Dear Editor One of the several mechanisms accounting for HIV-1-induced lymphodepletion resides in the capacity of the envelope glycoprotein complex (Env) expressed on HIV-1 infected cells to

interact with CD4 and a suitable coreceptor (CXCR4 or CCR5) expressed on non-infected cells, thereby triggering cell fusion.1 The vast majority of syncytium-inducing HIV-1 variants employs

CXCR4 as a coreceptor, and a strong correlation between CD4+ T cell decline and infection by syncytium-induced HIV-1 variants has been established. Formation of syncytia _in vitro_ is

generally followed by cell death, either by apoptosis or necrosis, depending on the cell types engaged in the process.2,3 _In vivo_, in lymphoid tissues from AIDS patients, syncytium

envelope (ADA, that does not interact with the CXCR4 co-receptor expressed on HeLa CD4) failed to form syncytia with HeLa CD4 cells and did not undergo apoptosis7 (Figure 1A). Fusion events

were monitored by means of two stable, non-toxic CellTracker fluorescent dies with which HeLa Env (CellTracker Green) or HeLa CD4 cells (CellTracker Orange) were pre-incubated. After 24 h of

co-culture, juxtaposed nuclei from both cell types could be clearly distinguished within a common cytoplasm, the center of syncytia (Figure 1A) whose overall organization roughly

recapitulates that of normal cells.1 After 48 h, however, nucleoplasm fusion (karyogamy) occurred in ∼50% of syncytia, as detectable by the blending of the two CellTracker dies within the

nucleus. Concomitantly, an increasing percentage of syncytia spontaneously exhibited apoptotic chromatin condensation, as detected with the Hoechst 33342 dye (Figure 1A,B). Karyogamy induced

by the ENV-CD4/CXCR4 interaction involved circumscript fusions of the nuclear envelopes and occurred in a fraction of cells which are lacking signs of nuclear chromatin condensation (Figure

1C). It thus may be uncoupled from apoptotic chromatin condensation, as also suggested by kinetic analyses (Figure 1B). What is then the functional relationship between syncytium formation,

karyogamy, and apoptosis? Freshly formed syncytia (24 h) driven into apoptosis by a short-term incubation (2 h) with staurosporin (STS) exhibited a similar degree of chromatin condensation

as syncytia undergoing apoptosis spontaneously (48 h). However, neither STS (Figure 1) nor other pro-apoptotic agents such as etoposide (not shown) did increase the frequency of syncytia

exhibiting karyogamy, indicating that apoptosis induced by exogenous stimuli obeys other principles than spontaneous syncytial apoptosis. The two pan-caspase inhibitors Boc-D.fmk and

Z-VAD.fmk (but not the chemically related cathepsin inhibitor Z-FA.fmk) suppressed nuclear chromatin condensation (Figure 1A,B) as well as the detachment of HeLa Env/HeLa CD4 syncytia from

the culture substrate (not shown). Moreover, inhibition of apoptosis by the caspase inhibitor Z-VAD.fmk allowed for the formation of much larger syncytia as compared to control HeLa Env/HeLa

CD4 cocultures (Figure 1D), suggesting that caspase activation with the consequent irreversible loss of cellular functions limits the recruitment of cells into growing syncytia. In strict

contrast to their effects on chromatin condensation (Figure 1A,B) and syncytial size (Figure 1D), Boc-D.fmk or Z-VAD.fmk failed to prevent nuclear fusion (Figure 1A,B), suggesting that,

karyogamy precedes in a caspase-independent fashion. To further investigate the impact of ongoing fusion events between individual cells/nuclei and existing syncytia (or fusion between

syncytia), the monoclonal antibody Leu 3a (which blocks the CDR2 region of the CD4 D1 domain) was added to cocultures. Blockade of CD4 by adding Leu3a from the beginning of cocultures

completely abolished cell fusion (Figure 1A). When added after 24 h of culture, Leu 3a led to a net reduction of the median number of nuclei per syncytium, strongly inhibited karyogamy

(Figure 1A), and, concomitantly, reduced the frequency of apoptotic nuclear events observed 72 h (Figure 1D) after initiation of coculture. Altogether, these data suggest that the size of

syncytia determines the probability of karyogamy and spontaneous apoptosis and that, inversely, apoptotic caspase activation limits ongoing syncytium formation. More importantly, these data

suggest also that both karyogamy and apoptosis may be mechanistically coupled to non-physiologial syncytium formation. To our knowledge, this is the first description of nuclear fusion in

somatic mammalian cells. The molecular mechanisms of nuclear fusion or karyogamy has previously only been explored in the context of sexual reproduction of unicellular fungi such as

_Saccharomyces cerevisiae_.8 It will be interesting to learn whether similar mechanisms also participate in cell fusion-induced karyogamy of somatic mammalian cells. Supported by a special

grant by the Ligue Nationale contre le Cancer, as well as by grants from ANRS, FRM, the European Commission (to G Kroemer). KF Ferri receives a fellowship from the French Ministry of

Science. E Jacotot receives an ANRS fellowship. M Geuskens is a senior research associate of the Belgian National Fund for Scientific Research. REFERENCES * Sylwester A _et al_. 1997 _J.

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AFFILIATIONS * Centre National de la Recherche Scientifique, UMR1599, Institut Gustave Roussy, 39 rue Camille-Desmoulins, Villejuif, F-94805, France K F Ferri, E Jacotot & G Kroemer *

Laboratory of Molecular Parasitology, Université, Libre de Bruxelles, Gosselies, B-6041, Belgium M Geuskens Authors * K F Ferri View author publications You can also search for this author

inPubMed Google Scholar * E Jacotot View author publications You can also search for this author inPubMed Google Scholar * M Geuskens View author publications You can also search for this

author inPubMed Google Scholar * G Kroemer View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to G Kroemer. RIGHTS AND

PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Ferri, K., Jacotot, E., Geuskens, M. _et al._ Apoptosis and karyogamy in syncytia induced by the HIV-1-envelope

glycoprotein complex. _Cell Death Differ_ 7, 1137–1139 (2000). https://doi.org/10.1038/sj.cdd.4400748 Download citation * Published: 09 November 2000 * Issue Date: 01 November 2000 * DOI:

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