Promyelocytic Leukemia-retinoic Acid Receptor Research Articles

Biological Responses to Arsenic Compounds

Arsenic is a metalloid that generates various biological effects on cells and tissues. Depending on the specific tissue exposed and the time and degree of exposure, diverse responses can be observed. In humans, prolonged and/or high dose exposure to arsenic can have a variety of outcomes, including the development of malignancies, severe gastrointestinal toxicities, diabetes, cardiac arrhythmias, and death. On the other hand, one arsenic derivative, arsenic trioxide (As(2)O(3)), has important antitumor properties. This agent is a potent inducer of antileukemic responses, and it is now approved by the Food and Drug Administration for the treatment of acute promyelocytic leukemia in humans. The promise and therapeutic potential of arsenic and its various derivatives have been exploited for hundreds of years. Remarkably, research focused on the potential use of arsenic compounds in the treatment of human diseases remains highly promising, and it is an area of active investigation. An emerging approach of interest and therapeutic potential involves efforts to target and block cellular pathways activated in a negative feedback manner during treatment of cells with As(2)O(3). Such an approach may ultimately provide the means to selectively enhance the suppressive effects of this agent on malignant cells and render normally resistant tumors sensitive to its antineoplastic properties.

Arsenic Trioxide Augments Chk2/p53-mediated Apoptosis by Inhibiting Oncogenic Wip1 Phosphatase

The oncogenic Wip1 phosphatase (PPM1D) is induced upon DNA damage in a p53-dependent manner and is required for inactivation or suppression of DNA damage-induced cell cycle checkpoint arrest and of apoptosis by dephosphorylating and inactivating phosphorylated Chk2, Chk1, and ATM kinases. It has been reported that arsenic trioxide (ATO), a potent cancer chemotherapeutic agent, in particular for acute promyelocytic leukemia, activates the Chk2/p53 pathway, leading to apoptosis. ATO is also known to activate the p38 MAPK/p53 pathway. Here we show that phosphatase activities of purified Wip1 toward phosphorylated Chk2 and p38 in vitro are inhibited by ATO in a dose-dependent manner. Furthermore, DNA damage-induced phosphorylation of Chk2 and p38 in cultured cells is suppressed by ectopic expression of Wip1, and this Wip1-mediated suppression can be restored by the presence of ATO. We also show that treatment of acute promyelocytic leukemia cells with ATO resulted in induction of phosphorylation and activation of Chk2 and p38 MAPK, which are required for ATO-induced apoptosis. Importantly, this ATO-induced activation of Chk2/p53 and p38 MAPK/p53 apoptotic pathways can be enhanced by siRNA-mediated suppression of Wip1 expression, further indicating that ATO inhibits Wip1 phosphatase in vivo. These results exemplify that Wip1 is a direct molecular target of ATO.

Coiled-coil domain of PML is essential for the aberrant dynamics of PML-RARα, 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.

Two cases of isolated symptomatic myocarditis induced by all-trans retinoic acid (ATRA)

Two cases of isolated symptomatic myocarditis induced by all-trans retinoic acid (ATRA)

Lentiviral PU.1 overexpression restores differentiation in myeloid leukemic blasts

PU.1, a transcription factor of the ETS family, plays a pivotal role in normal hematopoiesis, and particularly in myeloid differentiation. Altered PU.1 function is possibly implicated in leukemogenesis, as PU.1 gene mutations were identified in some patients with acute myeloid leukemia (AML) and as several oncogenic products (AML1-ETO, promyelocytic leukemia-retinoic acid receptor alpha, FMS-like receptor tyrosine kinase 3 internal tandem duplication) are associated with PU.1 downregulation. To demonstrate directly a role of PU.1 in the blocked differentiation of leukemic blasts, we transduced cells from myeloid cell lines and primary blasts from AML patients with a lentivector encoding PU.1. In NB4 cells we obtained increases in PU.1 mRNA and protein, comparable to increases obtained with all-trans retinoic acid-stimulation. Transduced cells showed increased myelomonocytic surface antigen expression, decreased proliferation rates and increased apoptosis. Similar results were obtained in primary AML blasts from 12 patients. These phenotypic changes are characteristic of restored blast differentiation. PU.1 should therefore constitute an interesting target for therapeutic intervention in AML.

クローン病を合併した残存小腸１２０ｃｍの急性前骨髄球性白血病患者における経口トレチノイン（ａｌｌ‐ｔｒａｎｓ ｒｅｔｉｎｏｉｃ ａｃｉｄ：ＡＴＲＡ）の体内動態

Tretinoin (All-trans retinoic acid : ATRA) is the drug of choice for the remission induction treatment of acute promyelocytic leukemia (APL). Its action against APL cells involves the degradation of promyelocytic leukemia-retinoic acid receptor α (PML-RARα) protein which represses their cellular differentiation.We treated a 44-year-old Japanese male patient with APL using orally administered ATRA. He had previously undergone multiple resections of the bowels due to Crohn’s disease and his residual intestine length was only 120 cm. In order to investigate the pharmacokinetics of ATRA in this patient, we measured the plasma concentrations. On the 11th day of orally administering ATRA twice a day, the maximum concentration (Cmax) and the area under the plasma concentration versus time curve (AUC0-12) of ATRA were 51.9μg/L and 460μg·h/L, respectively. These data are comparable with those for patients without gastrointestinal complications, indicating that ATRA was sufficiently absorbed in spite of the patient having such a short intestine. Complete remission was achieved by the course of ATRA treatment.

CCAAT/enhancer binding proteins alpha and epsilon cooperate with all-trans retinoic acid in therapy but differ in their antileukemic activities

AbstractCCAAT/enhancer binding proteins (C/EBPs) play critical roles in myelopoiesis. Dysregulation of these proteins likely contributes to the pathogenesis of myeloid disorders characterized by a block in granulopoiesis. In one such disease, acute promyelocytic leukemia (APL), a promyelocytic leukemia–retinoic acid receptor α (PML-RARα) fusion protein is expressed as a result of a t(15;17) chromosomal translocation. Treatment of PML-RARα leukemic cells with all-trans retinoic acid (ATRA) causes them to differentiate into mature neutrophils, an effect thought to be mediated by C/EBPs. In this study, we assess the potential for cooperativity between increased C/EBP activity and ATRA therapy. We demonstrate that although both C/EBPα and C/EBPϵ can significantly prolong survival in a mouse model of APL, they are not functionally equivalent in this capacity. We also show that forced expression of C/EBPα or C/EBPϵ in combination with ATRA treatment has a synergistic effect on survival of leukemic mice compared with either therapy alone.

Dimerization-induced corepressor binding and relaxed DNA-binding specificity are critical for PML/RARA-induced immortalization.

The pathogenesis of acute promyelocytic leukemia involves the transcriptional repression of master genes of myeloid differentiation by the promyelocytic leukemia-retinoic acid receptor alpha (PML/RARA) oncogene. PML-enforced RARA homodimerization allows the tighter binding of corepressors, silencing RARA target genes. In addition, homodimerization dramatically extends the spectrum of DNA-binding sites of the fusion protein compared with those of normal RARA. Yet, any contribution of these two properties of PML/RARA to differentiation arrest and immortalization of primary mouse hematopoietic progenitors was unknown. We demonstrate that dimerization-induced silencing mediator of retinoid and thyroid receptors (SMRT)-enhanced binding and relaxed DNA-binding site specificity are both required for efficient immortalization. Thus, enforced RARA dimerization is critical not only for triggering transcriptional repression but also for extending the repertoire of target genes. Our studies exemplify how dimerization-induced gain of functions converts an unessential transcription factor into a dominant oncogenic protein.

ATRA resolves the differentiation block in t(15;17) acute myeloid leukemia by restoring PU.1 expression

AbstractTightly regulated expression of the transcription factor PU.1 is crucial for normal hematopoiesis. PU.1 knockdown mice develop acute myeloid leukemia (AML), and PU.1 mutations have been observed in some populations of patients with AML. Here we found that conditional expression of promyelocytic leukemia-retinoic acid receptor α (PML-RARA), the protein encoded by the t(15;17) translocation found in acute promyelocytic leukemia (APL), suppressed PU.1 expression, while treatment of APL cell lines and primary cells with all-trans retinoic acid (ATRA) restored PU.1 expression and induced neutrophil differentiation. ATRA-induced activation was mediated by a region in the PU.1 promoter to which CEBPB and OCT-1 binding were induced. Finally, conditional expression of PU.1 in human APL cells was sufficient to trigger neutrophil differentiation, whereas reduction of PU.1 by small interfering RNA (siRNA) blocked ATRA-induced neutrophil differentiation. This is the first report to show that PU.1 is suppressed in acute promyelocytic leukemia, and that ATRA restores PU.1 expression in cells harboring t(15;17).

In vivo analysis of the role of aberrant histone deacetylase recruitment and RARα blockade in the pathogenesis of acute promyelocytic leukemia

The promyelocytic leukemia–retinoic acid receptor α (PML-RARα) protein of acute promyelocytic leukemia (APL) is oncogenic in vivo. It has been hypothesized that the ability of PML-RARα to inhibit RARα function through PML-dependent aberrant recruitment of histone deacetylases (HDACs) and chromatin remodeling is the key initiating event for leukemogenesis. To elucidate the role of HDAC in this process, we have generated HDAC1–RARα fusion proteins and tested their activity and oncogenicity in vitro and in vivo in transgenic mice (TM). In parallel, we studied the in vivo leukemogenic potential of dominant negative (DN) and truncated RARα mutants, as well as that of PML-RARα mutants that are insensitive to retinoic acid. Surprisingly, although HDAC1-RARα did act as a bona fide DN RARα mutant in cellular in vitro and in cell culture, this fusion protein, as well as other DN RARα mutants, did not cause a block in myeloid differentiation in vivo in TM and were not leukemogenic. Comparative analysis of these TM and of TM/PML −/− and p53 −/− compound mutants lends support to a model by which the RARα and PML blockade is necessary, but not sufficient, for leukemogenesis and the PML domain of the fusion protein provides unique functions that are required for leukemia initiation.

Optimizing treatment for elderly patients with acute promyelocytic leukemia: is it time to replace chemotherapy with all-trans retinoic acid and arsenic trioxide?

This review focuses on the treatment of acute promyelocytic leukemia (APL) in elderly patients and offers recommendations for improving outcomes. Nineteen percent of patients with APL are ≥60 years. Rates of response and survival are lower in elderly compared with younger patients, owing to a higher incidence of early deaths or deaths in remission. However, relapse-free survival rates are similar in both groups. Ongoing trials assess the role of reduced-intensity regimens. All-trans retinoic acid (ATRA) and concurrent arsenic trioxide is associated with high rates of response and molecular remission and low rates of induction deaths. We propose this combination as the treatment of choice in patients with APL, including the elderly. Patients with elevated leukocyte counts may also benefit from gemtuzumab ozogamicin therapy, with or without leukapheresis. Monitoring major organ function and toxicity is essential. Patients should be assessed for minimal residual disease using polymerase chain reaction testing for promyelocytic leukemia-retinoic acid receptor alpha. If molecular relapse is evident, treatment with ATRA and idarubicin, with or without gemtuzumab ozogamicin, is recommended.

Single-agent liposomal all-trans retinoic acid can cure some patients with untreated acute promyelocytic leukemia: an update of The University of Texas M. D. Anderson Cancer Center Series

The present study aimed to investigate single-agent liposomal all-trans retinoic acid (Lipo-ATRA) in untreated acute promyelocytic leukemia (APL). Induction therapy consisted of Lipo-ATRA 90 mg/m2 i.v. every other day. Patients in complete remission (CR) continued to receive Lipo-ATRA 90 mg/m2 i.v. three times a week for 9 months. Idarubicin was added only if a polymerase chain reaction test for promyelocytic leukemia-retinoic acid receptor alpha (sensitivity level, 10−4), performed every 3 months from CR, was positive. The results were compared with those of a historical control group treated with oral ATRA and idarubicin. Lipo-ATRA induced CR in 79% of patients; CR rates were 92% and 38% in patients with white blood cell (WBC) counts <10 × 109/L and >10 × 109/L, respectively. Ten of the 26 responders to Lipo-ATRA remain in first CR at a median of 6.4 years, despite never receiving idarubicin; all 10 had initial WBC counts <10 × 109/L. The 5-year survival rate was 76% for patients treated with Lipo-ATRA. Comparisons with oral ATRA+idarubicin as given at M. D. Anderson are confounded because of their historical nature and the absence of ATRA from post-remission therapy in the former group. Nonetheless, a multivariate Cox model identified higher WBC counts and older age, but not treatment (historical vs. Lipo-ATRA), as being predictive of shorter relapse-free and overall survival. Lipo-ATRA can cure patients presenting with WBC counts <10 × 109/L (low risk) without additional therapy, contrary to conventional ATRA, which, when given alone, probably cures no patients. The observation that patients can be cured of APL without the use of chemotherapy should encourage further study of ‘targeted’ therapy in APL and in other leukemias.

C/EBP Is Deregulated by PML-RAR in Acute Promyelocytic Leukemia.

Acute promyelocytic leukemia (APL) cells are blocked at the promyelocyte stage of myeloid differentiation. The majority of APL cells display the t(15;17) reciprocal chromosomal translocation leading to the expression of the fusion protein promyelocytic leukemia-retinoic acid receptor alpha (PML-RARa). Cells harboring this reciprocal translocation can be induced to differentiate after treatment with all-trans retinoic acid (at-RA) both in vivo and in vitro. During normal hematopoiesis, differentiation is regulated by several key transcription factors. One of them, CCAAT/enhancer binding protein alpha (C/EBPa), controls expression of genes regulating normal myeloid differentiation. Its disruption leads to a block of granulocytic differentiation. We thus hypothesize that C/EBPa could be deregulated in APL and therefore participate in the pathogenesis of APL. Using the U937PR9 cell line, which expresses an inducible PML-RARa, we observed that expression of PML-RARa induced a decrease of both C/EBPa mRNA and protein, leading to decreased C/EBPa DNA binding activity. Using a transient transfection assay with a C/EBPa promoter construct in presence or absence of PML-RARa, we are able to demonstrate that PML-RARa can repress C/EBPa promoter activity. This repression is specific to the fusion protein, as both PML and RARa have no effect upon the C/EBPa promoter. A computer search of the C/EBPa promoter sequence did not exhibit any evident RARE binding site, and therefore we are currently mapping the site(s) responsible for this repression. In conclusion, PML-RARa down regulates C/EBPa expression; this down regulation could participate in the pathogenesis of APL. This hypothesis is also supported by the observation that at-RA treatment of APL cell lines (NB4 and HT93) induces a rapid restoration of both C/EBPa RNA and protein. Thus, a decrease in both C/EBPa expression and activity could contribute to the differentiation block of APL cells by deregulating the normal myeloid differentiation program.

Benzodithiophenes Induce Differentiation and Apoptosis in Human Leukemia Cells

All-trans retinoic acid (ATRA) induces clinical remission in patients with t(15;17) acute promyelocytic leukemia (APL) carrying leukemogenic promyelocytic leukemia-retinoic acid receptor alpha (PML-RARalpha) fusion protein by overcoming PML-RARalpha transcriptional repression and inducing myeloid differentiation. To identify more potent chemical differentiation inducers, a screening assay was developed utilizing an ATRA-insensitive NB4 cell line (NB4-c) in which differentiation could be measured after 48 hours when primed with ATRA followed by other potential inducers. Over 300 cytostatic agents selected from the National Cancer Institute library were screened using this established method. Three compounds, NSC656243, NSC625748, and NSC144168, were identified to amplify ATRA-induced differentiation with acceptable cytotoxicity in NB4-c cells. In the absence of ATRA, these compounds also induced HL-60 and murine erythroleukemia cells to undergo partial differentiation. NSC656243, a benzodithiophene compound, was selected for further studies to examine the underlying mechanism of action. The differentiation effect of NSC656243 was associated with enhanced ATRA-mediated up-regulation of cell cycle regulatory proteins p21waf1 and p27kip1, retinoblastoma dephosphorylation, expression of RIG-E and RIG-G, and myelomonocytic differentiation-specific down-regulation of the myeloperoxidase (MPO) gene. Moreover, at 2- to 3-fold higher concentrations than those used to synergize with ATRA, NSC656243 induced apoptosis in NB4-c cells by reactive oxygen species-mediated pathways. The dual effects of benzodithiophenes (i.e., differentiation and apoptosis induction) support further development of these compounds as therapeutic agents for leukemia.

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.

Post-PCR Multiplex Fluorescent Ligation Detection Assay and Flow Cytometry for Rapid Detection of Gene-Specific Translocations in Leukemia

We describe a novel method to detect specific polymerase chain reaction (PCR) target amplicons, involving thermostable ligation of fluorescent and biotinylated oligonucleotides, microparticle bead capture of the ligated products, and flow cytometric analysis. This approach, termed fluorescent ligation detection reaction (f-LDR) is more rapid and cost-effective than oligoprobe Southern blot hybridization (SBH). A standard f-LDR protocol was developed to detect the leukemia-associated chimeric transcripts bcr-abl and promyelocytic leukemia-retinoic acid receptor a (PML-RARalpha) in 2 multiplex and multicolor assays. The f-LDR platform was 100% specific and demonstrated comparable or better sensitivity than standard oligoprobe SBH. The usefulness of f-LDR was evaluated in 94 posttherapy samples from 13 patients with acute promyelocytic leukemia with the PML-RARalpha gene fusion. The f-LDR method was highly concordant (93%) with oligoprobe SBH; essentially all discrepancies were noted to be due to the enhanced sensitivity of f-LDR. We conclude that f-LDR is a highly specific and sensitive post-PCR method with wide potential application.

Post-PCR Multiplex Fluorescent Ligation Detection Assay and Flow Cytometry for Rapid Detection of Gene-Specific Translocations in Leukemia

We describe a novel method to detect specific polymerase chain reaction (PCR) target amplicons, involving thermostable ligation of fluorescent and biotinylated oligonucleotides, microparticle bead capture of the ligated products, and flow cytometric analysis. This approach, termed fluorescent ligation detection reaction (f-LDR) is more rapid and cost-effective than oligoprobe Southern blot hybridization (SBH). A standard f-LDR protocol was developed to detect the leukemia-associated chimeric transcripts bcr-abl and promyelocytic leukemia-retinoic acid receptor a (PML-RARalpha) in 2 multiplex and multicolor assays. The f-LDR platform was 100% specific and demonstrated comparable or better sensitivity than standard oligoprobe SBH. The usefulness of f-LDR was evaluated in 94 posttherapy samples from 13 patients with acute promyelocytic leukemia with the PML-RARalpha gene fusion. The f-LDR method was highly concordant (93%) with oligoprobe SBH; essentially all discrepancies were noted to be due to the enhanced sensitivity of f-LDR. We conclude that f-LDR is a highly specific and sensitive post-PCR method with wide potential application.

PML-RARα is associated with leptin-receptor induction: the role of mesenchymal stem cell–derived adipocytes in APL cell survival

AbstractLeptin is secreted by bone marrow (BM) adipocytes and stromal cells and was shown to stimulate myeloid proliferation. We here report that primary acute promyelocytic leukemia (APL) cells express high levels of the leptin-receptor (OB-R) long isoform. In cells with regulated promyelocytic leukemia–retinoic acid receptor (PML-RARα) expression, inducing PML-RARα was found to increase OB-R levels. We then investigated the effects of leptin produced by BM adipocytes on APL cells using a coculture system with mesenchymal stem cell (MSC)–derived adipocytes. In PML-RARα–expressing cells, all-trans retinoic acid (ATRA)– and doxorubicin-induced apoptosis were significantly reduced by coculture with adipocyte-differentiated MSCs. This antiapoptotic effect required direct cell-to-cell interactions, was associated with phosphorylation of signal transducer and activator of transcription-3 (STAT3) and mitogen-activated protein kinase (MAPK), and was reduced by blocking OB-R. This report provides a mechanistic basis for the BM adipocyte–leukemia cell interaction and suggests that OB-R receptor blockade may have therapeutic use in APL.

Secondary near-tetraploidy with double der(15)t(15;17) in acute promyelocytic leukemia in relapse

Tetraploidy or near-tetraploidy is a rare cytogenetic abnormality in acute myelocytic leukemia. We report here a case of acute promyelocytic leukemia that showed near-tetraploidy with double der(15)t(15;17) the leukemia relapsed. At diagnosis, cytogenetic analysis failed to reveal any karyotypic abnormality; however, a promyelocytic leukemia–retinoic acid receptor α ( PML/ RARA) fusion transcript of the bcr3-type was detected with reverse transcriptase-polymerase chain reaction analysis, and a single PML/RARA fusion signal was observed with fluorescence in situ hybridization analysis. At the first relapse, the majority of leukemic cells showed pseudodiploid karyotypes with der(15)t(15;17), as well as additional chromosomal abnormalities, and exhibited a single PML/RARA fusion signal. A small fraction of leukemic cells, however, showed near-tetraploid karyotypes with double der(15)t(15;17), as well as some additional chromosomal abnormalities in common with the pseudodiploid clones, and exhibited double PML/RARA fusion signals. At the second and third relapses, leukemic cells with near-tetraploidy and double PML/RARA fusion signals became predominant. The PML/RARA fusion transcript of the bcr3 type was also observed at each relapse. In addition, Southern blot analysis of the RARA gene at diagnosis and at the second relapse showed a common rearranged band. Notably, giant, bizarre, and hypogranular promyelocytes expressing CD2, CD34, and HLA-DR appeared at the first relapse and became predominant at the second and third relapses. These observations indicate that the APL cells with near-tetraploidy and double der(15)t(15;17) clonally evolved from the pseudodiploid leukemic cells and exhibited the bizarre morphology and aberrant surface immunophenotypes.

C/EBPbeta: a major PML-RARA-responsive gene in retinoic acid-induced differentiation of APL cells.

In acute promyelocytic leukemia (APL), the translocation t(15;17) induces a block at the promyelocytic stage of differentiation in an all-trans-retinoic acid (ATRA)-responsive manner. Here we report that upon treatment with ATRA, t(15;17) cells (NB4) reveal a very rapid increase in protein level and binding activity of C/EBPbeta, a C/EBP family member, which was not observed in an ATRA-resistant NB4 cell line. We further provide evidence that ATRA mediates a direct increase of C/EBPbeta, only in PML-RARA (promyelocytic leukemia-retinoic acid receptor alpha)-expressing cells. In addition, transactivation experiments indicate that the PML-RARA fusion protein, but not PML-RARA mutants defective in transactivation, strongly transactivates the C/EBPbeta promoter. These results suggest that PML-RARA mediates ATRA-induced C/EBPbeta expression. Finally, we demonstrate the importance of C/EBPbeta in granulocytic differentiation. We show that not only does C/EBPbeta induce granulocytic differentiation of non-APL myeloid cell lines independent of addition of ATRA or other cytokines, but also that C/EBPbeta induction is required during ATRA-induced differentiation of APL cells. Taken together, C/EBPbeta is an ATRA-dependent PML-RARA target gene involved in ATRA-induced differentiation of APL cells.