PML-RAR expression causes RA-sensitive differentiation block and enhanced survival in vitro.

(A) Scheme of the experimental strategy. (B) Uninfected (Control) or sorted cells infected with the empty vector (Empty), or PML-RAR–expressing cells were plated in methylcellulose medium containing IL-3, IL-6, SCF, G-CSF, and GM-CSF, in the presence or in the absence of RA (1 μM). Colonies were pooled and analyzed for the presence of the myeloid differentiation markers MAC1 (▪) and GR1 (■). (C) Control, empty, or PML-RAR cells were plated as described. Colonies were pooled and 10 000 cells were reseeded in methylcellulose plates. This procedure was repeated twice, and in the third plating RA was also included in the medium. (D) Typical morphology of the colonies observed in control (× 50), or PML-RAR expressing cells (× 100). The “clusters” of PML-RAR–expressing cells are particularly evident from the second plating. Control colonies derive from cells infected with the empty vector. Identical results were obtained from uninfected cells. (E) Wright-Giemsa staining of PML-RAR pooled colonies (third plating), after cytospins. (Fi) Western blot analysis using an anti-RAR antibody of sorted cells from control and PML-RAR infections. The arrow indicates the PML-RAR protein. (Fii) DNA-PCR analysis of several individual colonies (lanes 3-8) obtained from growing PML-RAR expressing cells in semisolid medium. Lane 1: negative control, from one control colony; lane 2: positive control, from plasmid DNA. The arrow indicates the specific, amplified PCR product. (Gi) PML expression pattern in lin⁻ cells by immunofluorescence using anti-mPML antibodies (× 125). (Gii) PML-RAR expression pattern on pooled colonies by immunofluorescence using anti-hPML antibodies (× 125). Double immunofluorescence with anti-mPML antibodies showed delocalization of the normal PML signal from “nuclear bodies” to microspeckles colocalizing with hPML-RAR (data not shown).