Pathogenesis Of Acute Promyelocytic Leukemia Research Articles

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.

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.

Forced retinoic acid receptor α homodimers prime mice for APL-like leukemia

RARA becomes an acute promyelocytic leukemia (APL) oncogene by fusion with any of five translocation partners. Unlike RARalpha, the fusion proteins homodimerize, which may be central to oncogenic activation. This model was tested by replacing PML with dimerization domains from p50NFkappaB (p50-RARalpha) or the rapamycin-sensitive dimerizing peptide of FKBP12 (F3-RARalpha). The X-RARalpha fusions recapitulated in vitro activities of PML-RARalpha. For F3-RARalpha, these properties were rapamycin sensitive. Although in vivo the artificial fusions alone are poor initiators of leukemia, p50-RARalpha readily cooperates with an activated mutant CDw131 to induce APL-like disease. These results demonstrate that the dimerization interface of RARalpha fusion partners is a critical element in APL pathogenesis while pointing to other features of PML for enhancing penetrance and progression.

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.

Deregulation of Retinoic Inducible Gene---Rig-I Underlies the Pathogenesis of Acute Promyelocytic Leukemia.

numerous chromosomal translocations, all of which involve the gene encoding retinoid acid receptor a (RARa), resulting in the generation of corresponding oncogenic fusion proteins including PML-RARa and PLZF- RARa (hereafter referred as to X- RARa). However, through deregulating which downstream genetic program(s) X- RARas perturb the hematopoiesis has remained unclear. Here we report that Rig-I, a putative RNA helicase, was identified in RA-induced granulocytic differentiation of APL cell line NB4 cells. Interestingly, the up-regulation of Rig-I was also realized in RA-treated freshly isolated primary APL blasts harboring PML-RARa and HL-60 cell line, but not in RA-treated NB4 variant NB4-R2 (RA refractory) and APL blasts carrying PLZF-RARa, suggesting that the induced Rig-I activity is one essential step underlying the granulocytic differentiation of myeloid leukemia cells. In line with this, the knockout mice of Rig-I produced a phenotype of profound granulocytosis with moderate differentiation blockage; and conversely the overexpression of Rig-I inhibited in vivo hematopoietic reconstitution of transduced normal primitive bone marrow cells. Most importantly, the Rig-I expression was found significantly repressed in myeloid compartment of X-RARa transgenic mice, and that the retroviral transduction of Rig-I cDNA into murine APL blasts carrying hMRP8-PML/RARa greatly decreased their leukemogenesis reconstitution ability in sublethally-irradiated syngeneic recipients. Moreover, we found that Rig-I integrity is required for the full expression of TRAIL, whose expression is critical factor in determining the pool size of myeloid cells. Such, we characterize Rig-I as a crucial negative regulator of granulopoiesis and a potential candidate for developing molecule-based target therapy of myeloid leukemia.

The t(15;17)-Associated Fusion Protein PML/RAR Upregulates c-Kit Which Contributes to the Induction of the Leukemic Phenotype.

The pathogenesis of acute myeloid leukemia (AML) is strictly related to a block of terminal differentiation combined with an increased proliferation of hematopoietic progenitors (HP) in the bone marrow (BM). Furthermore an aberrant self-renewal of leukemic stem cells seems to be obligatory for the establishment of the leukemic clone in the BM. The block of differentiation is due to a deregulated function of differentiation-relevant transcription factors, whereas the proliferation is induced by aberrantly activated signaling pathways related to growth factor-dependent receptor kinases such as Flt3 or c-Kit (CD117). The aberrant self renewal can be attributed to specific pathways such as the Wnt-signaling, which play an important role also in the maintenance of the normal hematopoietic stem cell (HSC). The acute promyelocytic leukemia (APL) is characterized by the t(15;17) which leads to the expression of the PML/RAR fusion protein in the leukemic cells. PML/RAR mediates the APL phenotype given by a differentiation block at the promyelocytic level and an increased self renewal of the APL stem cells by the activation of the Wnt-signaling. The differentiation block, but not the aberrant self renewal, can be overcome by treatment with ATRA resulting ina high percentage of complete remissions. Nevertheless, if ATRA is used as a monotherapy a relapse is inevitable. APL blasts are frequently positive for constitutive activating Flt3 mutations and are constantly c-Kit-positive. Given the fact that c-Kit is a stem cell marker, the expression of c-Kit has to be considered aberrant and not related to the differentiation stage of the promyelocytes in APL.Therefore we investigated first the relationship between PML/RAR and the aberrant expression of c-Kit and then the role of aberrantly activated c-Kit for the pathogenesis of APL by studying its influence on the biology of early HSC. Here we report that i.) in contrast to the t(8;21)-associated AML-1/ETO, PML/RAR activated the c-Kit promotor in HP; ii.) the inhibition of the endogenous c-Kit kinase activity by imatinib or by AZD2171 abrogated the aberrant “replating efficiency“ of PML/RAR-positive HSC; iii.) activated c-Kit (c-Kit-D814H) accelerated cell cycle progression of Sca1+/lin- HSC; iv.) activated c-Kit blocked the differentiation of Sca1+/lin- HSC and increased their “replating efficiency“; v.) “colony forming unit-spleen“ (CFU-S) as well as “competitive repopulation“ assays“ (CRA) revealed that c-Kit-D814H strongly increased the “self renewal“-potential of Sca1+/lin- HSC; vi) c-Kit-D814H augmented the migration of Sca1+/lin- HSC into a 3D stromal spheroid model based on M2-10B4 cells, but did not have any influence on their adhesion (flow chamber on TNFalpha-stimulated HUVEC cells) as well as on their chemotaxis (SDF-1 gradient in transwell assays); vii.) c-Kit-D814H further increased the aberrant replating efficiency of PML/RAR- as well as of AML-1/ETO-positive HSC.Taken together our results strongly indicate not only that c-Kit importantly contributes to the leukemogenesis of APL, but that PML/RAR has a dual effect on c-Kit - the amplification of its expression at the promotor level as well as driving its expression in cells which normally do not have this proliferation stimulus. Thus it seems that there is a sort of positive feedback between PML/RAR and c-Kit which establishes c-Kit as a valuable therapeutic target for the treatment of APL-patients.

Gene expression profiling of acute promyelocytic leukaemia identifies two subtypes mainly associated with Flt3 mutational status

Acute promyelocytic leukaemia (APL) is a well-defined disease characterized by a typical morphology of leukaemic cells, the presence of t(15;17) translocation and the unique sensitivity to the differentiating effect of all-trans retinoic acid. Nevertheless, some aspects are variable among APL patients, with differences substantially related to morphological variants, peripheral leukocytes count, the presence of a disseminated intravascular coagulopathy, different PML/RARalpha isoforms (long, variable or short) and Fms-like tyrosine kinase 3 (Flt3) mutations. In order to better define this variability, we investigated the gene expression profiles of 18 APL cases revealing, besides a high uniformity in gene expression pattern, the presence of few robust differences among patients able to identify, by an unsupervised analysis, two major clusters of patients characterized by different phenotypes (hypogranular M3v vs classical M3) and by the presence or absence of Flt3 internal tandem duplications (ITDs). Further supervised analysis confirmed that Flt3 status was the APL parameter best associated with these two subgroups. We identified, between Flt3 wild-type and Flt3-ITDs subsets, 147 differentially expressed genes that were involved in the cytoskeleton organization, in the cell adhesion and migration, in the proliferation and the coagulation/inflammation pathways as well as in differentiation and myeloid granules constitution suggesting a role of Flt3 mutations in the pathogenesis and clinical manifestations of APL.

New rearrangement t(3;17)(q26.3;q12) in an AML patient with a poor outcome

In more than 90% of acute promyelocytic leukemia (APL) cases a reciprocal translocation t(15;17)(q22;q12) can be observed. The RARalpha gene on 17q12 is known to have other translocation partners than PML (in 15q22) in a minority of APL cases. Here, we describe a previously unrecorded chromosomal translocation involving the RARalpha gene and an unknown partner on chromosome 3. The chromosomal rearrangement was studied in detail by 24-color-FISH using whole chromosome painting probes plus multicolor banding. Thus, the breakpoint could be characterized as t(3;17) (q26;q12). In this case 10% of blasts showed AML-M3 characteristics although typical rearrangements with RARalpha were not detected by molecular methods. The characterization of the present and other comparable APL-cases with exceptional translocation partners of PML or RARalpha will help to enlighten the understanding of the pathogenesis of APL.

Molecular signature of retinoic acid treatment in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia characterized by a block of differentiation at the promyelocytic stage. APL patients respond to pharmacological concentrations of all-trans retinoic acid (RA) and disease remission correlates with terminal differentiation of leukemic blasts. The PML/RAR oncogenic transcription factor is responsible for both the pathogenesis of APL and for its sensitivity to RA. In order to identify physiological targets of RA therapy, we analysed gene expression profiles of RA-treated APL blasts and found 1056 common target genes. Comparing these results to those obtained in RA-treated U937 cell lines revealed that transcriptional response to RA is largely dependent on the expression of PML/RAR. Several genes involved in the control of differentiation and stem cell renewal are early targets of RA regulation, and may be important effectors of RA response. Modulation of chromatin modifying genes was also observed, suggesting that specific structural changes in local chromatin domains may be required to promote RA-mediated differentiation. Computational analysis of upstream genomic regions in RA target genes revealed nonrandom distribution of transcription factor binding sites, indicating that specific transcriptional regulatory complexes may be involved in determining RA response.

A sumoylation site in PML/RARA is essential for leukemic transformation

Pathogenesis of acute promyelocytic leukemia (APL) has been proposed to involve transcriptional repression through enhanced corepressors binding onto RARA moieties of PML/RARA homodimers. Unexpectedly, we show that the K160 sumoylation site in the PML moiety of PML/RARA is required for efficient immortalization/differentiation arrest ex vivo, implying that RARA homodimerization is insufficient to fully immortalize primary hematopoietic progenitor cells. Similarly, PML/RARAK160R transgenic mice develop myeloproliferative syndromes, but never APL. The Daxx repressor no longer binds PML/RARAK160R, but fusion of these two proteins restores the differentiation block ex vivo. Thus, transcriptional repression dependent on a specific sumoylation site in PML is critical for the APL phenotype, while forced RARA dimerization could control expansion of the myeloid compartment.

Neutrophil Elastase Is Important for PML-Retinoic Acid Receptor α Activities in Early Myeloid Cells

Expression of the PML-retinoic acid receptor alpha (PML-RARalpha) fusion protein is the initiating genetic event for acute promyelocytic leukemia (APL), but the molecular mechanisms responsible for disease initiation are not yet clear. Several observations have suggested that early myeloid cells are uniquely susceptible to transformation by PML-RARalpha. Recently, we have shown that the early myeloid-specific protease neutrophil elastase is important for APL development in the mouse. To better understand the role of neutrophil elastase for the pathogenesis of APL, we examined the consequences of PML-RARalpha expression in early myeloid cells with or without neutrophil elastase. We found that high-level PML-RARalpha expression was associated with cellular toxicity that was dependent on the expression of neutrophil elastase; a mutant form of PML-RARalpha that resisted neutrophil elastase cleavage was not toxic. When PML-RARalpha was expressed at very low levels in the early myeloid cells of mice, it induced myeloid expansion and delayed myeloid maturation; neutrophil elastase was also required for these activities. The activities of PML-RARalpha in early myeloid cells are therefore strongly influenced by the presence of neutrophil elastase. To assure physiologic relevance, PML-RARalpha functions should be evaluated in neutrophil elastase-expressing early myeloid cells.

Disruption of PLZP in Mice Leads to Increased T-Lymphocyte Proliferation, Cytokine Production, and Altered Hematopoietic Stem Cell Homeostasis

Deregulated function of members of the POK (POZ and Kruppel) family of transcriptional repressors, such as promyelocytic leukemia zinc finger (PLZF) and B-cell lymphoma 6 (BCL-6), plays a critical role in the pathogenesis of acute promyelocytic leukemia (APL) and non-Hodgkin's lymphoma, respectively. PLZP, also known as TZFP, FAZF, or ROG, is a novel POK protein that displays strong homology with PLZF and has been implicated in the pathogenesis of the cancer-predisposing syndrome, Fanconi's anemia, and of APL, in view of its ability to heterodimerize with the FANC-C and PLZF proteins, respectively. Here we report the generation and characterization of mice in which we have specifically inactivated the PLZP gene through in-frame insertion of a lacZ reporter and without perturbing the expression of the neighboring MLL2 gene. We show that PLZP-deficient mice display defects in cell cycle control and cytokine production in the T-cell compartment. Importantly, PLZP inactivation perturbs the homeostasis of the hematopoietic stem and/or progenitor cell. On the basis of our data, a deregulation of PLZP function in Fanconi's anemia and APL may affect the biology of the hematopoietic stem cell, in turn contributing to the pathogenesis of these disorders.

PML/RARα Mediates Resistance to Apoptosis and Down-Regulation of the NF-κB Pathway in Acute Promyelocytic Leukemia.

Acute promyelocytic leukemia (APL) is genetically characterized by the translocation t(15;17) that results in the fusion gene PML/RARα. The chimeric protein renders hematopoietic progenitor cells resistant to FAS-, TNF- and IFN-induced apoptosis and caspase-3 activation, supporting a role for apoptosis impairment in the pathogenesis of APL. To better investigate apoptosis deregulation in APL, we analyzed the expression profile of 53 newly diagnosed APL patients (27M/26F; median age: 41 yr. (range: 9–80); median WBC count x 109/L: 2.67 (range: 0.5–128); median platelet count x 109/L: 19 (range: 4.4–135); FAB subtype: 39 M3/14 M3v) using the OncoChip®, a cDNA microarray especially designed for analyzing genes involved in cancer which contains 6,386 genes. After array processing, a total of 371 and 249 known genes were found to be ≥2 fold down- or up-regulated, respectively. To verify the results of microarray analysis, six differentially expressed genes (JAG1, JUN, CDKN1C, FAS, TRAIL, TRAF6 and MMP9) were tested by real-time quantitative PCR analysis (Q-RT-PCR). As initially hypothesized, numerous genes involved in apoptotic pathways were deregulated. In particular, we found a significant down-regulation of genes involved in the activation of NF-κB and of genes related to TNF-mediated apoptosis (FAS, TRAIL, TNFSF13B), with 46 and 24 genes deregulated, respectively. Expression changes in other genes implicated in apoptosis were also identified, being of special interest those affecting to the BCL2 family. Thus, anti-apoptotic genes BCL2 and BCL11A were up-regulated while pro-apoptotic members of the family were down-regulated in the analyzed series. Genes involved in the regulation of p53-dependent apoptosis (such as APAF1, p53DINP1, ATM), as well as numerous genes involved in diverse mechanisms of DNA repair, were also inhibited. Finally, the protein kinase C-δ (PKCδ) and several interferon- and interleukin-related genes were also infraexpressed in APL. These findings suggest that inactivation of apoptotic pathways is a common event in the pathogenesis of APL and may have important implications in the design of therapeutic protocols. It was of particular interest the unexpected inhibition of the NF-κB pathway. It could be in accordance with the hypothesis that NF-κB is an inductor of apoptosis under some circumstances. The role of NF-κB in promoting or repressing cell death in APL should be further investigated.

Homodimerization of Retinoic Acid Receptor alpha through its Fusion Partners Underlies Pathogenesis of Acute Promyelocytic Leukemia.

In almost all cases of acute promyelocytic leukemia (APL), t(15;17)(q22;q12) fuses the promyelocytic leukemia (PML) gene with the retinoic acid receptor alpha (RARA) gene. In rare cases of APL, other genes are fused to RARA including PLZF, NPM, NuMA, and STAT5b. These chimeras are collectively referred to as X-RARA fusions. Common features of the X-RARα fusions include their ability to form dimers through the X domain proteins while retaining the ability to interact with rexinoid receptors (RXRs, the proteins with which RARα normally heterodimerizes). We previously created an artificial X-RARα: p50-RARα (a fusion of the dimerization interface of p50 NFκB with the portion of RARα found in naturally occurring X-RARα proteins). p50-RARα has effects similar to PML-RARα including (i) enhancing repression at retinoic acid response elements, due to impaired release of co-repressors at low levels of all-trans retinoic acid (ATRA), and (ii) inhibiting differentiation of myeloid cell lines. We generated transgenic mice expressing p50-RARα under the control of the MRP8 promoter. p50-RARα had a minimal effect on myelopoiesis and initiated myeloid leukemias at a rate of <5% in the first year of observation. Nevertheless, transduction of p50-RARA transgenic bone marrow with a retrovirus expressing an activated cytokine receptor (βcV449E) generated leukemias with features of APL including therapeutic responsiveness to ATRA. Complementing our work with the p50-RARA transgenic mice, retroviral co-transduction of normal bone marrow with βcV449E plus either p50-RARA or PML-RARA generated APL-like myeloid leukemias. Although retrovirally generated βc/p50-RARα and βc/PML-RARα leukemias were nearly identical, the PML fusion appeared associated with a subtle decrease in differentiation relative to the p50 fusion. In contrast to these X-RARα fusions, preliminary experiments suggest that RCRα (a homodimeric form of RARα in which the dimerization interface of RARα has been replaced by the dimerization domain of the homodimeric COUP-TF transcription factor) does not readily cooperate with βcV449E to cause leukemia. This finding lends support to the hypothesis that the decreased binding site selectivity of X-RARα fusions contributes to APL pathogenesis. Furthermore, we have generated a novel X-RARα fusion, F3-RARα, in which three copies of the F36M mutant of the Rapamycin-binding protein have been fused to RARα. This protein mimics the in vitro effects of PML-RARα on transcription & differentiation, and de-dimerization of F3-RARα by rapamycin reverses these effects. Additional experiments in vivo with F3-RARα are expected to further confirm the importance of abnormal transcription factor dimerization in leukemia pathogenesis.

Promyelocytic leukemia protein-induced growth suppression and cell death in liver cancer cells

The promyelocytic leukemia protein (PML), involved in the pathogenesis of acute promyelocytic leukemia, is a coactivator of p53 tumor suppressive functions. The ability of PML to inhibit growth and induce cell death in solid tumor cells, however, has not been determined. We therefore assayed the tumor suppressor activities of PML and compared them with those of p53 in four liver cancer cell lines. Following infection of cells with replication-deficient recombinant PML adenovirus, the exogenous PML localized in the nucleus and formed abnormally enlarged PML-nuclear bodies after 24 hours. In vitro growth curve analysis showed that the overexpressed PML initially induced a substantial G1 cell cycle arrest and triggered massive cell death in all tested cell lines, irrespective of their p53 status. PML-induced cell death decreased by about 30% in the presence of a broad caspase inhibitor, zVAD. The cell death effect of PML was higher than that induced by p53 over a longer period of time. As with p53, overexpression of PML was closely related to upregulation of p21 and decrease of cyclin D1 expression. Unexpectedly, retinoic acid (RA) antagonized rather than enhanced PML-triggered cell death. RA enhanced the expression of adenovirus-cytomegalovirus-promoted PML at both transcription and protein levels within 12 hours after treatment; however, the PML protein was significantly degraded in the presence of RA at days 3-5 postinfection. PML degradation was also observed in SK-BR3 breast cancer cells treated with RA. Taken together, our findings strongly support the hypothesis that PML acts as a strong independent cell death inducer and that RA conversely abolishes the therapeutic effects of the PML proteins through proteasomal degradation of the protein.

Acute promyelocytic leukemia: PML/RARalpha and the leukemic stem cell.

Acute promyelocytic leukemia (APL) is distinguished from other acute myeloid leukemias (AMLs) by cytogenetic, clinical, as well as biological characteristics. The hallmark of APL is the t(15;17), which leads to the expression of the PML/RARalpha fusion protein. PML/RARalpha is the central leukemia-inducing lesion in APL and is directly targeted by all trans retinoic acid (t-RA) as well as by arsenic, both compounds able to induce complete remissions. This review focuses on potential stem cell involvement in APL outlining the knowledge about the APL-initiating stem cell and the influence of PML/RARalpha on the biology of the hematopoietic stem cell. Moreover, the importance of the blockage of t-RA signaling by the PML/RARalpha for the pathogenesis of APL is discussed, taking the relevance of the t-RA signaling pathway for the global hematopoiesis into account.

Antibody binding capacity for evaluation of MDR-related proteins in acute promyelocytic leukemia: Onset versus relapse expression.

Multidrug resistance (MDR) remains a major obstacle for successful treatment in cancer, in particular in acute leukemia. In acute promyelocytic leukemia (APL), the high sensitivity to anthracyclines appears to be attributable to the low frequency of MDR proteins overexpression at onset even if 30% of patients still relapse and become resistant to therapy. In attempt to explain different blast cell sensitivity, we studied the expression of PGP, MRP1, MRP2, and LRP in 45 cases of APL, comparing onset of disease with relapse. PGP, LRP, and MRP on bone marrow or peripheral blood blast cells were evaluated by flow cytometry using the MRK-16, LRP-56, MRP-m6, and MRP2 antibodies and results expressed by the mean fluorescence index (MFI). The antibody binding capacity (ABC) for each MDR protein was also calculated. At diagnosis, only 2 of 45 patients overexpressed PGP and 1 overexpressed LRP. PGP and LRP overexpressing cases significantly grew up during disease progression and at second relapse mean PGP MFI and mean LRP MFI were significantly higher than at onset (P = 0.001 and P = 0.008, respectively). By analyzing ABC, the same trend was more evident because a significant increment of PGP and LRP was observed at second (P = 0.002 and P = 0.002, respectively), but even at first relapse (P = 0.018 and P = 0.002, respectively). No changes were demonstrated in MRP1 and MRP2 expression in any phase of disease considered. Our data confirm the low expression at diagnosis of proteins related to development of drug resistance in APL. The evidence of a relative easy induction of PGP and LRP, but not of MRP, can be useful in choosing drugs to employ for consolidation or rescue therapy.

Restoration of promyelocytic leukemia protein-nuclear bodies in neuroblastoma cells enhances retinoic acid responsiveness.

Neuroblastoma is the most common solid tumor of infancy and is believed to result from impaired differentiation of neuronal crest embryonal cells. The promyelocytic leukemia protein (PML)-nuclear body is a cellular structure that is disrupted during the pathogenesis of acute promyelocytic leukemia, a disease characterized by impaired myeloid cell differentiation. During the course of studies to examine the composition and function of PML-nuclear bodies, we observed that the human neuroblastoma cell line SH-SY5Y lacked these structures and that the absence of PML-nuclear bodies was a feature of N- and I-type, but not S-type, neuroblastoma cell lines. Induction of neuroblastoma cell differentiation with 5-bromo-2'deoxyuridine, all-trans-retinoic acid, or IFN-gamma induced PML-nuclear body formation. PML-nuclear bodies were not detected in tissue sections prepared from undifferentiated neuroblastomas but were present in neuroblasts in differentiating tumors. Expression of PML in neuroblastoma cells restored PML-nuclear bodies, enhanced responsiveness to all-trans-retinoic acid, and induced cellular differentiation. Pharmacological therapies that increase PML expression may prove to be important components of combined modalities for the treatment of neuroblastoma.

Neutrophil Elastase Cleaves PML-RARα and Is Important for the Development of Acute Promyelocytic Leukemia in Mice

The fusion protein PML-RARα, generated by the t(15;17)(q22;q11.2) translocation associated with acute promyelocytic leukemia (APL), initiates APL when expressed in the early myeloid compartment of transgenic mice. PML-RARα is cleaved in several positions by a neutral serine protease in a human myeloid cell line; purification revealed that the protease is neutrophil elastase (NE). Immunofluorescence localization studies suggested that the cleavage of PML-RARα must occur within the cell, and perhaps, within the nucleus. The functional importance of NE for APL development was assessed in NE deficient mice. Greater than 90% of bone marrow PML-RARα cleaving activity was lost in the absence of NE, and NE (but not Cathepsin G) deficient animals were protected from APL development. Primary mouse and human APL cells also contain NE-dependent PML-RARα cleaving activity. Since NE is maximally produced in promyelocytes, this protease may play a role in APL pathogenesis by facilitating the leukemogenic potential of PML-RARα.

Acute promyelocytic leukaemia in patients originating in Latin America is associated with an increased frequency of the bcr1 subtype of the PML/RARalpha fusion gene.

The PML/RARalpha fusion gene in acute promyelocytic leukaemia (APL) has three subtypes based on the breakpoint site of the PML gene: long (bcr1), short (bcr3) and variable (bcr2) subtypes. The PML/RARalpha fusion protein is involved in the pathogenesis of APL and the breakpoint site of the PML gene might be associated with aetiological factor(s). Because APL is over-represented in patients that originate in Latin America (Latinos), we evaluated whether the distribution of the PML/RARalpha fusion mRNA in this population is different to that reported in non-Latinos. Among 52 APL patients (28 from Mexico and Central America diagnosed in Los Angeles and 24 from Peru, South America), bcr1, bcr2 and bcr3 expression was 75%, 10% and 15% respectively. However, bcr1 breakpoints were significantly higher compared with non-Latino patients (340/654, 52%) reported in four studies. Often bcr1 and bcr2 are reported together; 862 (60%) of 1429 non-Latino APL patients reported in nine studies were either bcr1 or bcr2, compared with 44 (85%) in our 52 Latino patients. This difference was also statistically significant when our patients were compared to each of the individual studies from USA and Europe, but not for a small series from China and Japan. These results suggest that the overrepresentation of APL among Latin American patients can be accounted for by an increase of a single subtype--bcr1, and the breakage sites in the PML gene may not be random but possibly influenced by genetic and/or environmental factor(s).