Journal of Histochemistry and Cytochemistry, Vol. 46, 999-1006, September 1998, Copyright © 1998, The Histochemical Society, Inc.


ARTICLE

Nuclear RNA Is Extruded from Apoptotic Cells

Marco Biggiogeraa, Maria Grazia Bottonea, and Carlo Pellicciaria
a Dipartimento di Biologia Animale, Laboratorio di Istologia, and Centro di Studio per l'Istochimica del CNR, University of Pavia, Pavia, Italy

Correspondence to: Marco Biggiogera, Dipartimento di Biologia Animale, Laboratorio di Istologia, U. of Pavia, Piazza Botta 10, 27100 Pavia, Italy..


  Summary
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Summary
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Materials and Methods
Results
Discussion
Literature Cited

During spontaneous apoptosis of thymocytes there is extrusion of ribonucleoproteins (RNPs) from the cell. The aim of this investigation was to elucidate whether the RNP aggregates in apoptotic cells and bodies still contain RNA in an appreciable amount. We demonstrated by specific cytochemical techniques that the aggregates of nuclear RNPs extruded in the cytoplasm of spontaneously apoptotic thymocytes contain RNA in a sufficient amount to be detected cytochemically. These heterogeneous ectopic RNP-derived structures (HERDS) are formed by perichromatin fibrils, interchromatin granules, perichromatin granules, and nucleolar material. The RNA detected inside these clusters should therefore correspond to both mRNA and snRNA as well as to rRNA. We never observed DNA-contaning aggregates in the cytoplasm of apoptotic thymocytes. The presence of RNA in the HERDS that may be released from apoptotic cells suggests that the decrease in the amount of total RNA during apoptosis may be mostly linked to cellular extrusion rather than to degradation of RNA by RNase activities. Another interesting aspect of these results lies in the hypothesis of apoptosis as a possible cause for the presence of autoantibodies in the serum of patients with systemic autoimmune diseases. (J Histochem Cytochem 46:999–1005, 1998)

Key Words: apoptosis, RNA, ribonucleoproteins, thymocytes, osmium ammine, terbium, cytochemistry


  Introduction
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Introduction
Materials and Methods
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Discussion
Literature Cited

During the course of apoptosis, several changes occur at the nuclear level, among which the superstructural modifications of chromatin have been extensively investigated (reviewed in Earnshaw 1995 ). Chromatin margination and condensation are considered as features peculiar to apoptosis, and degradation of nuclear DNA has been found to occur in the great majority of apoptotic cell deaths. The molecular basis for these phenomena should be the activation of specific proteases and endogenous endonucleases during apoptosis (reviewed in Chinnaiyan and Dixit 1996 ).

Recent data (Biggiogera et al. 1997a , Biggiogera et al. 1997b ; Lafarga et al. 1997 ) also showed that the nuclear ribonucleoprotein (RNP)-contaning structures [i.e., perichromatin granules (PG), perichromatin fibrils (PF), interchromatin granules (IG), and the nucleolus] also undergo severe alterations during the course of both spontaneous and experimentally induced apoptosis. In particular, in early stages of spontaneous apoptosis of thymocytes, RNP-containing structures segregate from their normal nuclear locations and give rise to heterogeneous fibrogranular clusters, which are preferentially found in the loose chromatin nuclear areas. In later stages of apoptosis these clusters are extruded from the nucleus into the cytoplasm, where they are visible as heterogeneous ectopic RNP-derived structures (HERDS). HERDS are finally released at the cell surface in the form of membrane-bounded blebs (Biggiogera et al. 1997b ).

Throughout this process, the different RNP structures forming the HERDS can be recognized morphologically and still react with specific antibodies (Biggiogera et al. 1997a ). This evidence demonstrates that during apoptosis significant portions of nuclear RNPs resist proteolytic cleavage, although degradation of some RNA-associated proteins, such as the 70-kD component of the U1 small nuclear RNP (snRNP), has been reported to occur during induced apoptosis (Casciola-Rosen et al. 1994b ; Casiano et al. 1996 ).

The aim of the present investigation was to elucidate whether the HERDS in apoptotic cells and bodies still contain RNA in appreciable amounts. In fact, it has been hypothesized (Cidlowsky 1982 ; Delic et al. 1993 ) that the decrease in the amount of cellular (and nuclear) RNA might be essentially due to the activation of endogenous RNase activities. However, RNA-containing surface blebs have been found in apoptotic human keratinocytes after UV irradiation (Casciola-Rosen et al. 1994a ). This suggests that the lower amount of RNA in apoptotic cells might be mostly due to the extrusion of RNA in probable association with RNP proteins rather than to the intracellular degradation of RNA. It is worth mentioning that a massive release of undegraded (or only partially degraded) RNA and RNA-associated proteins from apoptotic cells might have implications for the onset of autoimmune diseases (Casciola-Rosen et al. 1994a ; van Venrooij and Pruijn 1995 ; Biggiogera et al. 1997a , Biggiogera et al. 1997b ).


  Materials and Methods
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Materials and Methods
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Cell Preparation and Culture Conditions
Thymuses from 2-month-old rats were mechanically disaggregated, and thymocytes resuspended at a density of 3 x 106 cells/ml in RPMI complete medium containing 10% fetal bovine serum, 2 mM glutamine, and 100 U/ml each of penicillin and streptomycin. Thymocytes were immediately harvested by centrifugation and rinsed in PBS before being fixed for electron microscopy (see below).

EUE cells (human embryonic epithelium, an established cell line: Terni and Lo Monaco 1958 ) were also used as a model system in which apoptosis occurs spontaneously under conditions of very high-density growth (personal observations). These adherent cells have a very large amount of cytoplasm, high RNA content, and prominent nucleoli, and are therefore especially suitable for investigating the changes in RNP distribution during apoptosis, even by light microscopy. Cells were grown on glass coverslips in DMEM medium contanining 10% newborn bovine serum, 2 mM glutamine and 100 U/ml each of penicillin and streptomycin. Two days after confluence was achieved, cells on coverslips were fixed with 70% ethanol at -20C for 15 min.

Ultrastructural Cytochemistry
Thymocytes were fixed with either 4% paraformaldehyde or 2.5% glutaraldehyde in 0.1 M Sörensen phosphate buffer (pH 7.4) at 4C for 2 hr, rinsed in the same buffer, and placed in 0.5 M NH4Cl solution in buffer for 30 min at 4C to block free aldehyde groups. The specimens were dehydrated in ethanol at room temperature (RT) and embedded in LR White resin (Multilab; Dalmuir, UK).

DNA Staining. Thin sections from formaldehyde-fixed material on gold grids were hydrolyzed with 5 N HCl for 20 min at RT, rinsed with distilled water, and stained with 0.2% osmium ammine (prepared according to Vazquez-Nin et al. 1995 ) for 60 min at RT (Cogliati and Gautier 1973 ; Olins et al. 1989 ).

DNA + RNA Staining. To stain simultaneously both nucleic acids, the grids were either (a) treated with the osmium ammine technique as above, with the omission of the hydrolysis step (Derenzini and Farabegoli 1990 ), or (b) floated on droplets of 100 µg/ml propidium iodide (PI) solution in water for 30 min at RT, rinsed with water, and floated onto 1% neutralized phosphotungstic acid (PTA, pH 7.0) for 15–30 min (Biggiogera and Flach Biggiogera 1989 ).

DNA Immunolabeling. Grids with thin sections from formaldehyde-fixed material were incubated on normal goat serum (NGS) for 3 min, then floated onto a solution of 0.1% bovine serum albumin and 0.05% Tween-20 in PBS containing the monoclonal anti-DNA antibody (clone AC-30-10; Progen, Heidelberg, Germany) diluted 1:50, for 17 hr at 4C. After rinsing with PBS–Tween and PBS, grids were incubated with NGS as above and then reacted with a goat anti-mouse IgG+IgM antibody conjugated with 12-nm colloidal gold (Jackson Labs; West Grove, PA) for 30 min at RT. As a control, some sections were incubated in the absence of the primary antibody and then treated with the colloidal gold-labeled secondary antibodies. Grids were finally stained with the EDTA technique or with terbium citrate (see below).

RNA Staining. The grids with the sections were floated onto 0.2 M terbium citrate, prepared according to Biggiogera and Fakan 1998 , for 1 hr at RT or at 37C, rapidly rinsed with water, and dried.

EDTA Staining for Nuclear RNP. The grids were stained with uranyl acetate for 1 min, rinsed and dried, then floated onto 0.2 M EDTA for 3–5 min and finally stained with lead citrate (Bernhard 1969 ).

Stained specimens were observed with a Zeiss EM900 electron microscope equipped with a 30-µm objective aperture and operating at 80 kV.

PI Staining, RNase Treatment, and Fluorescence Microscopy on EUE Cells
Fixed EUE cells on glass slides were rehydrated in PBS and stained with 1 µg/ml PI in 0.1 M Tris-HCl buffer (pH 7.2) for 30 min at RT. The slides were rinsed in the same buffer and covered with a coverslip in a drop of buffer.

Micrographs (Agfapan APX 100 film) were taken with a BX50 Olympus microscope equipped with an HBO 100-W mercury lamp, a BP 530–550-nm excitation filter, a DM 570 dichroic mirror, and a BA 590 barrier filter. The horizontal and vertical substage coordinates of the fields of interest were recorded to make it possible to take a second picture after treating PI-stained slides with 200 U/ml of RNase A in 0.1 M Tris-HCl buffer (pH 7.2) for 45 min at RT under continuous stirring. Before use, RNase was boiled for 10 min to remove residual DNase activity.

To check the specificity of the RNase digestion, some slides were incubated with the same buffer solution from which RNase was omitted. As an additional control, RNase-treated slides were digested with DNase I (500 U/ml in 0.1 M Tris-HCl buffer, pH 7.2, containing 10 mM MgCl2) for 2 hr at RT. After this treatment, PI fluorescence (both nuclear and cytoplasmic) was completely abolished (data not shown).


  Results
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By electron microscopy, EDTA staining of apoptotic thymocytes showed the presence of nuclear RNPs both in the nucleus and in the cytoplasm (Figure 1), consistent with previous observations (Biggiogera et al. 1997a , Biggiogera et al. 1997b ). This technique, however, is preferential only for RNPs (Bernhard 1969 ) and stains mainly the protein moiety of the RNA–RNP complexes. Therefore, it cannot give conclusive information on the presence of nucleic acids.



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Figure 1. After EDTA regressive staining for RNPs, a large aggregate of RNP-containing structures (arrowhead) and fibrogranular material is visible in the central area of the nucleus. Bar = 0.1 µm.

Figure 2. PI–PTA staining for DNA and RNA. The precipitates corresponding to the endproduct are visible on chromatin, ribosomes, and HERDS (arrowheads) in the cytoplasm. Bar = 0.5 µm.

Figure 3. Osmium ammine staining for both DNA and RNA. When the staining is performed in the absence of acid hydrolysis, chromatin, ribosomes, and the HERDS (arrow) in the cytoplasm are contrasted. N, nucleus. Bar = 0.5 µm.

Figure 4. Osmium ammine staining for specific DNA detection. Only chromatin is stained. Note that the HERDS present in the cytoplasm (arrow) exhibits only inherent contrast. N, nucleus. Bar = 0.5 µm.

Figure 5. Anti-DNA immunolabeling and EDTA staining. The gold grains are present on the bleached chromatin in the nucleus. The HERDS in the cytoplasm (arrow) is not labeled. Bar = 0.1 µm.

Figure 6. Anti-DNA immunolabeling and EDTA staining. The nucleus is heavily labeled by the anti-DNA probe. The RNP-containing areas (arrowheads) are not labeled. Bar = 0.5 µm.

Figure 7. Terbium staining for RNA. The HERDS in the cytoplasm (arrow) is clearly stained, whereas the condensed chromatin of the nuclear remnant exhibits only inherent contrast. N, nucleus. Bar = 0.1 µm.

Figure 8. Terbium staining for RNA. RNA-containing HERDS (arrowheads) are present within the blebs at the surface of an apoptotic thymocyte. The chromatin masses (c) display inherent contrast. Bar = 0.1 µm.

PI–PTA staining resulted in detection of both DNA and RNA (Biggiogera and Flach Biggiogera 1989 ). Figure 2 shows a thymocyte with large, positively stained HERDS in the cytoplasm.

When both nucleic acids were visualized with direct osmium ammine staining (Derenzini and Farabegoli 1990 ), the RNP-containing aggregates of IG, PG, and PF were stained in the cytoplasm of apoptotic thymocytes (Figure 3). These HERDS were never found in nonapoptotic thymocytes (not shown).

After hydrochloric acid hydrolysis, osmium ammine staining is specific for DNA (Cogliati and Gautier 1973 ). Under these conditions, no cytoplasmic positivity was ever observed in nonapoptotic and apoptotic thymocytes (Figure 4), with the exception of mitochondrial DNA (not shown). Similar results were obtained after immunolabeling DNA with specific antibodies (Figure 5 and Figure 6). The gold grains were present only on the nucleus (or on the nuclear fragments in karyorrhexic cells) and did not label HERDS (Figure 5). Moreover, in some nuclei RNP structures were still present and the anti-DNA labeling was confined to their periphery (Figure 6).

The presence of RNA within the HERDS observed in the cytoplasm (Figure 7) and in the blebs of apoptotic thymocytes (Figure 8) was confirmed by specific staining of RNA with terbium.

The presence of RNA-contaning aggregates in the cytoplasm of apoptotic cells was further demonstrated in PI-stained samples of EUE cells. PI is an intercalating fluorochrome reacting with both double-stranded DNA and RNA (reviewed in Shapiro 1988 ). Consistently, in fluorescence microscopy, we observed positive PI staining in both the nucleus and the cytoplasm of EUE cells (Figure 9a). The cytoplasmic staining was homogeneous in nonapoptotic cells, whereas in the apoptotic ones large PI-positive aggregates were found (arrows in Figure 9a). After digestion with RNase (Figure 9b), PI cytoplasm staining was abrogated, thus demonstrating that the majority of PI-positive cytoplasmic material (including the above mentioned HERDS) contained RNA.



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Figure 9. PI-stained EUE cells before (a) and after (b) treatment with RNase A. In a, PI stains both the nucleus and the cytoplasm; in the apoptotic cells (large arrowheads), small cytoplasmic bodies blebbing at the cell surface (small arrowheads) are also brightly fluorescent. After RNA digestion, the fluorescence of the nucleoli, cytoplasm, and cytoplasmic bodies was abolished. Bar = 50 µm. (c) Terbium staining of an apoptotic EUE cell. The blebs protruding at the surface of the cell still contain detectable amounts of RNA (arrowheads). Bar = 0.1 µm.

Staining with terbium citrate showed by electron microscopy (Figure 9c) that RNA-containing aggregates are also released at the surface of apoptotic EUE cells.


  Discussion
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Materials and Methods
Results
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The specific cytochemical techniques used demonstrate that the aggregates of nuclear RNPs extruded in the cytoplasm (HERDS) of spontaneously apoptotic thymocytes (Biggiogera et al. 1997a , Biggiogera et al. 1997b ) contain RNA. The amount of this RNA is sufficiently great to be detected cytochemically with both light and electron microscopy. This evidence points to the possibility that nuclear RNA might be uncleaved or only partially cleaved during apoptosis.

This RNA surely has different origins. In previous studies, we have shown that the HERDS still contain morphologically recognizable structures such as PF, IG, and PG (Biggiogera et al. 1997b ). Moreover, HERDS were labeled both by anti-hnRNP and anti-snRNP antibodies (Biggiogera et al. 1997a ), showing that the antigen markers for PF and IG were still present. Consequently, we could hypothesize that a part of the cytochemically detectable RNA should be present inside the PF, which represent the in situ morphological expression of extranucleolar transcription (Fakan 1994 ). It remains to be elucidated whether these are either still pre-mRNAs or are already processed mRNAs. Inside the clusters of IG, snRNA should be present, and the RNA in the PG can be expected to correspond to processed and stored mRNA. The HERDS should also contain RNA of nucleolar origin, because remnants of the preribosomal fraction deriving from the nucleolar granular component were sometimes found (Biggiogera et al. 1997b ).

We never observed the presence of DNA within HERDS in apoptotic thymocytes either after osmium ammine staining or after immunolabeling with anti-DNA antibodies.

All these findings appear to contradict recent observations by Reipert et al. 1996 pointing to the presence of large amounts of DNA (of mitochondrial origin) in the cytoplasm of apoptotic cells. In an attempt to account for this apparent discrepancy, one major point to be considered is the different cell model system used by Reipert et al. 1996 , i.e., a hemopoietic stem cell line, FDCP-Mix, undergoing etoposide-induced apoptosis. These cells precociously show massive autolytic activity targeted to mitochondria, which increase significantly in number after etoposide treatment. On the contrary, very few autolytic vacuoles can be observed in spontaneously apoptotic thymocytes or EUE cells (data not shown), whose mitochondria preserve their normal morphology up to the late apoptotic stages (Pellicciari et al. 1996 ).

Another possible explanation for these contradictory results may be found in the different cytochemical techniques (DNase–gold labeling and PI staining without RNase treatment) used by Reipert et al. 1996 to assess the presence of DNA. DNase–gold labels DNA but may also interact with actin (as pointed out also by the authors). Because of this intrinsic limit, osmium ammine staining is definitely preferable, because it allows detection of both RNA and DNA when used in the absence of hydrochloric acid hydrolysis but only DNA after HCl treatment. A further advantage of the osmium ammine technique is that nucleic acids can be stained throughout the thickness of the section (Derenzini et al. 1990 ), thus overcoming the limit of the surface-labeling techniques. We have already noted that PI binds to DNA and RNA (Shapiro 1988 ) and that consequently RNase treatment is mandatory to conclusively relate PI positivity to the presence of DNA alone.

Recently, Zweyer et al. 1997 reported the occurrence of "granular and threaded nuclear bodies" in several cell lines induced to undergo apoptosis by different stimuli. These nuclear bodies proved to contain some RNP components in association with the nuclear matrix proteins p125 and p160, the proliferating cell nuclear antigen PCNA, and the nuclear mitotic apparatus-associated protein NuMA, but no DNA nor H1 histones. According to the authors, these nuclear aggregates should represent structures in which "proteins from the disassembled nuclear matrix aggregate prior to be extruded from the nucleus." These fibrogranular aggregates are morphologically reminiscent of HERDS and contain RNPs, thus suggesting they could be similar (or even the same) structures, although Zweyer et al. 1997 did not provide evidence for the "granular and threaded nuclear bodies" in the cytoplasm of apoptotic cells.

Whatever their true nature, we can conclude that the catastrophic rearrangement of the nuclear components occurring during spontaneous and induced apoptosis leads to the formation of unusual, highly heterogeneous aggregates of macromolecular complexes (including RNA, RNP, and perhaps matrix-associated proteins), which originally occupied specific nuclear locations in nonapoptotic cells.

RNA is therefore released at the surface of apoptotic cells within HERDS. However, this is not the only mechanism by which RNA can be eliminated from apoptotic cells. Organelles, including RER cisternae and ribosomes, are also released in apoptotic blebs (Biggiogera et al. 1997b ). We can hypothesize that the decrease in the total RNA amount of apoptotic cells is largely due to cell blebbing rather than to cleavage by endogenous RNase activities, as suggested by Cidlowsky 1982 and Delic et al. 1993 for the 28S rRNA.

The extrusion of RNA-containing HERDS from the nuclei of apoptotic cells obviously results in an irreversible block of RNA maturation. This process would actually induce the arrest of protein synthesis in the cytoplasm, in parallel with the block of nuclear transcription through the endonucleolytic degradation of DNA, thus being adaptively effective in determining the interruption of these key functional processes in apoptotic cells. However, the massive release of RNA-contaning HERDS from apoptotic cells might also be potentially harmful.

In fact, RNP complexes have been found to be powerful autoantigens in systemic autoimmune diseases (Uchiumi et al. 1991 ; Casciola-Rosen et al. 1994a ; van Venrooij and Pruijn 1995 ). It has been suggested that the occurrence of autoantibodies targeted to RNP-containing autoantigens might be related to and/or be dependent on the fate of these intracellular complexes during apoptosis (van Venrooij and Pruijn 1995 ). The present ultrastructural results as well as recent data in the literature (Biggiogera et al. 1997a , Biggiogera et al. 1997b ) confirm that highly stable RNP- and RNA-containing structures are extruded from the nucleus and eventually released at the cell surface inside apoptotic bodies.

Therefore, in apoptotic cells, both RNAs and RNPs should be only partially cleaved by nucleases and proteases, while possibly undergoing oxidative modifications due to the presence of reactive oxygen species (as suggested by Casciola-Rosen et al. 1994a ). These events might produce previously cryptic peptides (or RNA–peptide complexes) that could be present in association with MHC Class II molecules by antigen-presenting cells, thus inducing an immune response in genetically susceptible individuals. This immune response may subsequently even spread to other portions of the "self" molecule to which the organism was previously tolerant (Lehmann et al. 1992 ).


  Acknowledgments

Supported by grants from the Italian M.U.R.S.T. (Fondi di Ateneo per la Ricerca, 40%).

We thank Ms Paola Veneroni for technical assistance. We are also indebted to Drs B. Carlhoun and M. Fornasiero for critical reading of the manuscript.

Received for publication November 17, 1997; accepted May 5, 1998.


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Materials and Methods
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Discussion
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