Acute myelogenous leukemias (AMLs) are genetically heterogeneous and characterized by chromosomal rearrangements that produce fusion proteins with aberrant transcriptional regulatory activities. Expression of AML fusion proteins in transgenic mice increases the risk of myeloid leukemias, suggesting that they induce a preleukemic state. The underlying molecular and biological mechanisms are, however, unknown. To address this issue, we performed a systematic analysis of fusion protein transcriptional targets. We expressed AML1/ETO, PML/RAR, and PLZF/RAR in U937 hemopoietic precursor cells and measured global gene expression using oligonucleotide chips. We identified 1,555 genes regulated concordantly by at least two fusion proteins that were further validated in patient samples and finally classified according to available functional information. Strikingly, we found that AML fusion proteins induce genes involved in the maintenance of the stem cell phenotype and repress DNA repair genes, mainly of the base excision repair pathway. Functional studies confirmed that ectopic expression of fusion proteins constitutively activates pathways leading to increased stem cell renewal (e.g., the Jagged1/Notch pathway) and provokes accumulation of DNA damage. We propose that expansion of the stem cell compartment and induction of a mutator phenotype are relevant features underlying the leukemic potential of AML-associated fusion proteins.

The pathogenesis of acute myeloid leukemia is associated with the appearance of oncogenic fusion proteins generated as a consequence of specific chromosome translocations. Of the two components of each fusion protein, one is generally a transcription factor, whereas the other partner is more variable in function, but often involved in the control of cell survival and apoptosis. As a consequence, AML-associated fusion proteins function as aberrant transcriptional regulators that interfere with the process of myeloid differentiation, determine a stage-specific arrest of maturation and enhance cell survival in a cell-type specific manner. The abnormal regulation of transcriptional networks occurs through common mechanisms that include recruitment of aberrant co-repressor complexes, alterations in chromatin remodeling, and disruption of specific subnuclear compartments. The identification and analysis of common and specific target genes regulated by AML fusion proteins will be of fundamental importance for the full understanding of acute myeloid leukemogenesis and for the implementation of disease-specific drug design.

The ability to generate tandem repeat s of a DNA sequence has proven importan t for a large variety of studies of DNA stru c ture and function. The most commonly use d method to produce tandem repeats involve s cloning of an oligomerized monomer s e quence that contains asymmetric overla p ping ends, but, in practice, this approach i s inefficient because of the circularization o f oligomers before they ligate into vector. D e scribed here is a method that circumvent s this problem by the use of two separat e oligomerization reactions, each containin g an initiator fragment onto which monome r polymerizes without circularization. Subs e quent mixing of the two reactions permit s circularization, generating a viable plasmi d containing the sum of the added repeat s from each reaction. A variation of thi s method is also demonstrated that permit s the synthesis of constructs with a define d number of repeats .

A functional genomic approach, based on systematic data gathering, was used to characterize a family of proteins containing a tripartite motif (TRIM). A total of 37 TRIM genes/proteins were studied, 21 of which were novel. The results demonstrate that TRIM proteins share a common function: by means of homo‐multimerization they identify specific cell compartments.

Acute promyelocytic leukaemia is characterized by translocations that involve the retinoic acid receptor α ( RAR α) locus on chromosome 17 and the PML locus on 15 or the PLZF locus on 11. The resulting abnormal translocation products encode for PML/RARα or PLZF/RARα fusion proteins. There is increasing experimental evidence that the APL-specific fusion proteins have similar biologic activities on differentiation and survival and that both components of the fusion proteins (PML or PLZF and RARα) are indispensable for these biological activities. The physiologic function of PML or PLZF or whether PML and PLZF contribute common structural or functional features to the corresponding fusion proteins is not known. We report here immunofluorescence studies on the cellular localization of PLZF and PLZF/RARα and compare it with the localization of PML and PML/RARα. PLZF localizes to nuclear domains of 0.3-0.5 microns, approximately 14 per cell in the KG1 myeloid cell line. These PLZF-bodies are morphologically similar to the domains reported for PML (PML-NBs). There is tight spatial relationship between about 30% of PLZ-NBs and PML-NBs: they partially overlap. However, PML and PLZF do not form soluble complexes in vivo . PLZF- and PML-NBs are functionally distinct. Adenovirus E4-ORF3 protein expression alters the structure of the PML-NBs and interferon increases the number of PML-NBs and neither has any effect on PLZF NBs. The localization of PLZF/RARα is different to that of PLZF and RARα. The nuclear distribution pattern of PLZF/RARα is one of hundreds of small dots (microspeckles) less than 0.1 micron. Expression of PLZF/RARα did not provoke disruption of the PML-NBs. Co-expression of PML/RARα and PLZF/RARα in U937 cells revealed apparent colocalization. Overall the results suggest that the PML- and PLZF-NBs are distinct functional nuclear domains, but that they may share common regulatory pathways and/or targeting sequences, as revealed by the common localization of their corresponding fusion proteins.

PML/RARα is the abnormal protein product of the Acute Promyelocytic Leukemia-specific 15;17 translocation. Both the PML and RARα components are required for the PML/RARα 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/RARα 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/RARα on survival derive from the activation of the incorporated PML sequence.

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.

Efficiency of presentation of a peptide epitope by a MHC class I molecule depends on two parameters: its binding to the MHC molecule and its generation by intracellular Ag processing. In contrast to the former parameter, the mechanisms underlying peptide selection in Ag processing are poorly understood. Peptide translocation by the TAP transporter is required for presentation of most epitopes and may modulate peptide supply to MHC class I molecules. To study the role of human TAP for peptide presentation by individual HLA class I molecules, we generated artificial neural networks capable of predicting the affinity of TAP for random sequence 9-mer peptides. Using neural network-based predictions of TAP affinity, we found that peptides eluted from three different HLA class I molecules had higher TAP affinities than control peptides with equal binding affinities for the same HLA class I molecules, suggesting that human TAP may contribute to epitope selection. In simulated TAP binding experiments with 408 HLA class I binding peptides, HLA class I molecules differed significantly with respect to TAP affinities of their ligands. As a result, some class I molecules, especially HLA-B27, may be particularly efficient in presentation of cytosolic peptides with low concentrations, while most class I molecules may predominantly present abundant cytosolic peptides.

Acute promyelocytic leukemia (APL) is characterized by a reciprocal 15;17 chromosomal translocation, which fuses the promyelocytic leukemia (PML) and retinoic acid receptor alpha (RAR(alpha) genes, leading to the expression of the PML/RAR alpha fusion oncoprotein. Immunocytochemical labeling of the wild-type PML protein with the PG-MB monoclonal antibody (MoAb) directed against the amino terminal portion of the human PML gene product, produces a characteristic nuclear speckled pattern that is due to localization of the protein into discrete dots (5 to 20 per nucleus), named PML nuclear bodies. The architecture of PML nuclear bodies appears to be disrupted in APL cells that bear the ttl 5;17), thus resulting in a change of the nuclear staining pattern from speckled (wild-type PML protein) to microgranular (PML-RAR alpha fusion protein), To assess whether the PG-MS MoAb could assist in the diagnosis of APL (M3), bone marrow and/or peripheral blood samples from 100 cases of acute nonlymphoid leukemias of different subtypes were blindly immunostained with the PG-M3 MoAb, using the immunoalkaline phosphatase (APAAP) or immunofluorescence technique as detection system. Notably, the abnormal (micropunctate) pattern of the PML/RAR alpha fusion protein (usually greater than or equal to 50 small granules/per nucleus) was observed in APL (M3) samples, but not in other types of acute nonlymphoid leukemias. Immunocytochemical labeling with PG-MB was particularly useful in the diagnosis of microgranular variant of APL (M3V) (three cases misdiagnosed as M4 and M5), and also to exclude a morphologic misdiagnosis of APL (six of 78 cases). In all cases investigated, immunocytochemical results were in agreement with those of reverse transcription-polymerase chain reaction (RT-PCR) for PML/RAR alpha. Because the epitope identified by PG-M3 is located in the aminoterminal portion of PML (AA 37 to 51), the antibody was suitable for recognizing APL cases characterized by breakpoint occurring at different sites of PML (bcr 1, bcr 2 and bcr 3). In conclusion, immunocytochemical labeling with PG-M3 represents a rapid, sensitive, and highly-specific test for the diagnosis of APL that bears the t(15; 17). This should allow an easy and correct diagnosis of this subtype of acute leukemia to any laboratory provided with a minimal equipment for immunocytochemistry work. (C) 1997 by The American Society of Hematology.

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Translocations involving the HRX/ALL1 locus at chromosomal region 11q23 are among the most frequent cytogenetic abnormalities in acute leukemias. 11q23 translocations involve different chromosome partners and lead to the formation of HRX/ALL1 fusion proteins. The HRX/ALL1 protein is a putative transcription factor that has been implicated in developmental regulation in mammals. We report here the cellular localization of the HRX/ALL1 protein as well as that of the HRX/ALL1-eps15 fusion protein, the result of the t(1;11) (p32-q23) translocation of acute myeloid leukemias. The HRX/ALL1 protein was localized to both the cytoplasm and the nucleus. The nuclear pattern was characterized by diffuse staining, perinuclear accumulation, and localization within nuclear bodies of variable size, morphology, and number. The HRX/ALL1-eps15 localized exclusively to the nucleus within bodies that were smaller and more numerous than the HRX/ALL1 nuclear bodies. HRX/ALL1 fusion with an unknown partner in leukemia blasts with 11q23 abnormalities had similar morphological features. Thus, the fusion with eps15 alters the cellular compartmentalization of HRX/ALL1, providing a putative mechanism for activation of HRX/ALL1 by 11q23 abnormalities.

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A set of 40 heteroaromatic systems was multivariately characterized by 13 descriptors derived by GRID. From this data matrix a second generation of Principal Properties for heteroaromatics was derived. Such Principal Properties are suitable for designing series of molecules of biological interest containing heteroaromatic moieties.

This paper describes a large scale application of an earlier proposed strategy, based on statistical design and OSAR modelling, for screening and ranking hazardous chemicals. Goal of this application has been to define a model for estimating the rate constant of the tropospheric reaction of hydrohalofluoro-, -chloro-, and -bromo-methanes, -ethanes, and -propanes with the OH radical (k(OH)) The addressed compounds were 390, and they were characterised with 11 variables drawn from the structural formula. The model was developed using a training set of 16 hydrohalocarbons, selected out by a D-optimal design from the 55 compounds for which experimental k(OH)'s were available. The predictive capacity of the resulting model, measured in terms of the parameter lower case (Standard Deviation of the Errors of Predictions) was 0.17 (with log k(OH) ranging, from -12.04 to -14.72). These data are confirmed <> by comparing the predicted with the actual log k(OH) of an <> validation set of compounds. Finally, the model was tentatively expanded to include the above-mentioned hydrohalo alkanes plus their fully halogenated homologues.

Crystallographic studies of MHC class I proteins with peptides bound and sequence analysis of the released peptides have revealed that the allele specificity is governed by the complementary interaction of the side chains of several amino acid residues of the bound peptide ("anchor residues") with the corresponding peptide side chain-binding pockets of MHC class I proteins. The role of the secondary anchor residues in the interaction with MHC class I molecules can be studied by a chemometric approach to Quantitative Structure-Activity Relationship (QSAR). In the present work, we have applied this approach to the study of the role of the secondary anchor residues in HLA-B27 peptide binding. Nine nonapeptides were designed by this method and synthesized. All of these carried a primary HLA-B27 anchor motif, Arg in P2 and Lys in P9, while position P3, P5, P6 and P7 were varied using selected representative amino acids. The relative binding affinity of these peptides was determined quantitatively by a direct binding assay, provisionally called HLA class I alpha chain refolding assay. The relative importance of the amino acid structural factors in each of the varied position was then estimated and used to design peptides optimized for their binding to HLA-B27 class I molecules. In conclusion, the paper has shown the usefulness of the chemometric strategy for studying peptides of interest in molecular immunology.