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Acute promyelocytic leukemia (APL) is characterized by the presence of the promyelocytic leukemia (PML)‑retinoic acid receptor‑α (RAR‑α) fusion protein. PML‑RARα can be cleaved by neutrophil elastase (NE) in several positions in cells in the promyelocytic stage, nuclear location signal (NLS)‑negative PML and NLS‑RARα may be the products of PML‑RARα by NE. The function of NLS‑RARα may be affected by the addition of NLS, which would alter its localization in cells, as the role of NLS is to identify proteins for transport to the nucleus. Preliminary experiments demonstrated that the overexpression of NLS‑RARα in HL‑60 cells could promote cellular proliferation and inhibit cellular differentiation. Following treatment with all‑trans retinoic acid (ATRA), the degree of cellular differentiation was enhanced. In the present study, the localization of NLS‑RARα was identified and its activity as a novel transcriptional factor was assessed, which may be critical in the development of APL. The location of NLS‑RARα was detected in the nucleus and cytoplasm by indirect immunofluorescence and western blot analysis, with expression in the nucleus revealed to be increased compared with that in the cytoplasm. Next, native‑PAGE was performed and NLS‑RARα and RXRα were revealed to form heterodimers in the nucleus. In addition, co‑immunoprecipitation revealed an interaction between NLS‑RARα and retinoid X receptor‑α (RXRα). An electrophoresis mobility shift assay (EMSA) indicated that NLS‑RARα could bind retinoic acid response elements (RAREs) in the presence of ATRA. Indeed, NLS‑RARα could bind RAREs just as WTRARα could, including the RAREs direct repeat‑2 (DR‑2) and DR‑5. In addition, results from a luciferase reporter gene assay demonstrated that NLS‑RARα could mediate the activity of RAREs that it bound. Together, these results indicated that NLS‑RARα may be a novel transcription factor that contributes to leukemogenesis by competitively binding RAREs as heterodimers with RXRα, just as PML‑RARα does, thus repressing the gene transcription essential for myeloid differentiation. These findings indicate the potential role of NLS‑RARα targeted therapy in APL. [ABSTRACT FROM AUTHOR]

Promyelocyte leukemia‑retinoic acid receptor α (PML‑RARα) is known as a fusion gene of acute promyelocytic leukemia (APL). Previous studies have reported that neutrophil elastase (NE) cleaves PML‑RARα in early myeloid cells, which leads to the removal of the nuclear localization signal (NLS) in PML and increases the incidence of APL. The resultant PML without the NLS is termed PML(NLS‑). The aim of the present study was to verify the existence and location of the PML(NLS‑) protein in NB4 cells. NB4 cells underwent electroporation with the pCMV‑HA‑NE plasmid to form NB4‑HA‑NE cells, which were then transplanted to produce tumors in nude mice and samples were collected from patients with APL. Western blot analysis, an immunofluorescence assay, confocal laser microscopy and immunohistochemistry were performed to detect the expression and localization of the PML(NLS‑) protein. The findings demonstrated that PML(NLS‑) was detectable in the cytoplasm of NB4‑HA‑NE cells, the tumors in nude mice and in neutrophils from patients with APL. This indicated that PML(NLS‑) may be an effective and novel target for the diagnosis of APL. [ABSTRACT FROM AUTHOR]

In the majority of acute promyelocytic leukemia (APL) cases, translocons produce a promyelocytic leukemia protein-retinoic acid receptor α (PML-RARα) fusion gene. Studies have reported that neutrophil elastase (NE) cleaves bcr-1-derived PML-RAα in early myeloid cells, leaving only the nuclear localization signal (NLS) of PML attached to RARα. NLS-RARα promotes cell growth and inhibits differentiation in response to ATRA. However, the mechanisms by which NLS-RARα affects cell biological characteristics are yet to be fully elucidated. The present study found that the location of RARαwas altered after it was cleaved by NE. Firstly, NE was overexpressed during the preparation of recombinant plasmid NB-4/pCMV6-NE-Myc to cleave PML-RARα. The total protein expression levels of myc and NE and expression levels of NLS-RARα in nucleoprotein were detected by western blotting. Location of NLS-RARα protein was detected by immunofluorescence and confocal laser scanning. Secondly, a nude mice model was constructed and NE protein, NLS-RARα and RARα protein assays, and the location of NLS-RARα and RARα proteins were assessed as described. The present results showed that, compared with the control groups, the location of NLS-RARα protein was predominantly detected in the nucleus, whereas RARα was mainly distributed in the cytoplasm. These findings were consistent with those of the nude mice model, and these may be used as a foundation to explain the occurrence mechanism of APL. [ABSTRACT FROM AUTHOR]

Neutrophil elastase (NE) is an early myeloid-specific serine protease, which is predominantly produced by promyelocytes. A previous study demonstrated that NE has an important role in the development of acute promyelocytic leukemia (APL). The process of APL was shown to be accelerated in animals that expressed abundant NE, whereas NE-deficient mice were protected from APL development; thus suggesting an important role for NE in the development of APL. The present study aimed to investigate the effects and possible mechanisms of NE. Up- and downregulation of NE in various leukemia cell lines was conducted in order to explore its significance in the occurrence and procession of leukemia, with the aim of identifying novel targeted therapeutic drugs for the treatment of leukemia. NE was overexpressed in cells following infection with an adenovirus, and Cell Counting kit-8 and flow cytometry results demonstrated that cell proliferation was promoted, and cell apoptosis was inhibited, as compared with the untreated cells. NE was downregulated in the cells by both RNA interference and treatment with GW311616A, a specific inhibitor of NE, following which cell growth was shown to be inhibited and apoptosis was induced. These results suggested that NE may promote the development of APL, therefore, NE may be a therapeutic target and its inhibitor GW311616A may be a potential therapeutic drug for leukemia. Furthermore, the apoptosis-associated protein B-cell lymphoma 2 (Bcl-2)-associated X protein was significantly increased, whereas Bcl-2 was markedly decreased in the cells with downregulated NE. Further experiments revealed that the probable apoptosis-associated signaling pathway was the phosphoinositide 3-kinase/AKT pathway. The present study is the first, to the best of our knowledge, to demonstrate that GW311616A, a specific NE inhibitor, may act as a potential targeted drug for leukemia, which may have a profound impact on the future of leukemia-targeted therapy. [ABSTRACT FROM AUTHOR]