Nuclear import of NLS- RARα is mediated by importin α/β

The promyelocytic leukemia-retinoic acid receptor α (PML/RARα) is hypothesized to play a vital role in the pathogenesis of acute promyelocytic leukemia (APL). A previous study has demonstrated that PML/RARα is cleaved by neutrophil elastase (NE) in early myeloid cells, which leads to an increase in the nuclear localization signal (NLS) in RARα and in the incidence of APL. In this study, we explored the effects of NLS-RARα on acute myeloid leukemia (AML) cells and studied the mechanism of its localization. LV-NLS-RARα recombinant lentivirus and negative control LV-NC lentivirus were transfected into HL-60 cells and U937 cells while mutant NLS-RARα were transfected into U937 cells, and all groups were treated with 1α, 25-dihydroxyvitamin D3(1,25D3). The results showed that NLS-RARα was located mainly in the nucleus while mutant NLS-RARα was located in the cytoplasm. Overexpression of NLS-RARα downregulated the expression of CD11b, CD11c, CD14, and three forms of CEBPβ compared to the overexpression of NC and mutant NLS-RARα. It was speculated that the abnormal localization of NLS-RARα was mediated via importin-α/β in the pathogenesis of APL. By producing point mutations in the two NLSs in NLS-RARα, we showed that the nuclear import of NLS-RARα was mainly dependent on the NLS of the RARα portion. Subsequently, we found that importin-α1 (KPNA2)/importin-β1 (KPNB1) participates in the nuclear transport of NLS-RARα. Taken together, abnormal localization of NLS-RARα blocks the differentiation of APL cells, and nuclear localization of NLS-RARα depends on NLS of the RARα portion and is mediated via binding with importin-α/β.     

NLS-RARα蛋白相互作用蛋白的筛选与验证

急性早幼粒细胞白血病(APL)具有特征性染色体易位,产生的PML-RARα融合基因在其发生发展中有重要作用．PML-RARα融合蛋白在细胞内被中性粒细胞弹性蛋白酶(neutrophil elastase,NE)切割为PML突变蛋白(核定位信号NLS缺失)和RARα突变体 (NLS-RARα,包含有PML的核定位信号),这两段蛋白质在APL的发生中可能具有重要作用．为进一步研究NLS-RARα的生物学功能,运用酵母双杂交技术在白血病cDNA文库中筛选与其作用的蛋白质．首先PCR技术扩增NLS-RARα编码序列,克隆至诱饵载体pGBKT7,测序鉴定后将其转化酵母AH109．免疫印迹检测到诱饵蛋白表达后,将含有诱饵载体的AH109与含有白血病cDNA文库的酵母Y187交配,在含有X-α-gal的营养缺陷性培养基上选择和筛选二倍体酵母．经回转实验和测序分析验证得到8个与NLS-RARα相互作用的蛋白质．为进一步验证这些相互作用,克隆其中的JTV-1蛋白,利用间接免疫荧光,GST pull-down和免疫共沉淀技术成功验证了它与NLS-RARα的相互作用．为进一步探讨APL的发生机制提供了新的线索．     

Study of the mechanisms of crocetin-induced differentiation and apoptosis in human acute promyelocytic leukemia cells

Crocetin, the major carotenoid in saffron, exhibits potent anticancer effects. However, the antileukemic effects of crocetin are still unclear, especially in primary acute promyelocytic leukemia (APL) cells. In the current study, the potential antipromyelocytic leukemia activity of crocetin and the underlying molecular mechanisms were investigated. Crocetin (100 µM), like standard anti-APL drugs, all-trans retinoic acid (ATRA, 10 µM) and As₂O ₃ (arsenic trioxide, 50 µM), significantly inhibited proliferation and induced apoptosis in primary APL cells, as well as NB4 and HL60 cells. The effect was associated with the decreased expressions of prosurvival genes Akt and BCL2, the multidrug resistance (MDR) proteins, ABCB1 and ABCC1 and the inhibition of tyrosyl-DNA phosphodiesterase 1 (TDP1), while the expressions of proapoptotic genes CASP3, CASP9, and BAX/BCL2 ratio were significantly increased. In contrast, crocetin at relatively low concentration (10 µM), like ATRA (1 µM) and As ₂O ₃ (0.5 µM), induced differentiation of leukemic cells toward granulocytic pattern, and increased the number of differentiated cells expressing CD11b and CD14, while the number of the immature cells expressing CD34 or CD33 was decreased. Furthermore, crocetin suppressed the expression of clinical marker promyelocytic leukemia/retinoic acid receptor-α ( PML/RARα) in NB4 and primary APL cells, and reduced the expression of histone deacetylase 1 ( HDAC1) in all leukemic cells. The results suggested that crocetin can be considered as a candidate for future preclinical and clinical trials of complementary APL treatment.     

Altered nuclear cofactor switching in retinoic-resistant variants of the PML-RARα oncoprotein of acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) results from a reciprocal translocation that fuses the gene for the PML tumor suppressor to that encoding the retinoic acid receptor alpha (RARα). The resulting PML-RARα oncogene product interferes with multiple regulatory pathways associated with myeloid differentiation, including normal PML and RARα functions. The standard treatment for APL includes anthracycline-based chemotherapeutic agents plus the RARα agonist all-trans retinoic acid (ATRA). Relapse, which is often accompanied by ATRA resistance, occurs in an appreciable frequency of treated patients. One potential mechanism suggested by model experiments featuring the selection of ATRA-resistant APL cell lines involves ATRA-resistant versions of the PML-RARα oncogene, where the relevant mutations localize to the RARα ligand-binding domain (LBD). Such mutations may act by compromising agonist binding, but other mechanisms are possible. Here, we studied the molecular consequence of ATRA resistance by use of circular dichroism, protease resistance, and fluorescence anisotropy assays employing peptides derived from the NCOR nuclear corepressor and the ACTR nuclear coactivator. The consequences of the mutations on global structure and cofactor interaction functions were assessed quantitatively, providing insights into the basis of agonist resistance. Attenuated cofactor switching and increased protease resistance represent features of the LBDs of ATRA-resistant PML-RARα, and these properties may be recapitulated in the full-length oncoproteins.     

Interplay of protein misfolding pathway and unfolded-protein response in acute promyelocytic leukemia

Protein misfolding has traditionally been linked to the pathogenesis of various neurodegenerative diseases. However, emerging evidence from various laboratories, including ours, suggests that protein misfolding may also play a fundamental role in some malignancies, particularly those caused by fusion oncoprotein generated from chromosomal translocation. Promyelocytic leukemia (PML) fused to the retinoic acid receptor (RAR) is a fusion oncoprotein linked to the transformation of acute promyelocytic leukemia (APL), and is not only a misfolded protein itself, but also promotes misfolding of nuclear receptor corepressor (N-CoR) protein, a corepressor essential for the growth-suppressive function of several tumor-suppressor proteins. PML–RAR promotes misfolding of N-CoR by inducing aberrant post-translational modification, which destabilizes its core and promotes instability. Misfolded N-CoR, thus, contributes to differentiation arrest and survival of APL cells through loss-of-function and aberrant gain-of-function properties. Therapeutic restoration of N-CoR conformation and function with conformation-modifying agents not only releases this differentiation arrest but also sensitizes APL cells to programmed cell death. These findings illustrate the potential of the misfolded N-CoR protein as a conformation-based drugable molecular target for APL, and highlights the promise of various conformation-modifying agents as novel therapeutics for APL. Protein conformational rearrangement, resulting from an inherited or acquired genetic alteration, could be a common pathological phenomenon contributing to transformation in different types of leukemias and solid tumors and, therefore, could serve as a common ground for designing a unifying diagnostic as well as therapeutic approach for a widely diverse disease such as cancer. To that end, APL could serve as a model for the development of a novel conformation-based therapeutic approach for other malignant diseases.     

Autophagy regulates myeloid cell differentiation by p62/SQSTM1-mediated degradation of PML-RARα oncoprotein

PML-RARα oncoprotein is a fusion protein of promyelocytic leukemia (PML) and the retinoic acid receptor-α (RARα) and causes acute promyelocytic leukemias (APL). A hallmark of all-trans retinoic acid (ATRA) responses in APL is PML-RARα degradation which promotes cell differentiation. Here, we demonstrated that autophagy is a crucial regulator of PML-RARα degradation. Inhibition of autophagy by short hairpin (sh) RNA that target essential autophagy genes such as Atg1, Atg5 and PI3KC3 and by autophagy inhibitors (e.g. 3-methyladenine), blocked PML-RARα degradation and subsequently granulocytic differentiation of human myeloid leukemic cells. In contrast, rapamycin, the mTOR kinase inhibitor, enhanced autophagy and promoted ATRA-induced PML-RARα degradation and myeloid cell differentiation. Moreover, PML-RARα co-immunoprecipitated with ubiquitin-binding adaptor protein p62/SQSTM1, which is degraded through autophagy. Furthermore, knockdown of p62/SQSTM1 inhibited ATRA-induced PML-RARα degradation and myeloid cell differentiation. The identification of PML-RARα as a target of autophagy provides new insight into the mechanism of action of ATRA and its specificity for APL.     

NLS-RARα contributes to differentiation block and increased leukemogenic potential in vivo

The fusion oncogene, promyelocytic leukemia (PML)-retinoic acid receptor-α (RARα), is crucial for acute promyelocytic leukemia (APL) pathogenesis. Previous studies have reported that PML-RARα is cleaved by neutrophil elastase (NE), an early myeloid-specific serine protease, leading to translocation of the nuclear localization signal (NLS) of the PML protein to the N-terminal of RARα. This study was designed to evaluate the value of NLS-RARα in the early diagnosis of APL. To investigate the potential functional role of NLS-RARα in leukemogenesis, HL-60 and U937 cell lines were transfected with NLS-RARα lentivirus and negative control (LVNC). The results showed that the induced expression of NLS-RARα down-regulated expressions of CD11b, CD11c, and CD14 compared to the LVNC group induced by 1α, 25-dihydroxyvitamin D₃(1,25(OH)₂D₃). This suggested that NLS-RARα overexpression inhibited granulocytic and monocytic differentiation of myeloid leukemia cells. In addition, Wright-Giemsa staining, flow cytometry, respiratory burst assay, and NBT reduction assay all confirmed the importance of NLS-RARα in differentiation. The mechanistic investigations revealed that induced NLS-RARα expression inhibited 1,25(OH)₂D₃-induced granulocytic differentiation by regulating the cell cycle regulators p19^INK4D, p21^WAF1/CIP1, cyclinD1, cyclin E1, and pRB. Furthermore, the cleaved protein NLS-RARα enhanced the oncogenicity of U937 cells in NOD/SCID mice. These findings collectively demonstrated that NLS-RARα blocked granulocytic and monocytic differentiation of myeloid leukemia cells by inhibiting the downstream targets of the RARα signal pathway and the cell cycle. This may provide a promising new target and method for diagnosing and treating APL.     

The PML-RAR alpha fusion mRNA generated by the t(15;17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR.

We have previously shown that the t(15;17) translocation specifically associated with acute promyelocytic leukemia (APL) fuses the retinoic acid receptor alpha (RAR alpha) locus to an as yet unknown gene, initially called myl and now renamed PML. We report here that this gene product contains a novel zinc finger motif common to several DNA-binding proteins. The PML-RAR alpha mRNA encodes a predicted 106 kd chimeric protein containing most of the PML sequences fused to a large part of RAR alpha, including its DNA- and hormone-binding domains. In transient expression assays, the hybrid protein exhibits altered transactivating properties if compared with the wild-type RAR alpha progenitor. Identical PML-RAR alpha fusion points are found in several patients. These observations suggest that in APL, the t(15;17) translocation generates an RAR mutant that could contribute to leukemogenesis through interference with promyelocytic differentiation.     

Differential gene expression in retinoic acid-induced differentiation of acute promyelocytic leukemia cells, NB4 and HL-60 cells

Acute promyelocytic leukemia (APL) is characterized by a specific chromosome translocation t(15;17), which results in the fusion of the promyelocytic leukemia gene (PML) and retinoic acid receptor alpha gene (RARalpha). APL can be effectively treated with the cell differentiation inducer all-trans retinoic acid (ATRA). NB4 cells, an acute promyelocytic leukemia cell line, have the t(15;17) translocation and differentiate in response to ATRA, whereas HL-60 cells lack this chromosomal translocation, even after differentiation by ATRA. To identify changes in the gene expression patterns of promyelocytic leukemia cells during differentiation, we compared the gene expression profiles in NB4 and HL-60 cells with and without ATRA treatment using a cDNA microarray containing 10,000 human genes. NB4 and HL-60 cells were treated with ATRA (10(-6)M) and total RNA was extracted at various time points (3, 8, 12, 24, and 48h). Cell differentiation was evaluated for cell morphology changes and CD11b expression. PML/RARalpha degradation was studied by indirect immunofluoresence with polyclonal PML antibodies. Typical morphologic and immunophenotypic changes after ATRA treatment were observed both in NB4 and HL-60 cells. The cDNA microarray identified 119 genes that were up-regulated and 17 genes that were down-regulated in NB4 cells, while 35 genes were up-regulated and 36 genes were down-regulated in HL60 cells. Interestingly, we did not find any common gene expression profiles regulated by ATRA in NB4 and HL-60 cells, even though the granulocytic differentiation induced by ATRA was observed in both cell lines. These findings suggest that the molecular mechanisms and genes involved in ATRA-induced differentiation of APL cells may be different and cell type specific. Further studies will be needed to define the important molecular pathways involved in granulocytic differentiation by ATRA in APL cells.     

Autophagy regulates myeloid cell differentiation by p62/SQSTM1-mediated degradation of PML-RARα oncoprotein

PML-RARα oncoprotein is a fusion protein of promyelocytic leukemia (PML) and the retinoic acid receptor-α (RARα) and causes acute promyelocytic leukemias (APL). A hallmark of all-trans retinoic acid (ATRA) responses in APL is PML-RARα degradation which promotes cell differentiation. Here, we demonstrated that autophagy is a crucial regulator of PML-RARα degradation. Inhibition of autophagy by short hairpin (sh) RNA that target essential autophagy genes such as Atg1, Atg5 and PI3KC3 and by autophagy inhibitors (e.g. 3-methyladenine), blocked PML-RARα degradation and subsequently granulocytic differentiation of human myeloid leukemic cells. In contrast, rapamycin, the mTOR kinase inhibitor, enhanced autophagy and promoted ATRA-induced PML-RARα degradation and myeloid cell differentiation. Moreover, PML-RARα co-immunoprecipitated with ubiquitin-binding adaptor protein p62/SQSTM1, which is degraded through autophagy. Furthermore, knockdown of p62/SQSTM1 inhibited ATRA-induced PML-RARα degradation and myeloid cell differentiation. The identification of PML-RARα as a target of autophagy provides new insight into the mechanism of action of ATRA and its specificity for APL.     

Coiled-coil domain of PML is essential for the aberrant dynamics of PML-RARalpha, resulting in sequestration and decreased mobility of SMRT

Promyelocytic leukemia-retinoic acid receptor α (PML-RARα) is the most frequent RARα fusion protein in acute promyelocytic leukemia (APL). Our previous study has demonstrated that, compared with RARα, PML-RARα had reduced intranuclear mobility accompanied with mislocalization. To understand the molecular basis for the altered dynamics of PML-RARα fusion protein, we performed FRAP analysis at a single cell level. Results indicated that three known sumoylation site mutated PML-RARα had same intracellular localization and reduced mobility as wild-type counterpart. The coiled-coil domain of PML is responsible for the aberrant dynamics of PML-RARα. In addition, we revealed that co-repressor SMRT co-localized with PML-RARα, resulting in the immobilization of SMRT while ATRA treatment eliminated their association and reversed the immobile effect of SMRT. Furthermore, co-activator CBP, co-localized with PML-RARα in an ATRA-independent way, was demonstrated as a high dynamic intranuclear molecule. These results would shed new insights for the molecular mechanisms of PML-RARα-associated leukemogenesis.     

Design of a sandwich-mode amperometric biosensor for detection of PML/RARα fusion gene using locked nucleic acids on gold electrode

In this study, a novel DNA electrochemical probe (locked nucleic acid, LNA) was designed and involved in constructing an electrochemical DNA biosensor for detection of promyelocytic leukemia/retinoic acid receptor alpha (PML/RARα) fusion gene in acute promyelocytic leukemia for the first time. This biosensor was based on a 'sandwich' sensing mode, which involved a pair of LNA probes (capture probe immobilized at electrode surface and biotinyl reporter probe as an affinity tag for streptavidin-horseradish peroxidase (streptavidin-HRP). Since biotin can be connected with streptavidin-HRP, this biosensor offered an enzymatically amplified electrochemical current signal for the detection of target DNA. In the simple hybridization system, DNA fragment with its complementary DNA fragment was evidenced by amperometric detection, with a detection limit of 74 fM and a linear response range of 0.1-10 pM for synthetic PML/RARα fusion gene in acute promyelocytic leukemia (APL). Otherwise, the biosensor showed an excellent specificity to distinguish the complementary sequence and different mismatch sequences. The new pattern also exhibited high sensitivity and selectivity in mixed hybridization system.