ATRA-induced upregulation of Beclin 1 prolongs the life span of differentiated acute promyelocytic leukemia cells

Acute promyelocytic leukemia (APL) results from a blockade of granulocyte differentiation at the promyelocytic stage. All-trans retinoic acid (ATRA) induces clinical remission in APL patients by enhancing the rapid differentiation of APL cells and the clearance of PML-RARα, APL’s hallmark oncoprotein. In the present study, we demonstrated that both autophagy and Beclin 1, an autophagic protein, are upregulated during the course of ATRA-induced neutrophil/granulocyte differentiation of an APL-derived cell line named NB4 cells. This induction of autophagy is associated with downregulation of Bcl-2 and inhibition of mTOR activity. Small interfering RNA-mediated knockdown of BECN1 expression enhances apoptosis triggered by ATRA in NB4 cells but does not affect the differentiation process. These results provide evidence that the upregulation of Beclin 1 by ATRA constitutes an anti-apoptotic signal for maintaining the viability of mature APL cells, but has no crucial effect on the granulocytic differentiation. This finding may help to elucidate the mechanisms involved in ATRA resistance of APL patients, and in the ATRA syndrome caused by an accumulation of mature APL cells.     

Neurodegeneration induces upregulation of Beclin 1

Autophagy, a bulk degradation of subcellular constituents, is activated in normal cell growth and development, and represents the major pathway by which the cell maintains a balance between protein synthesis and protein degradation. Autophagy was documented in several neurodegenerative diseases, and under stress conditions the autophagic process can lead to cell death (type II programmed cell death). Beclin 1 is a Bcl-2 interacting protein that was previously found to promote autophagy. We have used Beclin 1 protein as a marker for autophagy following traumatic brain injury in mice. We demonstrated a dramatic elevation in Beclin 1 levels near the injury site. Interestingly Beclin 1 elevation starts at early stages post injury (4 h) in neurons and 3 days later in astrocytes. In both cell types it lasts for at least three weeks. Neuronal cells, but not astrocytes, that overexpress Beclin 1 may exhibit damaged DNA but without changes in nuclear morphology. These observations may indicate that not all the Beclin1 overexpressing cells will die. The elevation of Beclin 1 at the site of injury may represent enhanced autophagy as a mechanism to discard injured cells and reduce damage to cells by disposing of injured components.     

miR-382-5p modulates the ATRA-induced differentiation of acute promyelocytic leukemia by targeting tumor suppressor PTEN

In acute promyelocytic leukemia (APL), all-trans retinoic acid (ATRA) treatment induces granulocytic differentiation and maturation. MicroRNAs play pivotal roles in formation of the leukemic phenotype. Previously, microRNA-382-5p (miR-382-5p) was upregulated in acute myeloid leukemia (AML) with t(15;17). In the present study, we found that miR-382-5p expression was elevated with ATRA-induced differentiation of APL. To investigate the potential functional role of miR-382-5p in APL differentiation, an APL cell line was transfected with miR-382-5p mimics, inhibitors, or negative control (NC). The results showed in APL cell line NB4 that miR-382-5p downregulation upon ATRA treatment was a key event in the drug response. Mechanistic investigations revealed that miR-382-5p targeted the ATRA-regulated tumor suppressor gene PTEN through direct binding to its 3′ UTR. Enforced expression of miR-382-5p or specific PTEN inhibitors inhibited ATRA-induced granulocytic differentiation via regulation of the cell cycle regulator cyclinD1. Conversely, PTEN overexpression promoted differentiation and enhanced sensitivity of NB4 cell line to physiological levels of ATRA. Finally, we found that PTEN overexpression restored PML nuclear bodies (NBs). Taken together, these results demonstrated that up-regulated miR-382-5p in NB4 cell line inhibited granulocytic differentiation through the miR-382-5p/PTEN axis, uncovering PTEN as a critical element in the granulocytic differentiation program induced by ATRA in APL.     

Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen prolongs the life span of primary human umbilical vein endothelial cells

Tumor spindle cells in all clinical types of Kaposi's sarcoma (KS) are infected with Kaposi's sarcoma-associated herpesvirus (KSHV). Although KSHV contains more than 80 genes, only a few are expressed in tumor spindle cells, including latency-associated nuclear antigen (LANA) and k-cyclin (kCYC). To assess the oncogenic potential of LANA and kCYC, primary human umbilical vein endothelial cells (HUVEC) and murine NIH 3T3 cells were stably transduced by using recombinant retroviruses expressing these genes or the known viral oncogene simian virus 40 large T antigen (LTAg). Interestingly, LANA-transduced HUVEC proliferated faster and demonstrated a greatly prolonged life span (mean +/- standard deviation, 38.3 +/- 11.0 passages) than untransduced cells and vector-transduced cells (<20 passages). By contrast, kCYC-transduced HUVEC did not proliferate faster or live longer than control cells. LANA- and kCYC-transduced HUVEC, but not LTAg-transduced HUVEC, retained the ability to form normal vessel-like structures in an in vitro model of angiogenesis. In cellular assays of transformation, LANA- and kCYC-transduced NIH 3T3 cells demonstrated minimal or no anchorage-independent growth in soft agar and no tumorigenicity when injected into nude mice, unlike LTAg-transduced NIH 3T3 cells. Lastly, gene expression profiling revealed down-regulation, or silencing, of a number of genes within LANA-transduced HUVEC. Taken together, these results suggest that KSHV LANA is capable of inducing prolonged life span, but not transformation, in primary human cells. These findings may explain why LANA-expressing spindle cells proliferate within KS tumors, yet most often do not demonstrate biologic characteristics of transformation or true malignant conversion.     

ImageStream promyelocytic leukemia protein immunolocalization: in search of promyelocytic leukemia cells

Acute promyelocytic leukemia (APL) is a hematological emergency in which a rapid diagnosis is essential for early administration of appropriate therapy, including all-trans retinoic acid before the onset of fatal coagulopathy. Currently, the following methodologies are widely used for rapid initial diagnosis of APL: 1) identification of hypergranular leukemic promyelocytes by using classical morphology; 2) identification of cells with diffuse promyelocytic leukemia (PML) protein distribution by immunofluorescence microscopy; 3) evidence of aberrant promyelocyte surface immunophenotype by conventional flow cytometry (FCM). Here, we show a method for immunofluorescent detection of PML localization using ImageStream FCM. This technique provides objective per-cell quantitative image analysis for statistically large sample sizes, enabling precise and operator-independent PML pattern recognition even in electronic and real dilution experiments up to 10% of APL cellular presence. Therefore, we evidence that this method could be helpful for rapid and objective initial diagnosis and the prompt initiation of APL treatment.     

The expression of Slit2 and Robo1 increased during retinoic acid syndrome in acute promyelocytic leukemia and impacted differentiated cell migration

Retinoic acid syndrome (RAS) is a serious complication developed during the induction therapy of acute promyelocytic leukemia (APL). Cytokines and differentiated cells migration play important roles in the development of RAS. Slit guidance ligand 2 (Slit2) and roundabout 1 (Robo1) involve in cell migration. Our study aimed to investigate the expression of Slit2 and Robo1 in APL and check whether they affected promyelocytes migration. 62 cases of newly diagnosed APL patients were involved and received all-trans retinoic acid (ATRA) and arsenic trioxide as induction therapy. Bone marrow cells (BMCs) were obtained on days 0 and 28, and promyelocytes and plasma were collected from day 1 to day 21. The expression of Robo1 in promyelocytes, and that of Slit2 and cytokines, including IL-8,IL-1β and others, in serum were monitored. 20 healthy individuals donated their cells as control. Of the 62 APL patients, 16 (25.81%) patients developed RAS. The expression of Robo1, Slit2 and IL-8 increased significantly with the development of RAS. In the 16 patients with RAS, levels of Slit2, Robo1 and IL-8 were higher during the development of RAS than before or after the RAS (P < 0.05). RhSlit2-N and rhIL-8 induced cells migration, and the migration induced by IL-8 was not inhibited by rhSlit2-N. Elevated Slit2 and Robo1 levels might be useful markers for the diagnosis and treatment of RAS. The levels of Slit2, Robo1 and IL-8 showed a positive correlation with the severity of RAS. Slit2 and IL-8 promoted the migration of differentiated cells.     

Triterpenoid CDDO-Im downregulates PML/RARalpha expression in acute promyelocytic leukemia cells

The triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) induces differentiation and apoptosis of diverse human tumor cells. In the present study, we examined the effects of the CDDO imidazolide imide (CDDO-Im) on the NB4 acute promyelocytic leukemia (APL) cell line and primary APL cells. The results show that CDDO-Im selectively downregulates expression of the PML/retinoic receptor alpha fusion protein by a caspase-dependent mechanism and sensitizes APL cells to the differentiating effects of all-trans retinoic acid (ATRA). CDDO-Im treatment of APL cells was also associated with disruption of redox balance and activation of the extrinsic apoptotic pathway. In concert with these results, CDDO-Im sensitizes APL cells to arsenic trioxide (ATO)-induced apoptosis. Our findings indicate that CDDO-Im may be effective in the treatment of APL by: (i) downregulation of PML/RARalpha; (ii) enhancement of ATRA-induced differentiation; and (iii) sensitization of ATO-induced APL cell death.     

Expression profiling of murine acute promyelocytic leukemia cells reveals multiple model-dependent progression signatures

Leukemia results from the expansion of self-renewing hematopoietic cells that are thought to contain mutations that contribute to disease initiation and progression. Studies of the gene expression profiles of human acute myeloid leukemia samples has allowed their classification based on the presence of translocations and French-American-British subtypes, but it is not yet clear whether their molecular signatures reflect the initiating mutations or mutations acquired during progression. To begin to address this question, we examined the expression profiles of normal murine promyelocyte-enriched samples, nontransformed murine promyelocytes expressing human promyelocytic leukemia-retinoic acid receptor alpha (PML-RARalpha) fusion gene, and primary acute promyelocytic leukemia cells. The expression profile of nontransformed cells expressing PML-RARalpha was remarkably similar to that of wild-type promyelocytes. In contrast, the expression profiles of fully transformed cells from three acute promyelocytic leukemia model systems were all different, suggesting that the expression signature of acute promyelocytic leukemia cells reflects the genetic changes that contributed to progression. To further evaluate these progression events, we compared two high-penetrance acute promyelocytic leukemia models that both commonly acquire an interstitial deletion of chromosome 2 during progression. The two models exhibited distinct gene expression profiles, suggesting that the dominant molecular signatures of murine acute promyelocytic leukemia can be influenced by several independent progression events.     

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.     

Targeted therapy: The new lease on life for acute promyelocytic leukemia, and beyond

Leukemia, a group of hematological malignancies characterized by abnormal proliferation, decreased apoptosis, and blocked differentiation of hematopoietic stem/progenitor cells, is a disease involving dynamic change in the genome. Chromosomal translocation and point mutation are the major mechanisms in leukemia, which lead to production of oncogenes with dominant gain of function and tumor suppressor genes with recessive loss of function. Targeted therapy refers to treatment strategies perturbing the molecules critical for leukemia pathogenesis. The t(15;17) which generates PML-RARα, t(8;21) that produces AML1-ETO, and t(9;22) which generates BCR-ABL are the three most frequently seen chromosomal translocations in myeloid leukemia. The past two to three decades have witnessed tremendous success in development of targeted therapies for acute and chronic myeloid leukemia caused by the three fusion proteins. Here, we review the therapeutic efficacies and the mechanisms of action of targeted therapies for myeloid leukemia and show how this strategy significantly improve the clinical outcome of patients and even turn acute promyelocytic leukemia from highly fatal to highly curable.     

PRAM-1 is a novel adaptor protein regulated by retinoic acid (RA) and promyelocytic leukemia (PML)-RA receptor alpha in acute promyelocytic leukemia cells

The t(15;17) translocation, found in 95% of acute promyelocytic leukemia, encodes a promyelocytic leukemia (PML)-retinoic acid receptor alpha (RARalpha) fusion protein. Complete remission of acute promyelocytic leukemia can be obtained by treating patients with all-trans retinoic acid, and PML-RARalpha plays a major role in mediating retinoic acid effects in leukemia cells. A main model proposed for acute promyelocytic leukemia is that PML-RARalpha exerts its oncogenic effects by repressing the expression of retinoic acid-inducible genes critical to myeloid differentiation. By applying subtraction cloning to acute promyelocytic leukemia cells, we identified a retinoic acid-induced gene, PRAM-1 (PML-RARalpha target gene encoding an Adaptor Molecule-1), which encodes a novel adaptor protein sharing structural homologies with the SLAP-130/fyb adaptor. PRAM-1 is expressed and regulated during normal human myelopoiesis. In U937 myeloid precursor cells, PRAM-1 expression is inhibited by expression of PML-RARalpha in the absence of ligand and de novo superinduced by retinoic acid. PRAM-1 associates with other adaptors, SLP-76 and SKAP-55HOM, in myeloid cell lines and with protein tyrosine kinase lyn. By providing the first evidence that PML-RARalpha dysregulates expression of an adaptor protein, our data open new insights into signaling events that are disrupted during transformation by PML-RARalpha and induced by retinoic acid during de novo differentiation of acute promyelocytic leukemia cells.     

Advances in the understanding and management of acute promyelocytic leukemia

Considerable progress has been made over the past decade in the understanding and management of acute promyelocytic leukemia (APL). At the laboratory level, molecular mechanisms underlying the arrest of differentiation that typically features in this malignancy, have been clarified and currently provide important models for addressing future investigation aimed at releasing the maturation block in other malignancies. In the clinic, advances in the management of APL have converted this rapidly fatal disease into the most frequently curable leukemia in adults. Use of retinoids in combinatorial protocols with anthracycline-based chemotherapy for front line treatment currently results in long-term survival and potential cure in at least 60% of newly diagnosed patients. Even after relapse, the disease is still curable in a high percentage of cases by various approaches including combinations of chemotherapy, retinoids, arsenic trioxide, stem cell transplantation and antibody-targeted chemotherapy. Genetic testing for identification of the disease-specific gene rearrangement and monitoring of residual disease have proved critical in establishing correct diagnosis and better evaluate the response to therapy at the molecular level. Current 'hot' issues for clinical investigation include: (i) better understanding and management of the severe coagulopathy present at diagnosis in most patients; (ii) the definition of risk categories to improve identification of patients at highest risk of relapse and (iii) the translation of successful differentiation therapy to other leukemia subsets.     

Over-expression of Mcl-1 impairs the ability of ATRA to induce growth arrest and differentiation in acute promyelocytic leukemia cells

Exposure of acute promyelocytic leukemia (APL) cells to all-trans retinoic acid (ATRA) increases levels of Mcl-1, however, the implication of ATRA-mediated expressions of Mcl-1 in these cells remains to be fully elucidated. This study found that exposure of NB4 and PL-21 cells to ATRA increased levels of Mcl-1 in association with phosphorylation of c-jun N-terminus kinases. Down-regulation of Mcl-1 by a small interfering (siRNA) or an inhibitor of JNK significantly potentiated the ability of ATRA to induce differentiation and apoptosis in these cells. On the other hand, the anti-leukemia effects of ATRA were blunted when Mcl-1 was forced expressed in NB4 and PL-21 cells as well as leukemia cells isolated from individuals with APL. Furthermore, down-regulation of Mcl-1 by an siRNA sensitized non-APL U937 and KG-1 leukemia cells to ATRA-mediated differentiation and apoptosis. Taken together, inhibition of Mcl-1 might be useful to potentiate the action of ATRA in APL as well as non-APL AML cells.