Opposite Effects of the Acute Promyelocytic Leukemia PML-Retinoic Acid Receptor α (RARα) and PLZF-RARα Fusion Proteins on Retinoic Acid Signalling

Abstract

Fusion proteins involving the retinoic acid receptor alpha (RAR alpha) and the PML or PLZF nuclear protein are the genetic markers of acute promyelocytic leukemias (APLs). APLs with the PML-RAR alpha or the PLZF-RAR alpha fusion protein are phenotypically indistinguishable except that they differ in their sensitivity to retinoic acid (RA)-induced differentiation: PML-RAR alpha blasts are sensitive to RA and patients enter disease remission after RA treatment, while patients with PLZF-RAR alpha do not. We here report that (i) like PML-RAR alpha expression, PLZF-RAR alpha expression blocks terminal differentiation of hematopoietic precursor cell lines (U937 and HL-60) in response to different stimuli (vitamin D3, transforming growth factor beta1, and dimethyl sulfoxide); (ii) PML-RAR alpha, but not PLZF-RAR alpha, increases RA sensitivity of hematopoietic precursor cells and restores RA sensitivity of RA-resistant hematopoietic cells; (iii) PML-RAR alpha and PLZF-RAR alpha have similar RA binding affinities; and (iv) PML-RAR alpha enhances the RA response of RA target genes (those for RAR beta, RAR gamma, and transglutaminase type II [TGase]) in vivo, while PLZF-RAR alpha expression has either no effect (RAR beta) or an inhibitory activity (RAR gamma and type II TGase). These data demonstrate that PML-RAR alpha and PLZF-RAR alpha have similar (inhibitory) effects on RA-independent differentiation and opposite (stimulatory or inhibitory) effects on RA-dependent differentiation and that they behave in vivo as RA-dependent enhancers or inhibitors of RA-responsive genes, respectively. Their different activities on the RA signalling pathway might underlie the different responses of PML-RAR alpha and PLZF-RAR alpha APLs to RA treatment. The PLZF-RAR alpha fusion protein contains an approximately 120-amino-acid N-terminal motif (called the POZ domain), which is also found in a variety of zinc finger proteins and a group of poxvirus proteins and which mediates protein-protein interactions. Deletion of the PLZF POZ domain partially abrogated the inhibitory effect of PLZF-RAR alpha on RA-induced differentiation and on RA-mediated type II TGase up-regulation, suggesting that POZ-mediated protein interactions might be responsible for the inhibitory transcriptional activities of PLZF-RAR alpha.

Formation of PML/RARα high molecular weight nuclear complexes through the PML coiled-coil region is essential for the PML/RARα-mediated retinoic acid response

“…This agrees with recent ®ndings showing that pharmacological concentrations of RA cause the release of the corepressorhistone deacetylase complex from PML/RARa Lin et al, 1998;He et al, 1998). Likewise, RA treatment of U937 cells induces, in the presence of PML/RARa expression, a dramatic increase in the activation of certain RA-target genes, for example transglutaminase type II and RARb (Ruthardt et al, 1997). Notably, the biological e ects of the mutants are in agreement with their RAdependent transcription factor action, since biologically active mutants have a transactivating activity similar to that of PML/RARa and increased with respect of that of RAR.…”

“…Expression of PML/RARa into di erent haematopoietic cell lines revealed that the fusion protein is capable of mediating the di erentiative response to RA (Grignani et al, 1993;Ruthardt et al, 1997). Here, we investigated the molecular mechanisms underlying the biological activity of the fusion protein on RAsignalling.…”

“…However, the degradation of PML/ RARa in RA-treated APL cells takes about 24 h, suggesting that the commitment to the di erentiation process occurs in the presence of PML/RARa expression and that the fusion protein might exert a direct e ect on the RA-signalling pathway in the presence of RA (Casini and Pelicci, 1998). Direct evidence for this hypothesis came from: (1) the demonstration that inhibition of PML/RARa degradation does not prevent RA-induced di erentiation ; (2) the identi®cation of RAresistant APL mutant cell clones with constitutive degradation of PML/RARa ; and (3) the demonstration that ectopic expression of PML/ RARa in cells that are poorly sensitive or resistant to RA restores RA-sensitivity (Grignani et al, 1993;Ruthardt et al, 1997). Notably, PML/RARa enhanced RA-response in U937 cells is also associated with the degradation of the fusion protein .…”

A function of p21 during promyelocytic leukemia cell differentiation independent of CDK inhibition and cell cycle arrest

“…Hybridization was performed in 50% formamide at 428C for 24 h with 32 P-labelled DNA probes followed by washes in 0.26SSC/0.1%SDS at 658C. Western blottings were performed on 100 mg of cellular lysates with a ± btubulin (Amersham), CP74 a-p21 (Dynlacht, 1997) and G3-245 a-RB (Pharmingen) monoclonal antibodies, and with the M2 a-CDK2, C-19 a-p27, H-432 a-cyclin A (Santa Cruz Inc), a-RARaF1 (Ruthardt et al, 1997) polyclonal antibodies. 500 mg of cell lysate were immunoprecipitated with 5 ml of M2 a-CDK2 antibody and the recovered immunocomplexes used for kinase assay using histone H1, as described (Xiong et al, 1993).…”

“…Several experimental ®ndings indicate that both the leukaemogenetic e ect of PML/RARa and its ability to mediate RA-sensitivity are due to interference of the fusion protein with the program of terminal di erentiation: (i) before the onset of leukemias, the PML/RARa transgenic mice show a pre-leukemic condition characterized by increased and poorly di erentiated hematopoietic precursors in the bonemarrow (Brown et al, 1997a); (ii) expression of PML/ RARa in hematopoietic precursor cell lines blocks di erentiation induced by physiological stimuli (Grignani et al, 1993); (iii) RA induces differentiation of PML/RARa-expressing cells, both in the patients (Grignani et al, 1994) and in transgenic mice (Brown et al, 1997a;Grisolano et al, 1997;LiZhen et al, 1997); (iv) expression of PML/RARa in resistant or poorly RA-sensitive cell lines restores a di erentiative response to RA (Grignani et al, 1993;Ruthardt et al, 1997). It appears, therefore, that PML/RARa blocks di erentiation at physiological concentrations of RA, while it favours it at pharmacological doses.…”