Flow cytometry analysis of cell population dynamics and cell cycle during HIV-1 envelope-mediated formation of syncytia in vitro.
Cell fusion occurs in physiological and pathological conditions and plays a role in regulation of cell fate. The analysis of cell population dynamics and cell cycle in cell-cell fusion experiments is necessary to determine changes in the quantitative equilibrium of cell populations and to identify potential bystander effects. Here, using cocultures of Jurkat HIV-1 envelope expressing cells and CD4(+) cells as a model system and flow cytometry for the analysis, the number, viability, and cell cycle status of the populations participating in fusion were determined. In 3-day cocultures, a sustained reduction of the number of CD4(+) cells was observed while they showed high viability and normal cell cycle progression; fusion, but not inhibition of proliferation or death, accounted for their decrease. In contrast, the number of Env(+) cells decreased in cocultures due to fusion, death, and an inherent arrest at G1. Most of syncytia formed in the first 6 h of coculture showed DNA synthesis activity, indicating that the efficient recruitment of proliferating cells contributed to amplify the removal of CD4(+) cells by syncytia formation. Late in cocultures, approximately 50% of syncytia were viable and a subpopulation still underwent DNA synthesis, even when the recruitment of additional cells was prevented by the addition of the fusion inhibitor T-20, indicating that a population of syncytia may progress into the cell cycle. These results show that the quantitative analysis of cellular outcomes of cell-cell fusion can be performed by flow cytometry.
Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad Universitaria, Distrito Federal, México.
Israel
I
Torres-Castro
César N
CN
Cortés-Rubio
Guadalupe
G
Sandoval
Edmundo
E
Lamoyi
Carlos
C
Larralde
Leonor
L
Huerta
D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000821:virology; D002453:Cell Cycle / Q000235:genetics*; D002459:Cell Fusion; D049109:Cell Proliferation / Q000235:genetics*; D018920:Coculture Techniques; D004247:DNA / Q000096:biosynthesis; D005434:Flow Cytometry; D015726:Giant Cells / Q000166:cytology* / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D019169:Jurkat Cells / Q000378:metabolism / Q000821:virology; D014759:Viral Envelope Proteins / Q000378:metabolism
D014759:Viral Envelope Proteins; D004247:DNA