Nov 7, 1994 - 1. Micrographs showing apoptotic cells in oligodendrocyte culture treated with ..... either protein kinase C (PKC), which represses cell death,.
The EMBO Journal vol.14 no.6 pp.1136-1144, 1995
Direct involvement of p53 in programmed cell death of oligodendrocytes
Orly Eizenberg, Anat Faber-Elman, Eyal Gottlieb', Moshe Oren', Varda Rotter2 and Michal Schwartz3 Departments of Neurobiology, 'Chemical Immunology and 2Cell Biology, The Weizmann Institute of Science, 76100 Rehovot, Israel 3Corresponding author Communicated by M.Oren
A covalent dimer of interleukin (IL)-2, produced in vitro by the action of a nerve-derived transglutaminase, has been shown previously to be cytotoxic to mature rat brain oligodendrocytes. Here we report that this cytotoxic effect operates via programmed cell death (apoptosis) and that the p53 tumor suppressor gene is involved directly in the process. The apoptotic death of mature rat brain oligodendrocytes in culture following treatment with dimeric IL-2 was demonstrated by chromatin condensation and internucleosomal DNA fragmentation. The peak of apoptosis was observed 16-24 h after treatment, while the commitment to death was already observed after 3-4 h. An involvement of p53 in this process was indicated by the shift in location of constitutively expressed endogenous p53 from the cytoplasm to the nucleus, as early as 15 min after exposure to dimeric IL-2. Moreover, infection with a recombinant retrovirus encoding a C-terminal p53 miniprotein, shown previously to act as a dominant negative inhibitor of endogenous wild-type p53 activity, protected these cells from apoptosis. Key words: apoptosis/dimeric interleukin-2/p53
Introduction Oligodendrocytes are post-mitotic cells that develop from rapidly dividing precursor cells (Temple and Raff, 1986; Gard and Pfeiffer, 1990; Hardy and Reynolds, 1991). The survival of oligodendrocytes depends on exogenous signaling tnolecules, such as growth factors, cytokines and neurotrophic factors (Noble et al., 1988; Richardson et al., 1988; Arakawa et al., 1990; McKinnon et al., 1990; Barres et al., 1992, 1993; Buchman and Davies, 1993; Louis et al., 1993). The death of oligodendrocytes due to insufficiency of survival factors was shown to be apoptotic in nature (Barres et al., 1992). Recent studies in our laboratory have shown that dimeric interleukin (IL)-2, produced enzymatically by nervederived transglutaminase (TGN; Eitan and Schwartz, 1993), is cytotoxic in vitro to mature oligodendrocytes (galactocerebroside-stained cells; Cohen et al., 1990; Sivron et al., 1991; Eitan et al., 1992), known to inhibit axonal growth (Schnell and Schwab, 1990; Sivron et al.,
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1994). In general, cytotoxicity may lead to cell death either by necrosis (as a result of injury, infection or other external stress) or through a more specific process of apoptosis (programmed cell death; Duvall and Wyllie, 1986). The latter is thought to be involved, among other functions, in restricting the sizes of immature progenitor cell pools in the absence of appropriate growth-promoting stimuli (Duke and Cohen, 1986; Rodriguez-Tarducy et al., 1990; Williams et al., 1990). Apoptotic cells exhibit a distinct morphology and often also a characteristic pattern of DNA fragmentation resulting from cleavage of nuclear DNA in intemucleosomal regions (Duvall and Wyllie, 1986). The aim of our study was to determine whether the death of oligodendrocytes induced by dimeric IL-2 is apoptotic and to identify signaling molecules involved in this process. Specifically, we examined whether p53, known to be involved in apoptotic death in other systems (Yonish-Rouach et al., 1991; Shaw et al., 1992; Clarke et al., 1993; Debbas and White, 1993; Lotem and Sachs, 1993; Lowe et al., 1993; Gottlieb et al., 1994), is also involved in the cell suicide program of oligodendrocytes.
Results Dimeric IL-2 causes apoptosis in oligodendrocytes Cultures enriched in mature rat brain oligodendrocytes were treated with a preparation containing dimeric IL-2, produced in vitro using recombinant human IL-2 and a fish optic nerve-derived preparation containing TGN (Eitan and Schwartz, 1993). The treated cells exhibited a typical apoptotic morphology, visualized by fluorescent staining with 4,6-diamidino-2-phenylindole (DAPI). Control cultures were: (i) treated with only the enzyme in the presence or absence of Ca2+, (ii) treated with only monomeric IL-2, or (iii) left untreated. Figure 1 shows an example of the results of one such experiment photographed 16 h after the initiation of treatment with dimeric IL-2. Cells with fragmented nuclei, indicative of an apoptotic process, are clearly visible. Apoptotic cells with fragmented nuclei and condensed chromatin were seen as early as 12 h after treatment with dimeric IL-2. To analyze the cultures quantitatively, the DAPI-stained apoptotic cells were counted at different times after treatment and their percentages relative to the total cell population were calculated at each time point. Figure 2 depicts a representative experiment in which the peak incidence of apoptotic cells was observed 16 h after the initiation of treatment, whereas significant cell death (3040%, average of three experiments), measured metabolically by a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Eitan et al., 1992), was seen after 40-48 h (data not shown). In control cultures (untreated cells and cells
p53 in apoptotic death of oligodendrocytes
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Treatments Fig. 2. Kinetics of apoptosis in dimeric IL-2-treated oligodendrocyte cultures. Apoptotic cells in the cultures described in Figure 1 were counted and their percentages calculated in relation to the total numbers of oligodendrocytes. The extent of cell death was determined in parallel cultures by the colorimetric assessment of the numbers of surviving cells (Eitan et al., 1992). The experiment was performed in duplicate and was repeated three times. For each type of treatment, 400-1100 cells were counted in each experiment. One-factor analysis of variance (ANOVA; DF = 3, F = 14.979, P = 0.0002) revealed, according to Fisher's test, a significant difference (at 95%) in the number of apoptotic cells at the peak time period in the dimeric IL-2treated cultures relative to untreated or other control cultures (results of two experiments were combined). m-IL-2, monomeric IL-2; d-IL-2, dimeric IL-2 produced in vitro (Eitan and Schwartz, 1993).
Fig. 1. Micrographs showing apoptotic cells in oligodendrocyte culture treated with dimeric IL-2. Enriched mature rat brain oligodendrocyte cultures were pretreated with dimeric IL-2. Cells were fixed, stained with DAPI and photographed at a magnification of x 1600. Phasecontrast micrograph shows the morphology of the cells (A). DAPIstained oligodendrocytes, same field as in (A), were photographed using a UV filter (B). Arrows point to cells with patched nuclei.
treated with monomeric IL-2 or with TGN, in the presence or absence of Ca2+), the percentage of apoptotic cells occurring spontaneously was much lower than in the dimeric IL-2-treated cultures at all time points analyzed (Figure 2). Of the total amount of IL-2 applied under these conditions, dimeric IL-2 comprised -25% (Eitan and Schwartz, 1993). As apoptosis above background levels was observed only in the presence of the preparation containing dimeric IL-2, it is concluded that this apoptosis was attributable to the dimeric IL-2. An analysis of DNA fragmentation in oligodendrocytes treated with dimeric IL-2 revealed a characteristic DNA ladder (Figure 3). The extent of DNA fragmentation detected 3 h after treatment (the first time point examined; data not shown) was similar to that in untreated cells. After 16 h of exposure to dimeric IL-2, the ladder became
prominent (Figure 3, lane 2). At 20 h of exposure to dimeric IL-2 an intensive ladder appeared (Figure 3, lane 3). At this time point, cell death measured colorimetrically was barely detectable. It thus seems that DNA fragmentation precedes cell death by a significant time interval. This is in agreement with observations in a variety of cell types undergoing induced apoptosis (Arends and Wyllie, 1991; Tomey and Cope, 1991).
Commitment to dimeric IL-2-induced apoptosis within a few hours The time course of commitment to cell death was examined by treating mature rat brain oligodendrocytes with dimeric IL-2, and at the indicated time points (Figure 4) washing and further incubating them in dimeric IL-2-free medium for up to 16 h after treatment (the time of peak apoptosis). Apoptotic cells were visualized by DAPI staining. For up to 2 h of incubation with dimeric IL-2, we could not discern a commitment of the cells to programmed death. However, from 3 to 4 h onwards (varying slightly among different experiments) the cells gradually became committed, and by 5 h the percentage of apoptotic cells was already similar to that in cultures treated continuously for 16 h (Figure 4). occurs
p53 in oligodendrocytes treated with dimeric IL-2 At different times after treatment with the preparation containing dimeric IL-2, oligodendrocytes were fixed and fluorescently stained with PAb421, a p53-specific 1137
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