PML is required for telomere stability in non-neoplastic human cells.

Telomeres interact with numerous proteins, including components of the shelterin complex, whose alteration, similarly to proliferation-induced telomere shortening, initiates cellular senescence. In tumors, telomere length is maintained by Telomerase activity or by the Alternative Lengthening of Telomeres mechanism, whose hallmark is the telomeric localization of the promyelocytic leukemia (PML) protein. Whether PML contributes to telomeres maintenance in normal cells is unknown. We show that in normal human fibroblasts the PML protein associates with few telomeres, preferentially when they are damaged. Proliferation-induced telomere attrition or their damage due to alteration of the shelterin complex enhances the telomeric localization of PML, which is increased in human T-lymphocytes derived from patients genetically deficient in telomerase. In normal fibroblasts, PML depletion induces telomere damage, nuclear and chromosomal abnormalities, and senescence. Expression of the leukemia protein PML/RARα in hematopoietic progenitors displaces PML from telomeres and induces telomere shortening in the bone marrow of pre-leukemic mice. Our work provides a novel view of the physiologic function of PML, which participates in telomeres surveillance in normal cells. Our data further imply that a diminished PML function may contribute to cell senescence, genomic instability, and tumorigenesis.

Common themes in the pathogenesis of acute myeloid leukemia.

The pathogenesis of acute myeloid leukemia is associated with the appearance of oncogenic fusion proteins generated as a consequence of specific chromosome translocations. Of the two components of each fusion protein, one is generally a transcription factor, whereas the other partner is more variable in function, but often involved in the control of cell survival and apoptosis. As a consequence, AML-associated fusion proteins function as aberrant transcriptional regulators that interfere with the process of myeloid differentiation, determine a stage-specific arrest of maturation and enhance cell survival in a cell-type specific manner. The abnormal regulation of transcriptional networks occurs through common mechanisms that include recruitment of aberrant co-repressor complexes, alterations in chromatin remodeling, and disruption of specific subnuclear compartments. The identification and analysis of common and specific target genes regulated by AML fusion proteins will be of fundamental importance for the full understanding of acute myeloid leukemogenesis and for the implementation of disease-specific drug design.

BCR-ABL mediates arsenic trioxide-induced apoptosis independently of its aberrant kinase activity.

In the prechemotherapy era arsenic derivatives were used for treatment of chronic myelogenous leukemia, a myeloproliferative disorder characterized by the t(9;22) translocation, the Philadelphia chromosome (Ph+). In acute promyelocytic leukemia response to arsenic trioxide (As2O3) has been shown to be genetically determined by the acute promyelocytic leukemia-specific t(15;17) translocation product PML/RARalpha. Hence, we reasoned that As2O3 might have a selective inhibitory effect on proliferation of BCR-ABL-expressing cells. Here, we report that: (a) As2O3 induced apoptosis in Ph+ but not in Ph- lymphoblasts; (b) enforced expression of BCR-ABL in U937 cells dramatically increased the sensitivity to As2O3; (c) the effect of As2O3 was independent of BCR-ABL kinase activity; and (d) As2O3 reduced proliferation of chronic myelogenous leukemia blasts but not of peripheral CD34+ progenitors. In summary, these data establish As2O3 as a tumor cell-specific agent, making its clinical application in Ph+ leukemia feasible.

The acute promyelocytic leukaemia specific PML and PLZF proteins localize to adjacent and functionally distinct nuclear bodies.

Acute promyelocytic leukaemia is characterized by translocations that involve the retinoic acid receptor alpha (RAR alpha) locus on chromosome 17 and the PML locus on 15 or the PLZF locus on 11. The resulting abnormal translocation products encode for PML/RAR alpha or PLZF/RAR alpha fusion proteins. There is increasing experimental evidence that the APL-specific fusion proteins have similar biologic activities on differentiation and survival and that both components of the fusion proteins (PML or PLZF and RAR alpha) are indispensable for these biological activities. The physiologic function of PML or PLZF or whether PML and PLZF contribute common structural or functional features to the corresponding fusion proteins is not known. We report here immunofluorescence studies on the cellular localization of PLZF and PLZF/RAR alpha and compare it with the localization of PML and PML/RAR alpha. PLZF localizes to nuclear domains of 0.3-0.5 microns, approximately 14 per cell in the KG1 myeloid cell line. These PLZF-bodies are morphologically similar to the domains reported for PML (PML-NBs). There is tight spatial relationship between about 30% of PLZ-NBs and PML-NBs: they partially overlap. However, PML and PLZF do not form soluble complexes in vivo. PLZF- and PML-NBs are functionally distinct. Adenovirus E4-ORF3 protein expression alters the structure of the PML-NBs and interferon increases the number of PML-NBs and neither has any effect on PLZF NBs. The localization of PLZF/RAR alpha is different to that of PLZF and RAR alpha. The nuclear distribution pattern of PLZF/RAR alpha is one of hundreds of small dots (microspeckles) less than 0.1 micron. Expression of PLZF/RAR alpha did not provoke disruption of the PML-NBs. Co-expression of PML/RAR alpha and PLZF/RAR alpha in U937 cells revealed apparent colocalization. Overall the results suggest that the PML- and PLZF-NBs are distinct functional nuclear domains, but that they may share common regulatory pathways and/or targeting sequences, as revealed by the common localization of their corresponding fusion proteins.

Immunocytochemical diagnosis of acute promyelocytic leukemia (M3) with the monoclonal antibody PG-M3 (anti-PML).

Acute promyelocytic leukemia (APL) is characterized by a reciprocal 15; 17 chromosomal translocation, which fuses the promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARalpha) genes, leading to the expression of the PML/RARalpha fusion oncoprotein. Immunocytochemical labeling of the wild-type PML protein with the PG-M3 monoclonal antibody (MoAb) directed against the amino terminal portion of the human PML gene product, produces a characteristic nuclear speckled pattern that is due to localization of the protein into discrete dots (5 to 20 per nucleus), named PML nuclear bodies. The architecture of PML nuclear bodies appears to be disrupted in APL cells that bear the t(15; 17), thus resulting in a change of the nuclear staining pattern from speckled (wild-type PML protein) to microgranular (PML-RARalpha fusion protein). To assess whether the PG-M3 MoAb could assist in the diagnosis of APL (M3), bone marrow and/or peripheral blood samples from 100 cases of acute nonlymphoid leukemias of different subtypes were blindly immunostained with the PG-M3 MoAb, using the immunoalkaline phosphatase (APAAP) or immunofluorescence technique as detection system. Notably, the abnormal (micropunctate) pattern of the PML/RARalpha fusion protein (usually >/=50 small granules/per nucleus) was observed in APL (M3) samples, but not in other types of acute nonlymphoid leukemias. Immunocytochemical labeling with PG-M3 was particularly useful in the diagnosis of microgranular variant of APL (M3V) (three cases misdiagnosed as M4 and M5), and also to exclude a morphologic misdiagnosis of APL (six of 78 cases). In all cases investigated, immunocytochemical results were in agreement with those of reverse transcription-polymerase chain reaction (RT-PCR) for PML/RARalpha. Because the epitope identified by PG-M3 is located in the aminoterminal portion of PML (AA 37 to 51), the antibody was suitable for recognizing APL cases characterized by breakpoint occurring at different sites of PML (bcr 1, bcr 2 and bcr 3). In conclusion, immunocytochemical labeling with PG-M3 represents a rapid, sensitive, and highly-specific test for the diagnosis of APL that bears the t(15; 17). This should allow an easy and correct diagnosis of this subtype of acute leukemia to any laboratory provided with a minimal equipment for immunocytochemistry work.