Characterization of a new monoclonal antibody (PG-M3) directed against the aminoterminal portion of the PML gene product: immunocytochemical evidence for high expression of PML proteins on activated macrophages, endothelial cells, and epithelia.
ABSTRACT PG-M3 is a new monoclonal antibody (MoAb) specifically directed against a peptide sequence located in the aminoterminal region of the human PML protein. PML gene fuses with the retinoic acid receptor alpha (RAR alpha) gene during the t(15; 17) chromosomal translocation of acute promyelocytic leukemia (APL). The epitope recognized by PG-M3 is species-specific and fixative-resistant and is shared by most PML isoforms and PML/RAR alpha fusion proteins. PML is consistently located within the nucleus, although a minority of cells (about 20%), both in vitro and in vivo, show positivity for PML also in the cytoplasm. The nuclear staining pattern of PG-M3 varies from speckled (cells other than APL) to micropunctate (APL cells). Although two physiologically expressed PML isoforms are detectable by immunocytochemistry only or predominantly in the cytoplasm of transfected cells, the cytoplasmic localization of PML is a property also shared by the PML isoforms that predominantly localize to the nuclei. Immunohistologic analysis of normal human tissues with the PG-M3 MoAb showed variable PML expression, with the highest levels of the protein in postmitotic, differentiated cell types, such as endothelial cells, epithelia, and tissue macrophages, especially activated ones. In keeping with this in vivo finding, PML appears strongly upregulated in the U937 promonocyte cell line after exposure to agents that induce monocyte/macrophage activation (interferon gamma) or maturation (vitamin D3 and transforming growth factor beta 1).
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ABSTRACT: Hantaviruses are negative strand RNA species that replicate predominantly in the cytoplasm. They also activate numerous cellular responses, but their involvement in nuclear processes is yet to be established. Using human umbilical vein endothelial cells (HUVECs), this study investigates the molecular finger-print of nuclear transcription factors during hantavirus infection. The viral-replication-dependent activation of pro-myelocytic leukemia protein (PML) was followed by subsequent localization in nuclear bodies (NBs). PML was also found in close proximity to activated Sp100 nuclear antigen and interferon-stimulated gene 20kDa protein (ISG-20), but co-localization with death-domain associated protein-6 (DAXX) was not observed. These data demonstrate that hantavirus triggers PML activation and localization in NBs in the absence of DAXX-PLM-NB co-localization. The results suggest that viral infection interferes with DAXX-mediated apoptosis, and expression of interferon-activated Sp100 and ISG-20 proteins may indicate intracellular intrinsic antiviral attempts.Virology 07/2013; 74. DOI:10.1016/j.virol.2013.05.024 · 3.28 Impact Factor
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ABSTRACT: Genetic diagnosis is currently considered the most reliable method to accurately identify patients with acute promyelocytic leukemia (APL) requiring tailored therapy including all- trans retinoic acid (ATRA). We investigated the clinical effectiveness of immunofluorescence techniques with the anti-PML monoclonal antibody PG-M3 for rapid and accurate diagnosis of APL. PML immunofluorescence staining was analyzed in 164 patients with acute myeloblastic leukemia (AML), including APL (110 patients) and non-APL subtypes (54 patients). All 54 patients with an AML phenotype, in whom tests for t(15;17) or its fusion gene PML/ RARα were negative, showed a speckled (macrogranular) nuclear pattern. Of the 110 genetically diagnosed APL patients, 108 showed a microgranular pattern that confirmed PG-M3 positivity. The remaining two patients were not evaluable for PG-M3 reactivity because of scarcity of cells. No patient with APL showed a normal pattern. The high sensitivity and specificity of immunolabeling using PG-M3 monoclonal antibody show that it is a highly efficient and reliable tool to identify PML/ RARα-positive patients with APL and that it should be standardized as a first-line diagnostic procedure. In addition, it is technically simple, fast, and cheap, only requiring small tissue samples and non-sophisticated equipment.Annals of Hematology 01/2004; 83(11):687-690. DOI:10.1007/s00277-004-0902-7 · 2.40 Impact Factor