Impaired calcineurin signaling in myeloid cells results in downregulation of pentraxin-3 and increased susceptibility to aspergillosis.

Treatment of post-transplant patients with immunosuppressive drugs targeting the calcineurin-nuclear factor of activated T cells (NFAT) pathway, including cyclosporine A or tacrolimus, is commonly associated with a higher incidence of opportunistic infections, such as Aspergillus fumigatus, which can lead to severe life-threatening conditions. A component of the A. fumigatus cell wall, ß-glucan, is recognized by dendritic cells (DCs) via the Dectin-1 receptor, triggering downstream signaling that leads to calcineurin-NFAT binding, NFAT translocation, and transcription of NFAT-regulated genes. Here, we address the question of whether calcineurin signaling in CD11c-expressing cells, such as DCs, has a specific role in the innate control of A. fumigatus. Impairment of calcineurin in CD11c-expressing cells (CD11ccrecnb1loxP) significantly increased susceptibility to systemic A. fumigatus infection and to intranasal infection in irradiated mice undergoing bone marrow transplant. Global expression profiling of bone marrow-derived DCs identified calcineurin-regulated processes in the immune response to infection, including expression of pentraxin-3, an important antifungal defense protein. These results suggest that calcineurin inhibition directly impairs important immunoprotective functions of myeloid cells, as shown by the higher susceptibility of CD11ccrecnbloxP mice in models of systemic and invasive pulmonary aspergillosis, including after allogeneic bone marrow transplantation. These findings are relevant to the clinical management of transplant patients with severe Aspergillus infections.

Automatic detection system for multiple region of interest registration to account for posture changes in head and neck radiotherapy.

Despite immobilization of head and neck (H and N) cancer patients, considerable posture changes occur over the course of radiotherapy (RT). To account for the posture changes, we previously implemented a multiple regions of interest (mROIs) registration system tailored to the H and N region for image-guided RT correction strategies. This paper is focused on the automatic segmentation of the ROIs in the H and N region. We developed a fast and robust automatic detection system suitable for an online image-guided application and quantified its performance. The system was developed to segment nine high contrast structures from the planning CT including cervical vertebrae, mandible, hyoid, manubrium of sternum, larynx and occipital bone. It generates nine 3D rectangular-shaped ROIs and informs the user in case of ambiguities. Two observers evaluated the robustness of the segmentation on 188 H and N cancer patients. Bland-Altman analysis was applied to a sub-group of 50 patients to compare the registration results using only the automatically generated ROIs and those manually set by two independent experts. Finally the time performance and workload were evaluated. Automatic detection of individual anatomical ROIs had a success rate of 97%/53% with/without user notifications respectively. Following the notifications, for 38% of the patients one or more structures were manually adjusted. The processing time was on average 5 s. The limits of agreement between the local registrations of manually and automatically set ROIs was comprised between ±1.4 mm, except for the manubrium of sternum (-1.71 mm and 1.67 mm), and were similar to the limits agreement between the two experts. The workload to place the nine ROIs was reduced from 141 s (±20 s) by the manual procedure to 59 s (±17 s) using the automatic method. An efficient detection system to segment multiple ROIs was developed for Cone-Beam CT image-guided applications in the H and N region and is clinically implemented in our department.

Validation of deformable registration in head and neck cancer using analysis of variance.

PURPOSE: Deformable image registration (DIR) is often validated based on a distance-to-agreement (DTA) criterion of automatically propagated anatomical landmarks that were manually identified. Due to human observer variability, however, the performance of the registration method is diluted. The purpose of this study was to evaluate an analysis of variance (ANOVA) based validation to account for such observer variation. METHODS: Weekly cone beam CTs (CBCTs) of ten head and neck cancer patients undergoing five weeks of radiotherapy were used. An expert identified 23 anatomical features (landmarks) on the planning CT. The landmarks were automatically propagated to the CBCT using multiregion-of-interest (mROI) registration. Additionally, two human observers independently localized these landmarks on the CBCTs. Subsequently, ANOVA was used to compute the variance of each observer on the pairwise distance (PWD). RESULTS: ANOVA based analysis demonstrated that a classical DTA approach underestimated the precision for the mROI due to human observer variation by about 25%. The systematic error (accuracy) of mROI ranged from 0.13 to 0.17 mm; the variability (1 SD) (precision) ranged from 1.3 to 1.5 mm demonstrating that its performance is dominated by the precision. CONCLUSIONS: The PWD-ANOVA method accounts for human observer variation allowing a better estimation of the of DIR errors.

A novel mutation in the SDHD gene responsible for familial paraganglioma. Medical and psychological implications.

Familial paragangliomas/pheochromocytomas are dominantly inherited disorders characterized by the development of highly vascularized tumors of the head and neck, derived from non-chromaffin cells of the extra-adrenal paraganglia, and tumors with endocrine activity, derived from chromaffin cells, usually located in the adrenal medulla and pre- and para-vertebral thoracoabdominal regions. Germline inactivating heterozygous mutations in one of the genes encoding for succinate dehydrogenase subunits B, C or D (SDHB, SDHC or SDHD) are responsible for hereditary paragangliomas (PGLs), accounting for nearly 70% of familial cases. Particularly in the SDHD gene, different types of mutations have been found, nevertheless, alterations other than point mutations and deletion leading to missense/nonsense/splicing mutations are extremely rare. Here we report a family with multiple cases of PGL which co-segregates with a novel SDHD gene mutation predictable to give rise to an abnormal gene product (CybS). The identification of the molecular event responsible for PGL in our family made genetic counseling particularly useful for younger first degree relatives at risk to develop this late-onset disease.

GITR modulates innate and adaptive mucosal immunity during the development of experimental colitis in mice.

BACKGROUND: Uncontrolled T cell activation and abnormal function of the innate immune system against normal enteric bacterial flora play a critical part in the pathogenesis of inflammatory bowel disease (IBD). Therefore, pharmacological strategies directed to restore the normal responsiveness of the immune system could be efficacious in the treatment of these pathological conditions. Glucocorticoid-induced tumour necrosis factor receptor (GITR)-related gene is a member of the tumour necrosis factor receptor superfamily that is constitutively expressed at high levels on regulatory T cells and at low levels on unstimulated T cells, B cells and macrophages. GITR triggering leads to activation of T effectors and reversal of suppressive function of regulatory T cells. AIM: To investigate the role of GITR in the development of experimental colitis in mice. RESULTS: Using GITR(-/-) mice, GITR deletion protected against 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis by reducing innate immune responses and effector T cell activity. Effector T cells isolated from GITR(-/-) mice were less effective than T cells isolated from GITR(+/+) mice to transfer colitis in immunodeficient mice. Blocking the GITR/ligand for GITR (GITRL) signal by giving soluble GITR prevented TNBS-induced colitis in normal GITR(+/+) and also in lymphocyte-deficient SCID mice. CONCLUSIONS: Collectively, these data suggest that GITR plays a critical part in regulating both acquired and innate mucosal immune responses during the development of experimental colitis in mice. Therefore, targeting the GITR/GITRL system signalling may represent a potential pharmacological tool for the treatment of IBD.

NO-mesalamine protects colonic epithelial cells against apoptotic damage induced by proinflammatory cytokines.

The activation of a self-amplifying cascade of caspases, of which caspase-8 is the apical protease, mediates Fas-, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-, and TNF-alpha-induced apoptosis in colon cell lines. Nitric oxide (NO) protects from apoptosis induced by Fas and TNF-alpha. We examined whether NCX-456, an NO-releasing derivative of mesalamine, protects colon epithelial cells from cytokine-induced apoptosis. Caco-2 and HT-29 cell lines express death factor receptors and are driven to apoptosis in response to incubation with Fas-agonistic antibody, TNF-alpha/interferon-gamma, and TRAIL. The two novel observations reported here are that 1) cotreatment of cells with NCX-456, but not mesalamine, resulted in concentration-dependent protection against death factor-induced apoptosis and inhibition of caspase activity, and 2) exposure to dithiothreitol, an agent that effectively removes NO from thiol groups, resulted in a 70% recovery of caspase activity, which is consistent with S-nitrosation as a major mechanism for caspase inactivation. These data suggest that caspase S-nitrosation represents a mechanism for protection of colonic mucosal epithelial cells from death factor-induced death.

An NO derivative of ursodeoxycholic acid protects against Fas-mediated liver injury by inhibiting caspase activity.

Caspases are key mediators in liver inflammation and apoptosis. In the present study we provide evidence that a nitric oxide (NO) derivative of ursodeoxycholic acid (UDCA), NCX-1000 ([2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)phenyl ester]), protects against liver damage in murine models of autoimmune hepatitis induced by i.v. injection of Con A or a Fas agonistic antibody, Jo2. Con A administration causes CD4(+) T lymphocytes to accumulate in the liver and up-regulates FasL expression, resulting in FasL-mediated cytotoxicity. Cotreating mice with NCX-1000, but not with UDCA, protected against liver damage induced by Con A and Jo2, inhibited IL-1beta, IL-18, and IFN-gamma release and caspase 3, 8, and 9 activation. Studies on HepG2 cells demonstrated that NCX-1000, but not UDCA, directly prevented multiple caspase activation induced by Jo2. Incubating HepG2 cells with NCX-1000 resulted in intracellular NO formation and a DTT-reversible inhibition of proapoptotic caspases, suggesting that cysteine S-nitrosylation was the main mechanism responsible for caspase inhibition. Collectively, these data suggest that NCX-1000 protects against T helper 1-mediated liver injury by inhibiting both the proapoptotic and the proinflammatory branches of the caspase superfamily.

IL-1 beta converting enzyme is a target for nitric oxide-releasing aspirin: new insights in the antiinflammatory mechanism of nitric oxide-releasing nonsteroidal antiinflammatory drugs.

Caspase-1, the IL-1beta converting enzyme (ICE), is required for intracellular processing/maturation of IL-1beta and IL-18. NO releasing nonsteroidal antiinflammatory drugs (NSAIDs) are a new class of NSAID derivatives that spare the gastric mucosa. Here, we tested the hypothesis that NCX-4016, a NO-aspirin derivative, inhibits proinflammatory cytokine release from endotoxin (LPS)-challenged monocytes. Our results demonstrated that exposing LPS-stimulated human monocytes to NCX-4016 resulted in a 40-80% inhibition of IL-1beta, IL-8, IL-12, IL-18, IFN-gamma, and TNF-alpha release with an EC(50) of 10-20 microM for IL-1beta and IL-18. Incubating LPS-primed monocytes with NCX-4016 resulted in intracellular NO formation as assessed by measuring nitrite/nitrate, intracellular cGMP concentration, and intracellular NO formation. Exposing LPS-stimulated monocytes to aspirin or celecoxib caused a 90% inhibition of prostaglandin E(2) generation but had no effect on cytokine release. NCX-4016, similar to the NO donor S-nitroso-N-acetyl-D-L-penicillamine, inhibited caspase-1 activity with an EC(50) of approximately 20 microM. The inhibition of caspase-1 by NCX-4016 was reversible by the addition of DTT, which is consistent with S-nitrosylation as the mechanism of caspase-1 inhibition. NCX-4016, but not aspirin, prevented ICE activation as measured by assessing the release of ICE p20 subunit. IL-18 immunoneutralization resulted in a 60-80% reduction of IL-1beta, IL-8, IFN-gamma, and TNF-alpha release from LPS-stimulated monocytes. Taken together, these data indicate that incubating human monocytes with NCX-4016 causes intracellular NO formation and suppresses IL-1beta and IL-18 processing by inhibiting caspase-1 activity. Caspase-1 inhibition is a new, cycloxygenase-independent antiinflammatory mechanism of NO-aspirin.

Natural killer cell-mediated lysis of autologous cells modified by gene therapy.

This study investigated the role of natural killer (NK) cells as effectors of an immune response against autologous cells modified by gene therapy. T lymphocytes were transduced with LXSN, a retroviral vector adopted for human gene therapy that carries the selectable marker gene neo, and the autologous NK response was evaluated. We found that (i) infection with LXSN makes cells susceptible to autologous NK cell-mediated lysis; (ii) expression of the neo gene is responsible for conferring susceptibility to lysis; (iii) lysis of neo-expressing cells is clonally distributed and mediated only by NK clones that exhibit human histocompatibility leukocyte antigen (HLA)-Bw4 specificity and bear KIR3DL1, a Bw4-specific NK inhibitory receptor; and (iv) the targets are cells from HLA-Bw4(+) individuals. Finally, neo peptides anchoring to the Bw4 allele HLA-B27 interfered with KIR3DL1-mediated recognition of HLA-B27, i.e., they triggered NK lysis. Moreover, neo gene mutations preventing translation of two of the four potentially nonprotective peptides reduced KIR3DL1(+) NK clone-mediated autologous lysis. Thus, individuals expressing Bw4 alleles possess an NK repertoire with the potential to eliminate autologous cells modified by gene therapy. By demonstrating that NK cells can selectively detect the expression of heterologous genes, these observations provide a general model of the NK cell-mediated control of viral infections.

Cooperation between the RING + B1-B2 and coiled-coil domains of PML is necessary for its effects on cell survival.

PML/RARalpha is the abnormal protein product of the Acute Promyelocytic Leukemia-specific 15;17 translocation. Both the PML and RARalpha components are required for the PML/RARalpha biological activities, namely its capacity to block differentiation and to increase survival of haematopoietic precursors. The physiological role of PML and its contribution to the function of the fusion protein are unknown. PML localizes to the cytoplasm and within specific nuclear bodies (NBs). In vitro, overexpression of PML correlates with suppression of cell transformation. The PML aminoterminal portion retained within the PML/RARalpha protein contains the RING finger, two newly defined cystein/histidine-rich motifs called B-boxes (B1 and B2) and a coiled-coil region. We report here that PML has a growth suppressive activity in all the cell lines tested, regardless of their transformed phenotype, and that the cellular basis for the PML growth suppression is induction of apoptotic cell death. Analysis of various nuclear and cytoplasmic PML isoforms showed that the PML growth suppressive activity correlates with its nuclear localization. Analysis of the localization and growth suppressive activity demonstrated that: (i) the Ring + B1-B2 and coiled-coil regions are both indispensable and sufficient to target PML to the NBs; (ii) individual deletions of the various PML domains have no effect on its growth suppressor activity; (iii) the Ring + B1-B2 region exerts a partial growth suppressor activity but its fusion with the coiled-coil region is sufficient to recapitulate the suppressive function of wild type PML. These results indicate that PML is involved in cell survival regulation and that the PML component of the fusion protein (Ring + B1-B2 and coiled-coil regions) retains intact biological activity, thereby suggesting that the effects of PML/RARalpha on survival derive from the activation of the incorporated PML sequence.

High-efficiency gene transfer and selection of human hematopoietic progenitor cells with a hybrid EBV/retroviral vector expressing the green fluorescence protein.

We report a retroviral expression vector (PINCO) that allows high-efficiency gene transfer and selection of hemopoietic progenitor cells (HPCs). The main characteristics of this vector are the presence outside the two long terminal repeats of the EBV origin of replication and the EBNA-1 gene and the presence in the retrovirus of the cDNA that encodes for the enhanced green fluorescence protein (GFP), controlled by a cytomegalovirus promoter. Transient transfection of PINCO in Phoenix packaging cells results in episomal propagation of the plasmid and generates viral titers as high as 10(7) colony-forming units/ml. Infection of established cell lines with the PINCO retrovirus yields more than 95% GFP-expressing cells. GFP expression remains stable for months in infected cell cultures and can easily be monitored by fluorescent microscopy or fluorescence-activated cell-sorting (FACS) analysis of living cells. The PINCO vector allows efficient expression of a second gene (thymidine kinase, Shc, and PML), and there is strict correlation between GFP and second gene expression levels in the infected cells. PINCO was used to infect human HPCs; infection efficiency was about 50%. GFP-positive cells can be FACS sorted to yield a homogeneous population of infected cells. FACS-sorted GFP-positive HPC cells have, with respect to unfractionated HPC cells, the same frequency of long-term culture initiating cells and an identical capacity to undergo multilineage and unilineage differentiation. The entire gene transfer procedure, from the transfection of the packaging cell line to the infection of target cells, requires less than a week. The high viral titer and the easy obtainment of homogeneously infected cell populations without drug selection procedures make PINCO an ideal vector for gene transfer of human primary hemopoietic cells.

Heterogeneous nuclear expression of the promyelocytic leukemia (PML) protein in normal and neoplastic human tissues.

The RING-finger promyelocytic leukemia (PML) protein is the product of the PML gene that fuses with the retinoic acid receptor-alpha gene in the t(15; 17) translocation of acute promyelocytic leukemia. Wild-type PML localizes in the nucleus with a typical speckled pattern that is a consequence of the concentration of the protein within discrete subnuclear domains known as nuclear bodies. Delocalization of PML from nuclear bodies has been documented in acute promyelocytic leukemia cells and suggested to contribute to leukemogenesis. In an attempt to get new insights into the function of the wild-type PML protein and to investigate whether it displays an altered expression pattern in neoplasms other than acute promyelocytic leukemia, we stained a large number of normal and neoplastic human tissues with a new murine monoclonal antibody (PG-M3) directed against the amino-terminal region of PML. As the PG-M3 epitope is partially resistant to fixatives, only cells that overexpress PML are detected by the antibody in microwave-heated paraffin sections. Among normal tissues, PML was characteristically up-regulated in activated epithelioid histiocytes and fibroblasts in a variety of pathological conditions, columnar epithelium in small active thyroid follicles, well differentiated foamy cells in the center of sebaceous glands, and hypersecretory endometria (Arias-Stella). Interferons, the PML of which is a primary target gene, and estrogens are likely to represent some of the cytokines and/or hormones that may be involved in the up-regulation of PML under these circumstances. In keeping with this concept, we found that PML is frequently overexpressed in Hodgkin and Reed-Sternberg cells of Hodgkin's disease, a tumor of cytokine-producing cells. Among solid tumors, overexpression of PML was frequently found in carcinomas of larynx and thyroid (papillary), epithelial thymomas, and Kaposi's sarcoma, whereas carcinomas of the lung, thyroid (follicular), breast, and colon were frequently negative or weakly PML+. We did not observe any changes in the levels of PML expression as the lesion progressed from benign dysplasia to carcinoma. Our immunohistological data are consistent with the hypothesized growth suppressor function of PML and strongly suggest that PML expression levels are likely to be modulated by a variety of stimuli, including cytokines and hormones.

Unusual split chimaerism after mismatched T-depleted BMT.

We report a case of haploidentical T-depleted BMT that engrafted durably after a highly immunosuppressive conditioning regimen. DNA polymorphism analysis showed that granulocytes and monocytes were donor type but T and B lymphocytes were host derived. Host tolerance to donor antigens was documented. The patient suffered from serious recurrent CMV infections until split chimaerism shifted to full donor type 2 years post-BMT. Seven years after BMT the patient remains in complete remission.

Effect of the acute promyelocytic leukemia PML/RAR alpha protein on differentiation and survival of myeloid precursors.

Acute promyelocytic leukaemia is characterized by an expansion of haematopoietic precursors arrested at the promyelocytic stage (1). The differentiation block can be reversed by retinoic acid, which induces blast differentiation both in vitro (2) and in vivo (3-4). Acute promyelocytic leukaemia is also characterized by a 15;17 chromosome translocation (5) with breakpoints within the retinoic acid alpha receptor (RAR alpha) gene on 17 and within the PML gene, that encodes a putative transcription factor of unknown function (6-7), on 15 (8-10). As a consequence of the translocation a PML/RAR alpha gene is formed. It is transcriptionally active and encodes a PML/RAR alpha fusion protein detectable in all APL cases (11-14). We expressed the PML/RAR alpha protein in U937 myeloid precursor cell line and show that they: 1) lose the capacity to differentiate under the action of different stimuli (vitamin D3, transforming growth factor beta 1); ii) acquire enhanced sensitivity to retinoic acid; iii) exhibit a higher growth rate that is due to a reduction in apoptotic cell death. These results provide the first evidence of biological activity of PML/RAR alpha and recapitulate critical features of the promyelocytic leukemia phenotype.

The acute promyelocytic leukemia-specific PML-RAR alpha fusion protein inhibits differentiation and promotes survival of myeloid precursor cells.

Acute promyelocytic leukemia is a clonal expansion of hematopoietic precursors blocked at the promyelocytic stage. The differentiation block can be reversed by retinoic acid, which induces blast maturation both in vitro and in vivo. Acute promyelocytic leukemia is characterized by a 15;17 chromosome translocation with breakpoints within the retinoic acid alpha receptor (RAR alpha) gene on 17 and the PML gene, which encodes a putative transcription factor, on 15. A PML-RAR alpha fusion protein is formed as a consequence of the translocation. We expressed the PML-RAR alpha protein in U937 myeloid precursor cells and showed that they lost the capacity to differentiate under the action of different stimuli (vitamin D3 and transforming growth factor beta 1), acquired enhanced sensitivity to retinoic acid, and exhibited a higher growth rate consequent to diminished apoptotic cell death. These results provide evidence of biological activity of PML-RAR alpha and recapitulate critical features of the promyelocytic leukemia phenotype.

Expression pattern of the RAR alpha-PML fusion gene in acute promyelocytic leukemia.

Two chimeric genes, PML-RAR alpha and RAR alpha-PML, are formed as a consequence of the acute promyelocytic leukemia (APL)-specific reciprocal translocation of chromosomes 15 and 17 [t(15;17)]. PML-RAR alpha is expressed as a fusion protein. We investigated the organization and expression pattern of the RAR alpha-PML gene in a series of APL patients representative of the molecular heterogeneity of the t(15;17) and found (i) two types of RAR alpha-PML mRNA junctions (RAR alpha exon 2/PML exon 4 or RAR alpha exon 2/PML exon 7) that maintain the RAR alpha and PML longest open reading frames aligned and are the result of chromosome 15 breaking at two different sites; and (ii) 10 different RAR alpha-PML fusion transcripts that differ for the assembly of their PML coding exons. A RAR alpha-PML transcript was present in most, but not all, APL patients.

Alternative splicing of PML transcripts predicts coexpression of several carboxy-terminally different protein isoforms.

The acute promyelocytic leukaemia (APL)-specific chromosome 15;17 translocation leads to the fusion of a newly identified putative transcription factor, PML, and the retinoic acid receptor alpha. We have characterized the structure of the PML genomic locus and preliminarily characterized its expression pattern. The PML locus spans a minimum of 35 kb and is subdivided into nine exons. The putative PML DNA binding site is encoded by exons 2 and 3. We isolated a large number of alternatively spliced PML transcripts that encode numerous PML isoforms. Two groups of isoforms were identified that differed either in their C-terminal region or in the length of their central region, but retained the putative DNA-binding and dimerization domains. RNAase protection experiments revealed that the different PML isoforms are equally expressed in established cell lines of different histological origin.

Genomic variability and alternative splicing generate multiple PML/RAR alpha transcripts that encode aberrant PML proteins and PML/RAR alpha isoforms in acute promyelocytic leukaemia.

The acute promyelocytic leukaemia (APL) 15;17 translocation generates a PML/RAR alpha chimeric gene which is transcribed as a fusion PML/RAR alpha mRNA. Molecular studies on a large series of APLs revealed great heterogeneity of the PML/RAR alpha transcripts due to: (i) variable breaking of chromosome 15 within three PML breakpoint cluster regions (bcr1, bcr2 and bcr3), (ii) alternative splicings of the PML portion and (iii) alternative usage of two RAR alpha polyadenylation sites. Nucleotide sequence analysis predicted two types of proteins: multiple PML/RAR alpha and aberrant PML. The PML/RAR alpha proteins varied among bcr1, 2 and 3 APL cases and within single cases. The fusion proteins contained variable portions of the PML N terminus joined to the B-F RAR alpha domains; the only PML region retained was the putative DNA binding domain. The aberrant PML proteins lacked the C terminus, which had been replaced by from two to ten amino acid residues from the RAR alpha sequence. Multiple PML/RAR alpha isoforms and aberrant PML proteins were found to coexist in all APLs. These findings indicate that two potential oncogenic proteins are generated by the t(15;17) and suggest that the PML activation pathway is altered in APLs.

Structure and origin of the acute promyelocytic leukemia myl/RAR alpha cDNA and characterization of its retinoid-binding and transactivation properties.

Acute promyelocytic leukemia (APL) is characterized by the 15;17 chromosomal translocation. Cloning experiments have established that the chromosome 17 breakpoint maps to the RAR alpha and the 15 to the myl locus. The resulting chimeric gene is transcribed as a myl/RAR alpha fusion mRNA. By isolating both normal myl and APL myl/RAR alpha cDNAs, we showed that the myl/RAR alpha mRNA encodes for a putative fusion protein with a molecular weight of about 103 kDa, which is made up of 530 amino acids derived from the myl N-terminus and 402 amino acids originating from the RAR alpha C-terminus. The protein includes the RAR alpha DNA and retinoid-binding regions but lacks the A portion of the N-terminal region (A/B region) which is thought to contain one of the RAR alpha transactivation domains. The myl/RAR alpha protein acted as a retinoid-inducible transcription factor with both ligand-independent repressor and ligand-dependent activator functions in transactivation experiments of a retinoic acid-responsive gene. Myl/RAR alpha exerted this dual function three times more effectively than RAR alpha and had about 10-fold greater affinity for RA than RAR alpha. Comparison of myl/RAR alpha genomic and cDNA sequences from the same case demonstrated that both chromosome 15 and 17 breakpoints occurred within introns and the myl and RAR alpha sequences are spliced in the same polyadenylated RNA.

RAR-alpha gene rearrangements as a genetic marker for diagnosis and monitoring in acute promyelocytic leukemia.

Acute promyelocytic leukemias (APLs) are characterized by a translocation that involves chromosomes 15 and 17. The translocation breakpoints have recently been identified and shown to involve the RAR-alpha gene on 17 and myl on 15. Here we report Southern blotting analysis of 26 APLs, including cases with normal karyotypes and atypical morphology, which showed RAR-alpha rearrangements in 92% cases, myl rearrangements in 73%, and either RAR-alpha or myl rearrangements in 100%. Despite a negative clinical and morphologic picture, DNA rearrangement analysis showed that neoplastic promyelocytes persisted in the bone marrow of two patients sampled after induction chemotherapy. Therefore, the RAR-alpha and myl rearrangements provide molecular markers for accurately diagnosing APLs and monitoring the course of the disease during and after chemotherapy.