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Cell death induction by the acute promyelocytic leukemia-specific PML/RARalpha fusion protein
P F Ferrucci1, F Grignani, M Pearson
1European Institute of Oncology, Department of Experimental Oncology, 20141 Milan, Italy.
Abstract:
PML/RARalpha is the abnormal protein product generated by the acute promyelocytic leukemia-specific t(15;17). Expression of PML/RARalpha in hematopoietic precursor cell lines induces block of differentiation and promotes survival. We report here that PML/RARalpha has a potent growth inhibitory effect on all nonhematopoietic cell lines and on the majority of the hematopoietic cell lines tested. Inducible expression of PML/RARalpha in fibroblasts demonstrated that the basis for the growth suppression is induction of cell death. Deletion of relevant promyelocytic leukemia (PML) and retinoic acid receptor (RARalpha) domains within the fusion protein revealed that its growth inhibitory effect depends on the integrity of the PML aminoterminal region (RING, B1, B2, and coiled coil regions) and the RARalpha DNA binding region. Analysis of the nuclear localization of the same PML/RARalpha deletion mutants by immunofluorescence and cell fractionation revealed that the biological activity of the fusion protein correlates with its microspeckled localization and its association to the nuclear matrix. The PML aminoterminal region, but not the RARalpha zinc fingers, is required for the proper nuclear localization of PML/RARalpha. We propose that the matrix-associated microspeckles are the active sites of PML/RARalpha and that targeting of RARalpha sequences to this specific nuclear subdomain through PML sequences is crucial to the activity of the fusion protein on survival regulation.
Insights
The PML/RARalpha fusion protein, linked to acute promyelocytic leukemia, inhibits growth in most cell types by inducing cell death. Its activity depends on specific regions of the PML and RARalpha proteins.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- Acute promyelocytic leukemia (APL) is characterized by the t(15;17) translocation, producing the PML/RARalpha fusion protein.
- PML/RARalpha expression in hematopoietic cells blocks differentiation and promotes survival, contributing to leukemogenesis.
Purpose of the Study:
- To investigate the growth inhibitory effects of the PML/RARalpha fusion protein on various cell lines.
- To elucidate the molecular mechanisms and structural requirements underlying PML/RARalpha-induced growth suppression and cell death.
Main Methods:
- Inducible expression of wild-type and mutant PML/RARalpha proteins in nonhematopoietic (fibroblast) and hematopoietic cell lines.
- Assays for cell growth inhibition and induction of cell death.
- Immunofluorescence and cell fractionation to analyze protein localization and nuclear matrix association.
Main Results:
- PML/RARalpha potently inhibits growth in nonhematopoietic and most hematopoietic cell lines, inducing cell death.
- Growth inhibitory activity requires the PML aminoterminal region (RING, B1, B2, coiled coil) and the RARalpha DNA binding domain.
- Proper nuclear localization and microspeckled, nuclear matrix-associated localization of PML/RARalpha correlate with biological activity.
Conclusions:
- The PML aminoterminal region is essential for nuclear localization of PML/RARalpha.
- Matrix-associated microspeckles are proposed as the active sites for PML/RARalpha function.
- Targeting RARalpha to these nuclear subdomains via PML sequences is critical for survival regulation.