Successful healing of hydroxyurea-related leg ulcers with topical granulocyte-macrophage colony-stimulating factor.

One of the conclusions of our report1 is that PML/RARa itself mediates RA-dependent differentiation and that its degradation by RAis not crucial for the differentiation process to occur. Naoe and Kitamura disagree. In general, we think that there is much direct and indirect evidence to support these conclusions: (1) PML/RARa is an RA-dependent transcriptional activator2,3; (2) PML/RARa mediates RA-induced differentiation in both APL and non-APL cells; in particular, PML/RARa restores RAsensitivity in RA-resistant cell lines carrying mutations of RARa4,5; (3) commitment to differentiation in the presence of RAoccurs within the first 12 hours, when PML/RARa is still detectable6; (4) inhibition of PML/RARa degradation by caspase inhibitors (another mediator of RA-induced degradation of the fusion protein) increases RA-induced differentiation inAPL cells7; and (5) PML/RARa re-expression restores RA-sensitivity in RA-resistant APL cells with constitutive degradation of the fusion protein.1 With respect to some specific criticisms of Naoe and Kitamura: (1) we did not sequence RARa and PML/RARa transcripts in NB4.007/6 cells. Therefore, as suggested, we cannot exclude that mutations of the PML/RARa coding sequence might affect fusion protein stability. However, we think that other mechanisms are more likely, because exogenous PML/RARa (GFP-PML/RARa) or PML/RARa from parental NB4 cells were equally degraded in NB4.007/6 cells in infection and in vitro degradation assay, respectively. (2) Immunostaining of NB4.007/6 cells showed a micropunctuated pattern, typical of PML/ RARa expression. Because we did not detect PML/RARa by Western blotting, this might reflect a better efficiency of our PG-M3 anti-PML monoclonal antibody on undenatured, versus denatured, antigens. Finally, Naoe and Kitamura mentioned the isolation of an arsenic-resistant/ RA-sensitive NB4 subline. Because the signaling pathway triggered by arsenic is yet undefined, we do not think that this model system can provide information as to the role of PML/RARa in RA-signaling.