Human Acute Promyelocytic Leukemia Cell Line Research Articles

Honokiol induces paraptosis-like cell death of acute promyelocytic leukemia via mTOR & MAPK signaling pathways activation

Acute promyelocytic leukemia (APL) is a blood system disease caused by the accumulation of a large number of immature blood cells in bone marrow. Although the introduction of all-trans retinoic acid (ATRA) and arsenic has reached a high level of complete remission rate and 5-year disease-free survival rate, the occurrence of various adverse reactions still severely affects the quality of life of patients. As a natural product, honokiol (HNK) has the advantages of low toxicity and high efficiency, and it is a potential drug for the treatment of cancer. Since cancer cells can escape apoptotic cell death through multiple adaptive mechanisms, HNK, a drug that induces cancer cell death in a nonapoptotic way, has attracted much interest. We found that HNK reduced the viability of human APL cell line (NB4 cells) by inducing paraptosis-like cell death. The process was accompanied by excessive reactive oxygen species (ROS), mitochondrial damage, endoplasmic reticulum stress, and increased microtubule-associated protein 1 light chain 3 (LC3) processing. The inactivation of proteasome activity was the main cause of misfolded and unfolded protein accumulation in endoplasmic reticulum, such as LC3II/I and p62. This phenomenon could be alleviated by adding cycloheximide (CHX), a protein synthesis inhibitor. We found that mTOR signaling pathway participated in paraptosis-like cell death induced by HNK in an autophagy-independent process. Moreover, the mitogen-activated protein kinase (MAPK) signaling pathway induced paraptosis of NB4 cells by promoting endoplasmic reticulum stress. In summary, these findings indicate that paraptosis may be a new way to treat APL, and provide novel insights into the potential mechanism of paraptosis-like cell death.

Inhibition of UBE2L6 attenuates ISGylation and impedes ATRA-induced differentiation of leukemic cells.

Ubiquitin/ISG15‐conjugating enzyme E2L6 (UBE2L6) is a critical enzyme in ISGylation, a post‐translational protein modification that conjugates the ubiquitin‐like modifier, interferon‐stimulated gene 15 (ISG15), to target substrates. Previous gene expression studies in acute promyelocytic leukemia (APL) cells showed that all‐trans‐retinoic acid (ATRA) altered the expression of many genes, including UBE2L6 (200‐fold) and other members of the ISGylation pathway. Through gene expression analyses in a cohort of 98 acute myeloid leukemia (AML) patient samples and in primary neutrophils from healthy donors, we found that UBE2L6 gene expression is reduced in primary AML cells compared with normal mature granulocytes. To assess whether UBE2L6 expression is important for leukemic cell differentiation—two cell line models were employed: the human APL cell line NB4 and its ATRA‐resistant NB4R counterpart, as well as the ATRA‐sensitive human AML HL60 cells along with their ATRA‐resistant subclone—HL60R. ATRA strongly induced UBE2L6 in NB4 APL cells and in ATRA‐sensitive HL60 AML cells, but not in the ATRA‐resistant NB4R and HL60R cells. Furthermore, short hairpin (sh)RNA‐mediated UBE2L6 depletion in NB4 cells impeded ATRA‐mediated differentiation, suggesting a functional role for UBE2L6 in leukemic cell differentiation. In addition, ATRA induced ISG15 gene expression in NB4 APL cells, leading to increased levels of both free ISG15 protein and ISG15 conjugates. UBE2L6 depletion attenuated ATRA‐induced ISG15 conjugation. Knockdown of ISG15 in NB4 APL cells inhibited ISGylation and also attenuated ATRA‐induced differentiation. In summary, we demonstrate the functional importance of UBE2L6 in ATRA‐induced neutrophil differentiation of APL cells and propose that this may be mediated by its catalytic role in ISGylation.

Fucoidan enhances the therapeutic potential of arsenic trioxide and all-trans retinoic acid in acute promyelocytic leukemia, in vitro and in vivo.

The morbidity and mortality associated with current therapies for acute promyelocytic leukemia (APL) remain a significant clinical concern, despite improvements in patient survival. Consequently, the development of adjuvant therapies that increase efficacy while reducing morbidities is important. Reducing the concentration of the toxic drugs in adjuvant therapy has the potential to reduce unwanted side effects. Therefore, this study aimed to determine the synergistic effects of fucoidan, an anti-tumor agent, with current APL therapies.When the human APL cell line, NB4, was treated in vitro with fucoidan plus ATO and ATRA at therapeutic and sub-therapeutic doses, there was an increase in sub-G0/G1 cells, annexin V/PI-positive-apoptotic cells and DNA fragmentation. This reduction in proliferation and increase in apoptosis was accompanied by enhanced myeloid differentiation as indicated by an increased expression of CD11b. This was not observed with the AML cell line Kasumi-1, suggesting specificity for APL.In vivo treatment of APL-bearing mice with fucoidan+ATRA or fucoidan+ATO delayed tumor growth, induced differentiation and increased tumor volume doubling time. The differentiated APL cells derived from the excised tumor mass exhibited decreased CD44 expression in fucoidan+ATRA treated mice. This could translate to decreased cell migration in APL patients.Our findings provide evidence supporting the use of fucoidan as an adjuvant therapeutic agent in the treatment of APL.

PCGF2 negatively regulates arsenic trioxide-induced PML-RARA protein degradation via UBE2I inhibition in NB4 cells

Arsenic trioxide (ATO) is a therapeutic agent for acute promyelocytic leukemia (APL) which induces PML-RARA protein degradation via enhanced UBE2I-mediated sumoylation. PCGF2, a Polycomb group protein, has been suggested as an anti-SUMO E3 protein by inhibiting the sumoylation of UBE2I substrates, HSF2 and RANGAP1, via direct interaction. Thus, we hypothesized that PCGF2 might play a role in ATO-induced PML-RARA degradation by interacting with UBE2I. PCGF2 protein was down-regulated upon ATO treatment in human APL cell line, NB4. Knockdown of PCGF2 in NB4 cells, in the absence of ATO treatment, was sufficient to induce sumoylation-, ubiquitylation- and PML nuclear body-mediated degradation of PML-RARA protein. Moreover, overexpression of PCGF2 protected ATO-mediated degradation of ectopic and endogenous PML-RARA in 293T and NB4 cells, respectively. In 293T cells, UBE2I-mediated PML-RARA degradation was reduced upon PCGF2 co-expression. In addition, UBE2I-mediated sumoylation of PML-RARA was reduced upon PCGF2 co-expression and PCGF2-UBE2I interaction was confirmed by co-immunoprecipitation. Likewise, endogenous PCGF2-UBE2I interaction was detected by co-immunoprecipitation and immunofluorescence assays in NB4 cells. Intriguingly, upon ATO-treatment, such interaction was disrupted and UBE2I was co-immunoprecipitated or co-localized with its SUMO substrate, PML-RARA. Taken together, our results suggested a novel role of PCGF2 in ATO-mediated degradation of PML-RARA that PCGF2 might act as a negative regulator of UBE2I via direct interaction.

Thrombin Generationand Fibrinolytic Activity in Microparticles In Acute Promyelocytic Leukemia

IntroductionThe coagulopathy in acute promyelocytic leukemia (APL) is unique among the leukemias and accounts for the major complications of hemorrhage and thrombosis. The pathogenesis is in large part the result of increased expression by the APL promyelocytes of the procoagulant TF, and the fibrinolytic components of tPA , PAI-1 and Annexin A2. Recently, microparticles derived from plasma of APL patients and from culture medium of the human APL cell line NB4 have been shown to contain antigens of procoagulant tissue factor (TF), and the fibrinolytic components of tPA , Annexin A2 and PAI-1 (Kwaan HC, Rego EM. Role of microparticles in the hemostatic dysfunction in acute promyelocytic leukemia. Seminars in thrombosis and hemostasis 2010;36:917-24;Kwaan et al:Microparticles in acute promyelocytic leukemia. 53rdASH Annual Meeting; Blood 2011 Abstract No.3346). Results of functional activities of these components and thrombin generation in the microparticles are presented in the present study. Material and MethodsBlood samples were collected in citrate vacutainers from 23 patients at the time of diagnosis and after induction therapy with ATRA at one month when there was molecular remission, under the approval of the respective institutional review boards. Control samples were collected from 37 healthy subjects. Preparation of Microparticlessodium citrated blood is centrifuged at 1500g for 20 minutes. The plasma portion is collected and centrifuged a second time at 3000g for 3 minutes to eliminate any extraneous platelets. 250µl of plasma is then centrifuged at 20,000g for 15 minutes at 4C. The resultant pellet contains the microparticles. [Standardization of platelet-derived microparticle enumeration by flow cytometry with calibrated beads: results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop. Lacroix R. Robert S. Poncelet P. Kasthuri RS. Key NS. Dignat-George F. ISTH SSC Workshop. Journal of Thrombosis & Haemostasis. 8(11):2571-4, 2010 Nov. ResultsConclusion Microparticles bearing TF have been shown to play a thrombogenic role in cancer. We had previously shown that there is an increase of microparticles containing procoagulants and fibrinolytic factors in APL and that these microparticles are mostly derived from CD33 positive (myeloid) cells. Present findings show that procoagulant and fibrinolytic factors are functional. The TF activity, thrombin generation are increased in the APL microparticles. It is noteworthy that thrombin generation is increased in the APL microparticles. In contrast, there is a complete absence of thrombin generation in the control microparticles. This suggests that the TF in microparticles, which are components of cell membranes, are not activated in healthy subjects but in APL. Disclosures:No relevant conflicts of interest to declare.

Abstract 4909: Calcitriol and arsenic trioxide synergistically inhibited proliferation of HL-60 cell line mainly through apoptosis pathway

Abstract Arsenic trioxide (As2O3) has shown substantial efficacy in the treatment of patients with acute promyelocytic leukemia (APL). However, since not all patients could achieve complete remission after As2O3 treatment, it is necessary to investigate a novel treatment method overcoming the resistance of current treatment. However, the therapeutic role of calcitriol against APL has not yet been fully elucidated. In our study, we investigated the anti-leukemic effect of calcitriol in combination with As2O3 on the human APL cell line HL-60. Inhibition of proliferation rate of HL-60 cells was measured by the trypan blue exclusion test was resulted in dose, time- dependent expressions. To evaluate the effects of combination treatment of As2O3 and calcitriol on the cell proliferation, we analyzed the combined effects of As2O3 and calcitriol using CalcuSyn software (BioSoft, Ferguson, MO, USA), which is based on the multiple drug-effect equation of Chou and Talalay (Chou TC, et al. Adv Enzyme Regul 1984): a combination index (CI) of <1, 1, or >1 is defined as synergistic, additive, or antagonistic, respectively. In all combinations, the CI values calculated were less than 1. To investigate the effects of these treatments on the apoptosis pathway, we analyzed the expression of Bax, Caspase-3, Bcl-2 with the Western blot and the RT-PCR. In the Western blot analysis, lower Bcl-2 and higher Bax and Caspase-3 expressions were observed in the As2O3 or calcitriol-treated groups compared to the control group. The combination treatment decreased Bcl-2 expression and increased Bax and Caspase-3 expressions more prominently than the single treatment using either As2O3 or calcitriol. In the RT-PCR analysis, these trends were validated again with the same results. These cells were then labeled with fluorescein-conjugated Annexin V-FITC and propidium iodide (PI), and analyzed with flow cytometry to differentiate whether the main cause of cell death was apoptosis or necrosis. To resultsze, calcitriol in combination with As2O3 synergistically inhibited proliferation of the human APL cell line HL-60. In addition, calcitriol combined with As2O3 more prominently activated the apoptosis pathway than single treatment using either calcitriol or As2O3. The main cause of cell death was also apoptosis. Our results suggest that these novel findings could be applied to improve the therapeutic outcomes of patients with APL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4909. doi:1538-7445.AM2012-4909

Activation of Notch Signaling Is An Early Event in the Development of PML-Rara-Induced Acute Promyelocytic Leukemia (APL)

Abstract 2468Acute promyelocytic leukemia (APL, FAB M3) is characterized by the presence of the t(15;17) translocation, resulting in production of the oncogenic transcription factor PML-RARA. We previously reported a gene expression signature of 1121 annotated genes that distinguish APL from the other FAB subtypes and normal promyelocytes (Payton et al, JCI 2009). Within this signature, we identified the Notch ligand JAG1 as being overexpressed in APL samples compared to both other AML subtypes (APL/AML=4.2, FDR<0.5%) and normal promyelocytes (APL/pros=5.66, FDR<0.5%). We then measured JAG1 expression in mouse and cellular models of APL. High levels of JAG1 mRNA and protein were found in both zinc treated PR9 cells (which contain a zinc inducible PML-RARA cassette), and in NB4 cells, a human APL cell line. APL cells derived from the mCG-PML-RARA knockin mouse overexpressed Jag1 mRNA compared to wildtype promyelocytes (APL/pros=10.4, p<0.01). Additionally, Jag1 protein was readily detected in up to 90% of murine APL cells by flow cytometry. Collectively, these results indicate that JAG1 overexpression is a common event in both human and murine APL pathogenesis.Hypothesizing that JAG1 overexpression could lead to Notch signaling in APL cells, we measured Notch signaling in human and murine APL cells. The Notch receptors Notch-1 and Notch-2, the components of the gamma secretase complex, and the transcriptional coactivators of Notch are highly expressed in nearly all human APL samples, suggesting that APL cells are capable of Notch signaling. Using bioinformatic approaches, we found that three previously defined activated Notch signatures were significantly enriched in APL cells (FDR=0.00) and also in PR9 and NB4 cells. Protein levels of cleaved Notch-1 increased in induced PR9 cells with the same kinetics as JAG1 protein, peaking at 16 hours post-induction. In addition, flow cytometry demonstrated the presence of cleaved Notch protein in PR9 and NB4 cells, and both primary and immortalized murine APL samples. To define the consequences of inhibiting Notch signaling in APL cells, we cultured primary murine APL cells in the presence of gamma secretase inhibitors; colony formation in 6/9 tumors was significantly inhibited (p<0.05). Growth of primary murine marrow and spleen cells was unaffected under identical culture conditions.Finally, we investigated the role of Notch signaling in APL pathogenesis in young mCG-PR mice prior to the development of leukemia. Marrow derived progenitors from PML-RARA mice replate serially in methylcellulose cultures, in contrast to wildtype marrow cells, which do not replate. In contrast to primary tumor cells, which were variably dependent on Notch signaling, preleukemic cells were uniformly dependent on Notch signaling for replating activity. In the presence of gamma secretase inhibitors, the replating phenotype is significantly decreased (p<0.01). In contrast, colony formation by wildtype cells is unaffected by inhibition of Notch signaling. Similarly, when Notch signaling in primary marrow cells was genetically inhibited by retroviral transduction of a dominant negative mastermind construct (DNMAML), colony formation by PML-RARA cells was significantly decreased at the first, second and third replatings compared to GFP control cells (p<0.01). Again, wildtype marrow colony formation was unaffected by DNMAML transduction. Importantly, wild type marrow cells transduced with a JAG1 expressing retrovirus did not acquire a replating phenotype, nor did animals transplanted with JAG1 expressing marrow develop leukemia. In conclusion, we have demonstrated overexpression of JAG1 and activation of Notch signaling in human and murine APL cells, and have shown that Notch signaling may play a role in the early pathogenesis of APL. Collectively, these results support a model in which PML-RARA induces the self-renewal of hematopoietic progenitors via a Notch signaling pathway, which may be a critical early step in APL pathogenesis. Disclosures:No relevant conflicts of interest to declare.

Discovery of Critical Functions of Histone Demethylase, PHF8, in Mediating ATRA Response in APL

Abstract 226 Introduction:Transcription deregulation plays a key role in acute leukemogenesis, which is mostly initiated by chimeric transcription factors such as PML–RARa that accounts for almost 100% of acute promyelocytic leukemia (APL). While APL is uniquely sensitive to all trans retinoic acid (ATRA) treatment and has been the paradigm of differentiation/epigenetic therapies, the underlying mechanisms remain largely unknown and are major interests in the field with a potential of extending the success to other hematological malignancies. Previously, others and we have shown that aberrant recruitment of histone deacetylases and polycomb repressive complexes by oligomeric PML-RARa are key for suppression of its downstream targets and promote cellular transformation. ATRA treatment disrupts their bindings and results in de-repression of target genes. However, the identification of the co-activator complex responsible for gene activation upon ATRA treatment remains a major hurdle that significantly hinders the progress of understanding mechanisms underlying the epigenetic therapy. Results and Discussion:Given the critical functions of JmjC-domains containing histone demethylases in mediating transcriptional regulation, we performed a systematic screening for differential interaction between JmjC-histone demethylases and PML–RARa upon ATRA treatment. We identified a highly specific interaction between PML–RARa and Plant Homeodomain Finger 8 (PHF8). To assess its effect on histone methylation, we detected specific reduction of H3K9me2 and enhancement of H3K4me3 in PHF8 transfected cells, consistent with its function as a transcriptional activator. This was further supported by results from chromatin immuno-precipitation (ChIP) assays in human APL cell line (NB4) showing that PHF8 differentially bound and mediated the same histone modifications in RARb promoter and activated its expression. To investigate the functional significance of this interaction, PHF8 was expressed in human NB4 cells harboring PML-RARa or murine primary bone marrow cells transformed by APL fusion proteins. Induction of PHF8 expression significantly decreased their in vitro transformation capacity in the presence of physiological concentrations of ATRA. Conversely, specific down-modulation of PHF8 expression by shRNAs reduced ATRA sensitivity of these cells, suggesting a critical function of PHF8 in mediating ATRA response. We hypothesized that PHF8 may be able to sensitize ATRA resistant cells to the treatment. Hence, we induced expression of PHF8 in the ATRA resistant variant of NB4 line, NB4-MR2. We were able to demonstrate that PHF8 sensitized NB4-MR2 cells to ATRA treatment in vitro. In contrast, NB4-MR2 cells expressing PHF8-F279S, a catalytically inactive mutant could not be sensitized to ATRA treatment, indicating that the enzymatic activity is critical for mediating the ATRA response. Most importantly, NB4-MR2 cells expressing wild type PHF8 were also sensitive to ATRA treatment in vivo and failed to induce leukemia in NOD/SCID mice, which would otherwise succumb to leukemia in a very brief latency.To gain further insights into the molecular regulation of PHF8 in ATRA response, we characterized the potential functions of CDK1-mediated phosphorylation of PHF8. It is known that in ATRA treated leukemic cells Cyclin A translocates into the nucleus where it interacts with CDK1. Activated CDK1 induces phosphorylation of PHF8 at two serine (S33)/ threonine (T84) phosphorylation sites. Our results showed that PHF8 in its phosphorylated form had a much higher binding affinity to PML-RARa. Consistently, ChIP analyses revealed that the binding of PHF8 to the RARb promoter and the resultant activation were significantly augmented when PHF8 was constitutively phosphorylated. Moreover, inhibition of PHF8 dephosphorylation by Okadaic Acid, sensitized NB4-MR2 cells to ATRA treatment. Conclusions:We discovered a novel function of the histone demethylase PHF8 in mediating therapeutic response in both ATRA sensitive and ATRA resistant cells. This function of PHF8 is critically dependent on two phosphorylation sites as well as its histone modification activity. Thus, therapeutic interventions such as phosphatase inhibitors that enhance the PHF8 activity might become useful tools for development of new epigenetic therapies sensitizing leukemic cells to ATRA treatment. Disclosures:No relevant conflicts of interest to declare.

Potential Role of Heme Oxygenase-1 in Arsenic Trioxide and Hemin-Mediated Differentiation of Human Promyelocytic Leukemia Cells

Acute promyelocytic leukemia (APL) is a myeloid-derived leukemia characterized by a block of differentiation at the promyelocytic stage of development. APL is associated with a reciprocal translocation of chromosomes 15 and 17 involving the retinoic acid receptor-alpha (RARα) locus and the promyelocytic leukemia locus (PML) forming a new fusion gene, PML/RARα. The fusion protein prevents the cells from differentiating by repressing transcription of target genes necessary for differentiation. Therapy with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) have improved the clinical outcomes in APL. ATRA differentiates APL cells by promoting degradation of PML/RARα and relieving transcriptional repression. ATO induces apoptosis and differentiation of APL cells through separate pathways involving activation of apoptotic pathways and degradation of the PML/RARα protein. The heme-catabolizing protein heme oxygenase-1 (HO-1) which produces carbon monoxide, biliverdin/bilirubin and releases Fe/ferritin can be cytoprotective and anti-apoptotic. HO-1 message and protein are markedly upregulated in response to hemin. ATO also can induce HO-1 expression and has been shown to induce differentiation of leukemic cells. We hypothesize that induction of HO-1 mediates APL differentiation in response to ATO and hemin. We utilized a human APL cell line, NB4, to test our hypothesis in vitro using NBT reduction to assess cell differentiation. ATRA (p < 0.005 at both 72 and 96 hours), ATO (p < 0.005 at 72 hours, and p < 0.05 at 96 hours), and hemin (p < 0.05 at both 72 and 96 hours, n=3) were shown to differentiate NB4 cells compared to untreated groups. RT-PCR indicated HO-1 expression increased in response to ATRA, ATO, and hemin, while biliverdin reductase (BVR), a signaling protein that degrades biliverdin to bilirubin, was only induced by hemin. Inhibition of HO-1 activity with tin-protoporphyrin significantly (p<0.05, n=2) blocked differentiation induced by ATO but had no effect on ATRA–treated cells. We speculate that HO-1 and its downstream metabolic products play critical roles in ATO-induced differentiation of APL cells.

Epigenomic changes during leukemia cell differentiation: analysis of histone acetylation and cytosine methylation using CpG island microarrays.

Dysregulation of epigenetic control is an important participant in carcinogenesis. The PML/RAR alpha translocation in acute promyelocytic leukemia (APL) is an example where the resultant fusion protein recruits histone deacetylase complexes to target genes resulting in their inappropriate transcriptional repression. All-trans-retinoic acid (ATRA) acts as a ligand that relieves this repression and produces an epigenetic transcriptional reprogramming of the cancer cell. CpG island microarrays were used to analyze the DNA methylation and histone acetylation state of the human APL cell line NB4 before and after differentiation with ATRA as well as normal peripheral blood mononuclear cells (PBMC). Over 70 CpG islands within 1 kb of transcription start of a known gene are aberrantly methylated in NB4 cells compared with PBMC; however, no changes in cytosine methylation were detected following ATRA-induced differentiation. With respect to histone H4 acetylation, over 100 single-copy CpG islands within 1 kb of transcription start of a known human gene became hyperacetylated following ATRA-induced differentiation. One CpG island was aberrantly methylated in NB4 cells, but became hyperacetylated and was induced following ATRA treatment and was associated with the HoxA1 gene, suggesting it may be a target gene of ATRA in APL. In addition to single-copy sequences, a selective increase in acetylation was detected in satellite DNA when compared with other high-copy sequences, such as Alu or rDNA. In summary, ATRA stimulates complex epigenomic changes during leukemic cell differentiation, and monitoring these changes may help to identify new targets of epigenetic dysfunction.

In vivo Effects of a Histone Deacetylase Inhibitor, FK228, on Human Acute Promyelocytic Leukemia in NOD/Shi‐scid/scid Mice

Histone acetylation and deacetylation are closely linked to transcriptional activation and repression, respectively. In acute promyelocytic leukemia (APL), histone deacetylase inhibitors (HDACIs) have a synergistic effect with all‐iraws retinoic acid (ATRA) in vitro to induce differentiation. Here we report in vitro and in vivo effects of a HDACI, FK228 (formerly FR901228 or depsipeptide), on the human APL cell line NB4. FK228 had a strong and irreversible cytotoxicity compared with another HDACI, trichostatin A. In vivo administration of ATRA or FK228 alone partly inhibited the growth of established tumors of NB4 subcutaneously transplanted in NOD/Shi‐scid/scid mice, and the combination was synergistically effective. Histopathological examination revealed that the combination induced apoptosis and differentiation as well as histone acetylation. Intravenous injection of NB4 in NOD/Shi‐scid/scid mice followed by combination treatment significantly prevented leukemia death, whereas single administration did not. These findings suggest that FK228 is a promising agent to enhance ATRA‐sensitivity in the treatment of APL.

Ligand-inducible interaction of the DRIP/TRAP coactivator complex with retinoid receptors in retinoic acid–sensitive and –resistant acute promyelocytic leukemia cells

Retinoic acid (RA) signaling is mediated by its nuclear receptors RXR and RAR, which bind to their cognate response elements as a heterodimer, RXR/RAR, and act in concert with coregulatory factors to regulate gene transcription on ligand binding. To identify specific cofactors that interact with the RXR/RAR heterodimer in acute promyelocytic leukemia (APL) cells, a double cistronic construct was used that allowed coexpression of the RXR LBD (ligand binding domain) with the RAR LBD as an affinity matrix to pull down interacting proteins from nuclear extracts prepared from a human APL cell line, NB4. A group of proteins was detected whose interaction with RXR/RAR is ligand inducible. The molecular weight pattern of these proteins is similar to that of a complex of proteins previously identified as DRIP or TRAP, which are ligand-dependent transcription activators of VDR and TR, respectively. The RXR/RAR-interacting proteins from NB4 were confirmed to be identical to the DRIP subunits by comparative electrophoresis, Western blot analysis, and in vitro protein interaction assay. In addition to RXR/RAR, the DRIP component can interact directly with the APL-specific PML-RARα fusion protein. The same DRIP complex is present in RA-resistant APL cells and in a variety of cancer cell lines, supporting its global role in transcriptional regulation.

Ligand-inducible interaction of the DRIP/TRAP coactivator complex with retinoid receptors in retinoic acid–sensitive and –resistant acute promyelocytic leukemia cells

AbstractRetinoic acid (RA) signaling is mediated by its nuclear receptors RXR and RAR, which bind to their cognate response elements as a heterodimer, RXR/RAR, and act in concert with coregulatory factors to regulate gene transcription on ligand binding. To identify specific cofactors that interact with the RXR/RAR heterodimer in acute promyelocytic leukemia (APL) cells, a double cistronic construct was used that allowed coexpression of the RXR LBD (ligand binding domain) with the RAR LBD as an affinity matrix to pull down interacting proteins from nuclear extracts prepared from a human APL cell line, NB4. A group of proteins was detected whose interaction with RXR/RAR is ligand inducible. The molecular weight pattern of these proteins is similar to that of a complex of proteins previously identified as DRIP or TRAP, which are ligand-dependent transcription activators of VDR and TR, respectively. The RXR/RAR-interacting proteins from NB4 were confirmed to be identical to the DRIP subunits by comparative electrophoresis, Western blot analysis, and in vitro protein interaction assay. In addition to RXR/RAR, the DRIP component can interact directly with the APL-specific PML-RARα fusion protein. The same DRIP complex is present in RA-resistant APL cells and in a variety of cancer cell lines, supporting its global role in transcriptional regulation.

A Retinoid-Resistant Acute Promyelocytic Leukemia Subclone Expresses a Dominant Negative PML-RARα Mutation

AbstractThe unique t(15; 17) of acute promyelocytic leukemia (APL) fuses the PML gene with the retinoic acid receptor α (RARα) gene. Although retinoic acid (RA) inhibits cell growth and induces differentiation in human APL cells, resistance to RA develops both in vitro and in patients. We have developed RA-resistant subclones of the human APL cell line, NB4, whose nuclear extracts display altered RA binding. In the RA-resistant subclone, R4, we find an absence of ligand binding of PML-RARα associated with a point mutation changing a leucine to proline in the ligand-binding domain of the fusion PML-RARα protein. In contrast to mutations in RARα found in retinoid-resistant HL60 cells, in this NB4 subclone, the coexpressed RARα remains wild-type. In vitro expression of a cloned PML-RARα with the observed mutation in R4 confirms that this amino acid change causes the loss of ligand binding, but the mutant PML-RARα protein retains the ability to heterodimerize with RXRα and thus to bind to retinoid response elements (RAREs). This leads to a dominant negative block of transcription from RAREs that is dose-dependent and not relieved by RA. An unrearranged RARα engineered with this mutation also lost ligand binding and inhibited transcription in a dominant negative manner. We then found that the mutant PML-RARα selectively alters regulation of gene expression in the R4 cell line. R4 cells have lost retinoid-regulation of RXRα and RARβ and the RA-induced loss of PML-RARα protein seen in NB4 cells, but retain retinoid-induction of CD18 and CD38. Thus, the R4 cell line provides data supporting the presence of an RARα-mediated pathway that is independent from gene expression induced or repressed by PML-RARα. The high level of retinoid resistance in vitro and in vivo of cells from some relapsed APL patients suggests similar molecular changes may occur clinically.

The PML/RARα Oncoprotein Is a Direct Molecular Target of Retinoic Acid in Acute Promyelocytic Leukemia Cells

Acute promyelocytic leukemia (APL) is characterized by the translocation, t(15;17) and the expression of a PML/RAR alpha fusion protein that is diagnostic of the disease. There is evidence that PML/RAR alpha protein acts as a dominant negative inhibitor of normal retinoid receptor function and myeloid differentiation. We now show that the PML/RAR alpha fusion product is directly downregulated in response to retinoic acid (tRA) treatment in the human APL cell line, NB4. tRA treatment induces loss of PML/RAR alpha at the protein level but not at the level of mRNA, as determined by Northern blots, by Western blots, and by ligand binding assays and in binding to RA-responsive DNA elements. We present evidence that this regulation is posttranslational. This evidence suggests that tRA induces synthesis of a protein that selectively degrades PML/RAR alpha. We further show that this loss of PML/ RAR-alpha is not limited to the unique APL cell line. NB4, because PML/RAR alpha protein is selectively downregulated by tRA when expressed in the transfected myeloid cell line U937. The loss of PML/RAR alpha may be directly linked to tRA-induced differentiation, because in a retinoid-resistant subclone of NB4, tRA does not decrease PML/RAR alpha protein expression. In NB4 cells, the specific downregulation of the fusion protein decreases the ratio of PML/RAR alpha to wild-type RAR alpha. Because the ratio of expression of PML/RAR alpha to wild-type RAR alpha and PML may be important in maintaining the dominant negative block of myelocytic differentiation, these data suggest a molecular mechanism for restoration by tRA normal myeloid differentiation in APL cells.

Establishment of a Human Acute Promyelocytic Leukemia-Ascites Model in SCID Mice

Acute promyelocytic leukemia (APL) is an interesting model for cancer research because of the presence of the specific PML-RARalpha fusion gene associated with the clinical response to retinoic acid differentiation therapy. To better understand and improve differentiation induction with retinoic acid, we have established a human APL-ascites model in SCID mice using the NB4 human APL cell line. NB4 (1 x 10(6) cells) were transplanted into the peritoneum (IP) of SCID mice for 1 month. NB4 ascites cells (A-NB4) appeared, which were then engrafted in SCID mice periodically for 18 passages at an interval of 3 to 4 weeks with a 100% success rate of tumor induction. The mean survival times of SCID mice transplanted with 1 x 10(6) A-NB4 cells was 21.6 +/- 2.3 days. Analysis of the biologic characteristics of ninth passage NB4 ascitic cells was performed and they were found to have the morphologic, immunologic, cytogenetic, and molecular features of cultured NB4 cells. Furthermore, A-NB4 cells were capable of differentiating when treated with all-trans retinoic acid (ATRA), as manifested by enhanced NBT reduction and CD11b expression. In vivo treatment with ATRA in SCID mice for 4 days also increased NBT reduction by A-NB4 cells. ATRA treatment significantly prolonged survival time in the group after transplantation (28.1 +/- 6.8 to 29.1 +/- 8.4 days) compared with the control (P < .001). Furthermore, treatment with adriamycin, an effective chemotherapeutic drug in APL, had a strong growth suppressive effect on A-NB4 cells. These results demonstrate that this SCID-APL (NB4 ascites cells) model is a useful preclinical system for evaluating new or known drugs in the treatment of APL.