0.05): (A) and (a), (A) and (b), (A) and (c). For the values (A) and (a) P = 0.000374; for (A) and (b) P = 1.62809E−05; for (A) and (c) P = 0.017711. (B) and (d), (B) and (e), (B) and (f). For the values (B) and (d) P = 0.004563; for (B) and (e) P = 0.005315; for (B) and (f) P = 0.014374. (a) and (a’), (b) and (b’), (d) and (d’), (e) and (e’). For the values (a) and (a’) P = 0.005028; for (b) and (b’) P = 2.02256E−05; for (d) and (d’) P = 0.014589; for (e) and (e’) P = 0.001935. The pairs of values which do not differ statistically significantly (t test, P > 0.05): (A’) and (a’), (A’) and (b’) (A) and (A’), (A) and (B), (A’) and (B′); (B) and (B′)

Fig. 1

Frequency distribution of the number of FISH-NORs in parent L929 fibroblasts and in B4 and D6 clones in early (a) and late (b) cultures. Horizontal axes show the number of NORs, vertical axes are the percentage of metaphases with a particular number of NORs. Green columns—L929 fibroblasts before cloning (a) and after long-term culturing (b); blue columns—B4 clone and red columns—D6 clone at the early (12th–15th) (a) and the late (47th–50th) (b) passages. The numbers of metaphases used to create the graphs are indicated in Table 1 as n (in italic) and in Supplementary material

Fig. 2

Frequency distribution of the number of Ag-NORs in parent L929 fibroblasts and in B4 and D6 clones in early (a) and late (b) cultures. Horizontal axes—the number of NORs; vertical axes—the percentage of metaphases with a particular number of NORs. Green columns—L929 fibroblasts before cloning (a) and after long-term culturing (b); blue columns—B4 clone and red columns—D6 clone at the early (12th–15th) (a) and the late (47th–50th) (b) passages. The numbers of metaphases used to create the graphs are indicated in Table 1 as n (in italic) and in Supplementary material

A long-term culturing of parental fibroblasts did not affect significantly the quantitative characteristics of their NORs despite certain differences between the parent fibroblasts before and after cloning can be observed (Table 1, Figs. 1b and 2b). In general, these observations were consistent with the results on the stability of the NOR number in human HeLa cells over culturing time (Smirnov et al. 2006).

Figure 3 illustrates a metaphase of parental cells, where we succeeded to detect prominent FISH- and Ag-NORs simultaneously (see “Materials and methods” for details). In this metaphase, eight FISH-NORs (a, arrows, a’) are distributed between six chromosomes (designated as af). The NOR-carrying chromosomes include a metacentric (chromosome a) and a very short (minute) telocentric (chromosome, b) which possess double NORs (i.e., two NORs per each sister chromatid), two metacentrics with single NORs (chromosomes c, d) and two telocentric, which also have single NORs (chromosomes e, f). Chromosomes with double NORs were stably present in 95–100% metaphases examined, and therefore, they were considered as the marker NOR-carrying chromosomes in the cells used. Such chromosomes are absent from the standard mouse karyotype (Suzuki et al. 1990; Britton-Davidian et al. 2012), but they have been described in mouse RAG cells originated in a spontaneous adenocarcinoma (Nielsén et al. 1979). The standard mouse karyotype also does not contain metacentric chromosomes which were regularly observed by us and other authors in established mouse cell cultures (Nielsén et al. 1979; Andreeva et al. 1987; Sorokina et al. 1988; Davisson and Akeson 1993).
Fig. 3

Simultaneous detection of FISH- and Ag-NORs in a metaphase of the parental L929 fibroblasts used for cloning. (a, b) General chromosome views; NORs are indicated by arrows; (a’, b’) NOR-carrying chromosomes at a higher magnification; (a, a’) superimposition of FISH signals (red) and DAPI staining of chromosomes (blue); (b, b’) Ag-NORs. NOR-carrying chromosomes are designated by letters af (in the italic font style). The chromosome d with a FISH-positive but an Ag-negative NOR is marked by asterisks. Scale bars, 5 μm

On chromosomes, the majority of FISH signals were resolved as paired dots, i.e., one dot per sister chromatid, but on the marker telocentric (chromosome b), the signals were regularly so intense that fused to a common “patch” (Fig. 3a’). It is noticeable that NORs carried by different chromosomes (e.g., by chromosomes a, e, and f) vary in size that supports observations on an unequal distribution of rDNA repeats between NOR-carrying chromosomes in various mouse cells (Kurihara et al. 1994; Britton-Davidian et al. 2012). In this particular metaphase, one can also see that a FISH-positive NOR located on chromosome d is Ag-negative, e.g., incompetent. This NOR is indicated by an asterisk in Fig. 3a’, b’.

It is important to mention that L929 fibroblasts we used had a rather low mode number of chromosomes (52–55, Table 1) unlike the L929 strains which karyotype was described by other authors (62–64 chromosomes, Andreeva et al. 1987; Sorokina et al. 1988). The average number of chromosomes remained stable for at least 6 months of cell culturing (Table 1).

The number of NORs in the early clones (12–15 passages)

The data described above show that L929 fibroblasts used for cloning were heterogeneous in terms of the FISH- and Ag-NOR numbers. One could assume that their clones, as progenies of single cells, would be uniform. However, cloned fibroblasts turned out to be nonuniform in the NOR numbers already at the early (12th–15th) passages or after 1.5–2 months of culturing. Nevertheless, the statistical analysis showed that all early clones contained on average significantly less numbers of FISH- and Ag-NORs (t test, P < 0.05) than the parental cells used for cloning (Table 1). For example, the mean number of FISH-NORs in the early B4 clone (6.65 ± 0.12) significantly differed from that in the parental cells (7.40 ± 0.15). The significant differences also were observed between the parental and early clones in the average numbers of Ag-NORs (Table 1).

The histograms in Fig. 1a demonstrate the frequency distribution of FISH-NORs in the early B4 (blue columns) and D6 (red columns) clones. The clones, on contrary to the parental fibroblasts (green columns), contain rare (B4 clone) or none (D4 clone) metaphases with eight FISH-NORs, while metaphases with nine NORs are not observed in both clones. In addition, metaphases with eight or nine Ag-NORs are absent (Fig. 2a).

Figure 4a, a’ illustrates a metaphase in the early D6 clone after detection of FISH-NORs. In this metaphase, seven rDNA sites are seen on five chromosomes. Unlike a “parental” metaphase (Fig. 3), this metaphase does not contain NOR-carrying metacentrics but has more—three—NOR-bearing telocentrics (chromosomes ce). At a higher magnification (Fig. 4a’), one can see lateral asymmetry of an NOR on the marker metacentric (chromosome a) and a perfect resolution of double FISH-NORs on the marker telocentric (chromosome b).
Fig. 4

FISH-NORs (a, a’, red) and Ag-NORs (b, b’) in metaphases observed in early clones. (a, b) General chromosome views; NORs are indicated by arrows; (a’, b’) the NOR-carrying chromosomes at higher magnifications. In a and a’, chromosomes are stained with DAPI (blue). The letters in italic designate chromosomes with NORs. Scale bars, 5 μm

In Fig. 4b, b’, a metaphase with Ag-NORs from the early B4 clone is shown. The metaphase contains seven NORs and five NOR-bearing chromosomes that may correspond to chromosomes ac and e, f of a metaphase obtained from parental cells (Fig. 3b’). Differences in the Ag-staining intensities of different NORs are also visible.

The number of NORs in the late clones (47–50 passages)

Two clones (B4 and D6) were further cultured for another 4 months and examined at the 47th–50th passages as the late clones. The results of their cytogenetic analysis evidenced that the average number of FISH-NORs per metaphase significantly (t test, P < 0.05) increased as compared to the corresponding early clones and became similar to that in the parental fibroblasts (Table 1). For example, in D6 clone, the average number of FISH-NORs changed from 6.57 ± 0.08 to 7.30 ± 0.14 or by about 10%. In both clones, the Ag-NOR numbers also increased significantly as compared to the early clones and reached the parental level (Table 1). These data indicate that a majority of the novel NORs were competent.

The FISH-NOR frequency distribution in the late B4 and D6 clones is illustrated in Fig. 1b, and that for Ag-NORs is shown in Fig. 2b. One can see that in both clones, the divergence in the FISH- and Ag-NOR numbers increased over time and became similar to that in the continuously cultured parental fibroblasts.

To define all possible NOR sites in late clones, we used metaphases which contained the maximal—nine—number of NORs assuming that Ag-positive NORs also indicated the presence of rDNA sites. In metaphases shown in Figs. 5 and 6, nine NOR sites are distributed between seven chromosomes (a–g). It is noteworthy that a FISH-NOR is located on an end of a large metacentric chromosome (chromosome c) (Fig. 5a’). Telomeric NORs were observed only in the late D6 clone where their frequency did not exceed 3–5%. One also can notice that the marker NOR-carrying chromosome b forms associations with (Fig. 5a’) or is translocated to (Fig. 6a’) another telocentric chromosome, which apparently prevents its lost during mitosis.
Fig. 5

FISH-NORs (arrows) in a metaphase of the late D6 clone. (a) General chromosome view; (a’) a higher magnification of NOR-carrying chromosomes. The letters in italic designate chromosomes with NORs. Red—FISH signals; blue—DAPI staining of chromosomes. Scale bars, 5 μm

Fig. 6

Ag-NORs (arrows) in a metaphase of the late B4 clone. (a) A general view of chromosomes; (a’) a higher magnification of the NOR-carrying chromosomes. The letters in italic designate chromosomes with NORs. Scale bars, 5 μm

Uneven distribution of rDNA repeats between sister chromatids

In human lymphocytes, active NORs exhibit the phenomenon called lateral asymmetry that appears as uneven Ag-signals on the sister chromatids comprising a single metaphase chromosome. The phenomenon was explained by an unequal distribution of active ribosomal genes between sister NORs (Strobel et al. 1981; Heliot et al. 2000). Here, we also observed lateral asymmetry of Ag-NORs as it is shown in Fig. 6a’ for chromosome e but the majority of Ag-NORs looked rather symmetrical (for example, NORs on chromosomes a, c, f, g in Fig. 6a’). On opposite, lateral asymmetry of FISH-NORs was prominent in all metaphases examined. For example, it is well visible on all chromosomes shown in Fig. 4a’.

Uneven distribution of rDNA repeats between sister chromatids was further confirmed on serial optical sections obtained with the aim of a confocal microscope (Fig. 7). Using ImageJ software, we measured the areas of fluorescent signals in sister chromatids of 17 metaphase chromosomes to estimate rough differences in the signal sizes and the number of rDNA copies in sister NORs by assuming that their rDNAs are equally accessible to FISH probes. This approximate analysis showed that sister chromatids of metaphase chromosomes may differ in the number of rDNA copies by 1.60 ± 0.07-fold (Table 2 and Supplementary material).
Fig. 7

A laterally asymmetric FISH-NOR (red) on a metaphase chromosome as visualized with a conventional epifluorescence microscope (a) and on serial optical sections obtained with a confocal laser scanning microscope (1–7). NORs on the sister chromatids are indicated by arrows, and the larger NOR is marked by asterisks. Scale bars, 1 μm

Table 2

Areas of asymmetrical sister FISH-NORs (in arbitrary units) measured at the same magnification in 17 NOR-bearing chromosomes from seven different metaphases and ratios of a larger NOR square (SqL) to a smaller NOR square (SqS). The last line shows the mean and SEM (standard error of the mean)

Chromosomes (metaphases)

Larger NOR area (SqL)

Smaller NOR area (SqS)

Ratio SqL/SqS

1 (1)

137

76

1.80

2 (1)

166

75

2.21

3 (1)

170

116

1.47

4 (2)

132

102

1.29

5 (2)

180

156

1.15

6 (2)

156

111

1.41

7 (2)

208

138

1.51

8 (2)

210

111

1.89

9 (3)

231

150

1.54

10 (3)

102

63

1.62

11 (4)

207

126

1.64

12 (5)

198

153

1.29

13 (6)

201

94

2.14

14 (7)

181

113

1.60

15 (7)

146

86

1.70

16 (7)

225

166

1.36

17 (7)

238

146

1.63

Mean ± SEM

182 ± 9

117 ± 8

1.60 ± 0.07

Discussion

The molecular mechanisms, which are responsible for rDNA (in)stability, are currently under intense investigations which were exhaustively surveyed in several recent reviews (Lam and Trinkle-Mulcahy 2015; McStay 2016; Kobayashi and Sasaki 2017; Tsekrekou et al. 2017; Tiku and Antebi 2018). According to the “rDNA theory of aging,” the rDNAs and nucleolus act to preserve genome stability, trigger cell senescence, and regulate evolutional adaptability (Kobayashi 2008). Numerous gene products that may contribute to the maintenance of rDNA stability in yeasts are identified (Kobayashi and Sasaki 2017). However, in mammals, mechanisms which may be involved in the rDNA/NOR stability remain more poorly studied. Here, we examined a content of the NOR stability in actively dividing mammalian cells such as mouse L929 fibroblasts by exploring the cloning, rDNA-FISH, and Ag-NOR staining techniques.

Like normal mouse cells (Kurihara et al. 1994; Britton-Davidian et al. 2012), parental L929 fibroblasts were highly divergent in the NOR number that was particularly evident when NOR parameters were examined statistically significant (Table 1, Figs. 1a and 2a). Unexpectedly, already at the early passages (or after 1.5–2 months of post-cloning), the cloned fibroblasts differed in the number of FISH-NORs. Nonetheless, they were prominently more homogeneous and contained significantly less FISH-NORs than the parental fibroblasts (Fig. 1, Table 1). However, when the early clones were continuously cultured for a longer time period (i.e., during additional 4 months), they restored the NORs’ quantitative parameters to the contents which were intrinsic to the parental cells. Based on these observations, we concluded that at least in established mammalian cell strains, the number of chromosomal rDNA sites can be maintained at the population level. The same conclusion was also made regarding the number of Ag-positive, i.e., competent, NORs (Fig. 2, Table 1). Taking into account that in L929 fibroblasts, the duration of cell cycle is 20–24 h (König et al. 1975), we approximated that the NOR recovery process required 150–200 cell generations. It is noteworthy to mention that like other researchers, who employ the in situ hybridization and Ag-NOR staining methods in cells with multiple NORs, we cannot exclude that detection of minor rDNA sites remained below the levels provided by the protocols used. However, in our hands, inclusion of an additional probe in a hybridization mixture (see “Materials and methods”) virtually did not increase the number of detected FISH-NORs.

In order to go insight the mechanisms of a high NOR divergence in L929 fibroblasts, we thoroughly examined their locations on chromosomes. The summarized observations allowed us to conclude that the NOR sites were stably present on the marker metacentric (in 100% metaphases) and the marker minute telocentric (95–97%) chromosomes, which had double NORs. These NORs were always Ag-positive, i.e., contained transcribed rDNA repeats. In addition, single NORs were present on two (in 57–65% metaphases) or three (43–35%) telocentric chromosomes. The presence of NORs on nonmarker metacentrics was most irregular: metaphases might contain none (Fig. 4a; in ~ 40% metaphases), one (Fig. 4b; ~ 50%), or two (Figs. 5 and 6; < 10%) such chromosomes. We suppose that various combinations of the NOR-carrying chromosomes were a main reason for alterations of the FISH-NOR number in L929 fibroblasts.

In mammalian dividing cells, the major mechanisms, which cause changes in global chromosome content, are mistakes in segregation of chromosomes or their fragments during mitosis (Potapova and Gorbsky 2017; Worrall et al. 2018). In a case of an NOR-bearing chromosome mis-segregation, the daughter cells will differ from a mother cell in terms of the rDNA copy numbers. Since both, a lower and a higher, rDNA dosages may be fatal, cells will utilize recombination mechanisms to restore or to remove (in alternative, to inactivate) rDNA repeats in order to produce a required number of ribosomes. As a consequent, additional rDNA sites may appear on chromosomes. The untypical presence of FISH-NORs on a chromosome end (Fig. 5, chromosome c) argues in favor of this assumption. A rare occurrence of such NORs is in line with an idea that in mice, telomerically positioned NORs are unstable in their locations on chromosomes (Britton-Davidian et al. 2012). Lateral asymmetry of chromosomal FISH-NORs regularly observed by us in L929 cells evidences in favor of unequal recombination events which may occur between sister chromatids (Kobayashi and Sasaki 2017) and lead to an uneven distribution of rDNA copies between the daughter cells in post-metaphase.

Irrespective to the aberrant karyotype, in L929 fibroblasts, NORs (except of the telomeric ones) were always located adjacently to centromeric heterochromatin. In mouse nuclei, centromeric heterochromatin is known to form numerous distinct structures, called chromocentres, which always co-sediment with nucleoli isolated from hepatocytes, but they can be purified as a separate fraction (Zatsepina et al. 2008). Recent studies showed that besides satellite and telomeric DNAs (Nishibuchi and Déjardin 2017), purified mouse chromocenters also contain rDNA sequences (Ostromyshenskii et al. 2018). A highly unstable nature of constitutive heterochromatin and its regular associations with nucleoli and chromosomal NORs (Németh and Längst 2011) may enhance a rate of rDNA recombination in mouse cells. It is interesting that the Ki-67 antigen that functions as an rRNA processing factor in nucleoli also organizes heterochromatin in mouse fibroblasts (Sobecki et al. 2016).

Altogether, our results support the idea that in mouse cells, NORs are most variable chromosomal loci and that in established mammalian strains, cells possess the mechanisms, which maintain the number and activity of NORs at the population level.

Notes

Acknowledgements

The author is grateful to Prof. Ingrid Grummt (German Cancer Research Center, Heidelberg, Germany) for a kind provision of the rDNA plasmids, and to Drs. Maria Chaplina, Iana Riabukha, and Anastasiya Moraleva (Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, Russia) for a valuable assistance in some experiments (MC) and statistical evaluation of the selected data (IR, AM).

Author contribution

The author designed the study, participated in the majority of experiments, performed a statistical analysis, wrote the paper, and prepared the illustrations.

Funding

The study was supported by the Russian Foundation for Basic Research (grant 16-04-01199).

Supplementary material

10577_2018_9598_MOESM1_ESM.xlsx (141 kb)
ESM 1 (XLSX 141 kb)

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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Shemyakin–Ovchinnikov Institute of Bioorganic ChemistryRussian Academy of SciencesMoscowRussian Federation

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