ARTICLE |
Correspondence to: Marco Biggiogera, Dipartimento di Biologia Animale, Laboratorio di Istologia, U. of Pavia, Piazza Botta 10, 27100 Pavia, Italy..
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Summary |
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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 46:9991005, 1998)
Key Words: apoptosis, RNA, ribonucleoproteins, thymocytes, osmium ammine, terbium, cytochemistry
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Introduction |
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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
Recent data (
Throughout this process, the different RNP structures forming the HERDS can be recognized morphologically and still react with specific antibodies (
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 (
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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:
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
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 (
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 PBSTween 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
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 35 min and finally stained with lead citrate (
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 530550-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).
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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 (
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PIPTA staining resulted in detection of both DNA and RNA (
When both nucleic acids were visualized with direct osmium ammine staining (
After hydrochloric acid hydrolysis, osmium ammine staining is specific for DNA (
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
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Staining with terbium citrate showed by electron microscopy (Figure 9c) that RNA-containing aggregates are also released at the surface of apoptotic EUE cells.
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Discussion |
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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 (
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 (
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
Another possible explanation for these contradictory results may be found in the different cytochemical techniques (DNasegold labeling and PI staining without RNase treatment) used by
Recently,
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 (
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 (
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
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Acknowledgments |
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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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