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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]

Neutrophil elastase (NE) is a neutrophil.derived serine proteinase with specificity for a broad range of substrates. NE has been reported to be associated with the pathogenesis of several conditions, particularly that of pulmonary diseases. Previous studies have shown that NE can cleave the pro.myelocyte ..retinoic acid receptor.alpha chimeric protein and is important for the development of acute pro.myelocytic leukemia. To further elucidate the role of NE in acute pro.myelocytic leukemia, the present study successfully constructed a lentiviral vector containing the NE gene (LV5.NE), which was transfected into NB4 acute pro.myelocytic leukemia cells. The effects of NE overexpression in NB4 cells were detected using a Cell-Counting Kit.8 assay, flow cytometry and western blot analysis. The results showed that NE significantly promoted the proliferation of NB4 cells, inhibited cell apoptosis and apoptotic signaling, and led the activation of Akt. In an additional experiment, a vector expressing small hairpin RNA targeting NE was constructed to assess the effects of NE knockdown in U937 cells. Western blot analysis revealed that apoptotic signaling was increased, while Akt activation was decreased following silencing of NE. The results of the present study may indicate that NE activates the phosphoinositide-3 kinase/Akt signaling pathway in leukemia cells to inhibit apoptosis and enhance cell proliferation, and may therefore represent a molecular target for the treatment of pro.myelocytic leukemia. [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]

Effects of the enzymes in Actinomucor elegans extract and the enzyme Alcalase 2.4L on debittering the soybean protein hydrolysates were investigated. When the protein was treated only with the latter, a strong bitterness formed; but it decreased if the protein was treated with both the enzymes. The more the enzymes were used, weaker was the bitterness tasted. SDS-PAGE profile and ESI-MS spectrum of the hydrolysates evidenced that the Alcalase could convert the protein into peptides rapidly, while the enzymes in the A. elegans extract were able to further degrade some peptides which were difficult or unable to be hydrolyzed by the Alcalase. Further systematic analysis of the peptidases showed that the Alcalase exhibited a significant endopeptidase activity towards NBZ-Phe-pNA substrate ( p < 0.01), whereas many exopeptidases in the A. elegans extract had the carboxypeptidase activity towards N-CBZ-Ile-Leu ( p < 0.01). It is concluded that those exopeptidases presented in the A. elegans extract can benefit by decreasing the bitterness of the soybean protein hydroysate. They are also capable of being used with the Alcalase in a single-step enzymatic reaction to prepare the bitterless protein hydrolysate, which may be an efficient application for food industry. [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]