1 Department of Infection, Virology Section, St Thomas's Hospital, Lambeth Palace Road, London SE1 7EH, UK
2 Enteric, Respiratory and Neurological Virus Laboratory, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT, UK
Correspondence
Li Jin
ljin{at}phls.org.uk
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ABSTRACT |
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MAIN TEXT |
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Apoptosis is a genetically controlled form of cell death characterized in the early stages by cleavage and subsequent activation of caspases, and later by internucleosomal DNA fragmentation (Cohen, 1997; Kerr et al., 1972
; Kiess & Gallaher, 1998
; Young et al., 1997
). RV-induced apoptosis was first described in 1998 (Pugachev & Frey, 1998
). This and other studies have demonstrated that RV cytopathic effect (CPE) is due to caspase-dependent apoptosis and that RV replication is required for this process (Duncan et al., 1999
, 2000
; Hofmann et al., 1999
; Megyeri et al., 1999
). These studies were carried out in susceptible cell lines, as animal models have not proved sufficiently reliable for the study of symptomatic RV infection and pathogenesis (Chantler et al., 2000
). Human cell lines that display RV-induced CPE have not been established.
The mechanisms by which RV induces apoptosis, the cellular signalling pathways activated and the time-course of these biological changes have not been elucidated. This paper describes the time-course of early apoptotic biochemical cascades induced in RK13 cells by a wild-type (wt) strain of RV, RN, and an attenuated vaccine strain, Cendehill. RN was isolated from an infant with congenital rubella in London, UK, in 1986 and passaged only five times in Vero cells (Bosma et al., 1996; Frey et al., 1998
). The Cendehill strain was obtained from SmithKline and French (Peetermans & Huygelen, 1967
). Cendehill was chosen as it is more attenuated than the current vaccine strain, RA27/3 (Banatvala & Best, 1989
; Chantler et al., 1993
; Zygraich et al., 1971
). RK13 cells were infected with RN and/or Cendehill strains of RV at an m.o.i. of 3 p.f.u. per cell. Mock-infected and etoposide-treated cells (50 µM) (Sigma) were used as negative and positive apoptosis controls, respectively. Virus-infected cells were also treated with z-VAD-fmk [zValAlaAsp(OMe)CH2F (20 µM)] (Sigma), a cell permeable, irreversible, pan-caspase inhibitor as a control for caspase-dependant apoptosis. The presence of virus was confirmed by indirect immunofluorescence, as described previously (Best & O'Shea, 1995
).
Detection of early apoptotic caspase cascades was carried out using a DEVD (AspGluValAsp)-specific caspase activity assay (Promega) and Western blot analysis of caspase-3 cleavage. The DEVD assay was used to measure the total enzyme activity of the downstream active caspase subunits (caspases 3 and 7), whereas Western blotting (Fig. 1b, c) was used to detect the total amount of cleaved active caspase-3 subunit produced by upstream caspases (caspases 8 and 9) (Cohen, 1997
). For the DEVD assay, cell lysates were collected at 1296 h post-infection (p.i.). Lysates (2 µl) were incubated with 32 µl caspase assay buffer [312·5 mM HEPES pH 7·5, 31·25 % (w/v) sucrose and 0·3125 % (w/v) CHAPS], 2 µl DMSO, 1 µl 100 mM DTT and 2 µl 10 mM caspase colorimetric substrate Ac-DEVD-pNA (AcetylAspGluValAspp-nitroalanine), and incubated at 37 °C for 4 h. Caspase activity was measured by cleavage of the Ac-DEVD-pNA substrate (specific for effector caspases 3 and 7) to pNA, the absorbance of which was measured at 405 nm. Activation of DEVD-specific caspases could be seen as early as 24 h p.i. with RN- and Cendehill-infected (but not etoposide-treated) cells (Fig. 1a
). The main peak of caspase activity in etoposide-treated and RN- and Cendehill-infected cells occurred at 72 h p.i., whereas levels in mock-infected cells remained more or less constant over time. Caspase activity dropped rapidly to the level of that seen in mock-infected cells by 84 h p.i. but then began to rise again at 96 h p.i.
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Apoptosis, induced by the two strains of RV, was measured by TUNEL assay, DNA fragmentation analysis and by counting dead floating cells from 24 to 96 h p.i. TUNEL assays were performed using the DeadEnd Fluorometric TUNEL system, according to the manufacturer's instructions (Promega). FITC emission of degraded cellular DNA was examined using an Olympus fluorescence microscope. The percentage of apoptotic cell death was calculated by counting the percentage of fluorescing cells at a magnification of 60x (Fig. 2a). The nuclei of approximately 800 cells per sample were counted on slide projections and the means of four different cell counts were taken. The percentages of fluorescing apoptotic cells in the monolayer were similar at each time-point for RN-infected, Cendehill-infected and etoposide-treated cells (Table 1
a). The maximum percentage of apoptotic cells in the monolayer at 96 h p.i. was 1·19 % for mock-infected cells, 23 % for etoposide-treated cells and 22 and 17 % for RN- and Cendehill-infected cells, respectively (Table 1
b). TUNEL-positive cells were often found in regions of CPE, where cells had begun to round up and come off the plate, suggesting that these cells, and floating cells, had begun to initiate the cell death programme (Fig. 2a
, panels iii and iv). TUNEL-positive cells could not be detected prior to 24 h p.i., even though some cells appear to show the beginnings of CPE (data not shown).
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The results of counting dead floating cells in the supernatant (identified by trypan blue exclusion staining) mirrored the monolayer results, with RN and etoposide producing marginally more floating cells than Cendehill at 24, 72 and 96 h p.i. (data not shown). RV in supernatant from RN- and Cendehill-infected cells used for TUNEL assays was titrated by plaque assay, as described previously (Albrecht et al., 1981). The growth rate (p.f.u. h-1) of RN was approximately 3·6 times greater than that of Cendehill, which suggests an increased rate of replication.
DNA fragmentation analysis was used to detect the presence of internucleosomal fragments in RN- and Cendehill-infected RK13 cells. DNA ladder fragments were extracted from monolayer and floating cell populations using a DNA Isolation kit, according to the manufacturer's instructions (Oncogene Research Products). Nucleic acids were precipitated using 3 M sodium acetate and 2-propanol, followed by ethanol precipitation, and resuspended in 10 mM Tris pH 7·5 and 1 mM EDTA. DNA was separated on 1·5 % agarose gels and visualized by ethidium bromide staining. DNA laddering characteristic of apoptosis was seen in the floating cells but not in RV-infected monolayers (Fig. 2b, c). Chromatin in the detached floating cells was completely fragmented at 24 h p.i. for both strains of RV and this continued to increase over time (Fig. 2b
). The cellular DNA extracted was proportional to the total number of floating cells in the medium at each time-point p.i. The intensity of DNA laddering at 48, 72 and 96 h p.i. was slightly greater with RN than Cendehill, which corresponds to the small increase in the number of floating cells produced by RN. In contrast, the cells in the RN-, Cendehill- and mock-infected monolayers did not show DNA fragmentation (Fig. 2c
). The lack of DNA laddering in the monolayer indicated that the majority of attached cells were not in the late stages of apoptosis. This suggests that detachment is either part of the execution phase of apoptosis or is required for its completion. A phenomenon called anoikis' has been described for adenovirus E1a protein, a process whereby apoptosis is induced by cell detachment from the extracellular matrix (Frisch & Francis, 1994
).
This is the first report of the time-course of early biochemical apoptotic events during RV-induced apoptosis. The induction of apoptotic cascades in RV-infected RK13 cells occurred as early as 12 h p.i., with both wt (RN) and attenuated (Cendehill) strains, and is much earlier than suggested by previous studies (Duncan et al., 1999). This rapid activation of apoptotic mechanisms is in agreement with current knowledge on the apoptotic cascade, with caspase activation as the leading event (Au et al., 1999
). The cyclic pattern of caspase activity seen in RV-infected cells resembles that of chemical teratogens (e.g. etoposide), suggesting similar mechanisms of activation of the caspase cascade (Au et al., 1999
; Benjamin et al., 1998
). However, in this study, assays incorporating the use of apoptotic inhibitor z-VAD-fmk suggest that additional cellular pathways may be involved during RV-induced apoptosis.
The low passage wt strain, RN, was expected to be more virulent than Cendehill, one of the most attenuated stains available. However, our results demonstrate that attenuation of the Cendehill strain of RV has not destroyed its ability to induce apoptosis. The slight increase in growth rate, number of floating cells in the supernatant, percentage of TUNEL-positive cells in the monolayer and caspase activity in RN-infected RK13 cells can be attributed to the greater replication rate and/or efficiency of the wt strain rather than any difference in apoptotic mechanism. The attenuated strain RA27/3 has also been shown to induce apoptosis (Domegan & Atkins, 2002). Attenuated vaccine strains of RV appear to pose little threat to the developing foetus. This suggests that virus attenuation may affect other biological properties, such as replication rate or virus assembly and release, which would limit the teratogenic effects during infection in pregnancy (Best & Banatvala, 2000
). The cellular mechanisms that result in the disruption of organogenesis during congenital RV infection are still unknown. Further studies are required to elucidate the biochemical pathways involved in the development of RV-induced cell death.
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ACKNOWLEDGEMENTS |
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Received 20 August 2002;
accepted 23 January 2003.