PML, a growth suppressor disrupted in acute promyelocytic leukemia.

Abstract

The nonrandom chromosomal translocation t(15;17)(q22;q21) in acute promyelocytic leukemia (APL) juxtaposes the genes for retinoic acid receptor a (RARa) and the putative zinc finger transcription factor PML. The breakpoint site encodes fusion protein PML-RARa, which is able to form a heterodimer with PML. It was hypothesized that PML-RARa is a dominant negative inhibitor of PML. Inactivation of PML function in APL may play a critical role in APL pathogenesis. Our results demonstrated that PML, but not PML-RARa, is a growth suppressor. This is supported by the following findings: (i) PML suppressed anchorage-independent growth of APL-derived NB4 cells on soft agar and tumorigenicity in nude mice, (ii) PML suppressed the oncogenic transformation of rat embryo fibroblasts by cooperative oncogenes, and (iii) PML suppressed transformation of NIH 3T3 cells by the activated neu oncogene. Cotransfection of PML with PML-RARoa resulted in a significant reduction in PML's transformation suppressor function in vivo, indicating that the fusion protein can be a dominant negative inhibitor of PML function in APL cells. This observation was further supported by the finding that cotransfection of PML and PML-RARot resulted in altered normal cellular localization of PML. Our results also demonstrated that PML, but not PML-RARao, is a promoter-specific transcription suppressor. Therefore, we hypothesized that disruption of the PML gene, a growth or transformation suppressor, by the t(15;17) translocation in APL is one of the critical events in leukemogenesis.Acute promyelocytic leukemia (APL) represents a clonal proliferation and expansion of the hematologic precursors at the promyelocyte stage of myeloid differentiation. A nonrandom chromosomal translocation, t(15;17), can be found in over 95% of patients with APL (3, 42), suggesting that this translocation plays a critical role in leukemogenesis. In recent attempts to explore this role further, genes involved in this translocation have been cloned and characterized (4,7,17,37,48). The t(15;17) breakpoint occurs within the second intron of the retinoic acid (RA) receptor ox (RARox) gene and within two major sites of the PML (or MYL) gene (1,8,10,28,47 (VDR) to its target sequence and prevent vitamin D3-dependent activation of VDR-responsive genes. Furthermore, it was shown that transfection of PML-RARot into U937 human myeloid leukemia cells inhibits its ability to respond to vitamin D3 and transforming growth factor 13 and so induce differentiation. These results strongly suggest that the fusion protein encoded from the breakpoint of the t(15;17) translocation plays a critical role in APL pathogenesis. Recently, Doucas et al. (19) reported that PML-RARot, instead of being an inhibitor, is an RA-dependent activator of the transcription factor AP-1. However, since, as Kastner et al. (38)

Modulation of Fos-mediated AP-1 transcription by the promyelocytic leukemia protein

“…The addition of 1 mM ATRA had no signi®cant e ect on the stimulatory e ect of PML, but completely abolished the PMLRARa e ects (compare lanes 4 and 9 in Figure 2). Cotransfection of a short isoform of PML (Mu et al, 1994) also showed the same e ects on the GAL4/Fos-mediated transcriptional activity. Expression of either PML, PMLRARa or Fos (without the GAL4 DNA-binding domain) had no e ect on the basal activity of the GAL4 target reporter plasmid.…”

“…The induction of Fos/Jun-mediated AP-1 transcription activity in response to PML as shown in this study may therefore correlate with its demonstrated growth suppressor function. Interestingly, deletion analysis showed that the RING-®nger and B1 box motifs of PML, which are required for its inhibition of neu-induced transformation and localization in PODs (Mu et al, 1994;Le et al, 1996), are also essential for its induction of Fos-mediated transcriptional activity. These observations could provide further evidence to support the hypothesis that disruption of PML's function as a result of its fusion to RARa may be responsible for the increase in proliferation and appearance of the leukemia phenotype in APL (Mu et al, 1994).…”

“…Interestingly, deletion analysis showed that the RING-®nger and B1 box motifs of PML, which are required for its inhibition of neu-induced transformation and localization in PODs (Mu et al, 1994;Le et al, 1996), are also essential for its induction of Fos-mediated transcriptional activity. These observations could provide further evidence to support the hypothesis that disruption of PML's function as a result of its fusion to RARa may be responsible for the increase in proliferation and appearance of the leukemia phenotype in APL (Mu et al, 1994). Further investigations are necessary to identify the site of interaction of PML with the AP-1 complex and factors by which PML mediates its stimulatory e ects on Fos-mediated transcriptional activity.…”

“…PML encodes a nuclear phosphoprotein with a growth and transformation suppressor function (Mu et al, 1994;Chang et al, 1995;Liu et al, 1995). PML is a primary target for interferons, and its expression is signi®cantly induced by both classes of interferons (Stadler et al, 1995).…”

“…Recent investigations have provided evidence supporting the involvement of PML in the regulation (repression and activation) of transcription. In transient transfection experiments, PML was shown to inhibit transcription from the epidermal growth factor receptor (EGFR) and human multi-drug resistance 1 (MDR1) promoters (Mu et al, 1994), and is able to repress transcription from GAL4-responsive promoters when directed to DNA via the DNA-binding domain of GAL4, a yeast transcription factor (Vallian et al, 1997). Conversely, another report indicated that both PML and the translocation fusion product PMLRARa are capable of inducing the transcriptional activity of steroid hormone receptors, and altering their subcellular distribution (GuiochonMantel et al, 1995).…”