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Biomedical subjects

L Trakhtenbrot

Publications and source records attributed to L Trakhtenbrot.

At least 19 recordsLinked to original sources
PubMed2006

P53 in blind subterranean mole rats--loss-of-function versus gain-of-function activities on newly cloned Spalax target genes.

A tumor suppressor gene, p53, controls cellular responses to a variety of stress conditions, including DNA damage and hypoxia, leading to growth arrest and/or apoptosis. Recently, we demonstrated that in blind subterranean mole rats, Spalax, a model organism for hypoxia tolerance, the p53 DNA-binding domain contains a specific Arg174Lys amino acid substitution. This substitution reduces the p53 effect on the transcription of apoptosis genes (apaf1, puma, pten and noxa) and enhances it on human cell cycle arrest and p53 stabilization/homeostasis genes (mdm2, pten, p21 and cycG). In the current study, we cloned Spalax apaf1 promoter and mdm2 intronic regions containing consensus p53-responsive elements. We compared the Spalax-responsive elements to those of human, mouse and rat and investigated the transcriptional activity of Spalax and human Arg174Lys-mutated p53 on target genes of both species. Spalax and human-mutated p53 lost induction of apaf1 transcription, and increased induction of mdm2 transcription. We conclude that Spalax evolved hypoxia-adaptive mechanisms, analogous to the alterations acquired by cancer cells during tumor development, with a bias against apoptosis while favoring cell arrest and DNA repair.

Animals↗
PubMed2006

Gene expression profiles of AML derived stem cells; similarity to hematopoietic stem cells.

Tumors contain a fraction of cancer stem cells that maintain the propagation of the disease. The CD34(+)CD38(-) cells, isolated from acute myeloid leukemia (AML), were shown to be enriched leukemic stem cells (LSC). We isolated the CD34(+)CD38(-) cell fraction from AML and compared their gene expression profiles to the CD34(+)CD38(+) cell fraction, using microarrays. We found 409 genes that were at least twofold over- or underexpressed between the two cell populations. These include underexpression of DNA repair, signal transduction and cell cycle genes, consistent with the relative quiescence of stem cells, and chromosomal aberrations and mutations of leukemic cells. Comparison of the LSC expression data to that of normal hematopoietic stem cells (HSC) revealed that 34% of the modulated genes are shared by both LSC and HSC, supporting the suggestion that the LSC originated within the HSC progenitors. We focused on the Notch pathway since Jagged-2, a Notch ligand was found to be overexpressed in the LSC samples. We show that DAPT, an inhibitor of gamma-secretase, a protease that is involved in Jagged and Notch signaling, inhibits LSC growth in colony formation assays. Identification of additional genes that regulate LSC self-renewal may provide new targets for therapy.

Cell Cycle↗
PubMed2005

Evolutionary regulation of the blind subterranean mole rat, Spalax, revealed by genome-wide gene expression.

We applied genome-wide gene expression analysis to the evolutionary processes of adaptive speciation of the Israeli blind subterranean mole rats of the Spalax ehrenbergi superspecies. The four Israeli allospecies climatically and adaptively radiated into the cooler, mesic northern domain (N) and warmer, xeric southern domain (S). The kidney and brain mRNAs of two N and two S animals were examined through cross-species hybridizations with two types of Affymetrix arrays (mouse and rat) and muscle mRNA of six N and six S animals with spotted cDNA mouse arrays. The initial microarray analysis was hypothesis-free, i.e., conducted without reference to the origin of animals. Principal component analysis revealed that 20-30% of the expression signal variability could be explained by the differentiation of N-S species. Similar N-S effects were obtained for all tissues and types of arrays: two Affymetrix microarrays using probe oligomer signals and the spotted array. Likewise, ANOVA and t test statistics demonstrated significant N-S ecogeographic divergence and region-tissue specificity in gene expression. Analysis of differential gene expression between species corroborates previous results deduced by allozymes and DNA molecular polymorphisms. Functional categories show significant N-S ecologic putative adaptive divergent up-regulation of genes highlighting a higher metabolism in N, and potential adaptive brain activity and kidney urine cycle pathways in S. The present results confirm ecologic-genomic separation of blind mole rats into N and S. Gene expression regulation appears to be central to the evolution of blind mole rats.

Adaptation, Physiological↗
PubMed2004

Multilineage hematopoietic engraftment after allogeneic peripheral blood stem cell transplantation without conditioning in SCID patients.

Successful stem cell transplantation for patients with severe combined immunodeficiency (SCID) from matched family donors without conditioning results in engraftment of T lymphocytes. B lymphocytes engraft in only 50% of the cases, while myelopoiesis and erythropoiesis remain of host origin. Full hematopoietic engraftment was reported in one case after bone marrow transplantation without conditioning for a SCID patient. We studied three SCID patients who were transplanted with unmodified mobilized peripheral blood from HLA-identical family sex-mismatched members. They received megadoses of stem cells (18-23 x 10(6)CD34/kg). In contrast to the expected mixed chimerism that usually occurs in the absence of conditioning, we found in our patients 100% donor cell engraftment based on fluorescence in situ hybridization (FISH) and microsatellite techniques. Subset analysis of the engrafted cells using a multiparametric system enabling a combined analysis of morphology, immunophenotyping and FISH showed that both T and B lymphocytes and myeloid cells were of donor origin in two patients, while T lymphocytes and myeloid cells were of donor origin in the third. In the two cases with ABO incompatibility, erythroid engraftment was evidenced by blood group conversion from recipient to donor type. Multilineage donor engraftment is possible in SCID patients even without conditioning.

B-Lymphocytes↗
PubMed2003

Tetraploid myeloid cells in donors of peripheral blood stem cells treated with rhG-CSF.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is frequently used to mobilize CD34+ cells in healthy donors and patient with malignant diseases prior to peripheral blood stem cell (PBSC) harvest. To analyze the effects of rhG-CSF on morphology and genotype of white blood cells, a novel multiparametric cell scanning system that combines morphologic, immune and genotypic analyses of the same cells was used. We report here that tetraploid myeloid cells are present in the peripheral blood of donors treated with rhG-CSF. The tetraploidy was detected in up to 0.6% of differentiated myeloid cells and all observed CD34+ cells were diploid. Thus, short treatment with rhG-CSF of PBSC donors induces numerfical chromosomal alterations in a small subset of mature myeloid cells.

Case-Control Studies↗
PubMed2002

Chimerism testing and detection of minimal residual disease after allogeneic hematopoietic transplantation using the bioView (Duet) combined morphological and cytogenetical analysis.

Recurrent disease remains a major obstacle to cure after allogeneic transplantation. Various methods have been developed to detect minimal residual disease (MRD) after transplantation to identify patients at risk for relapse. Chimerism tests differentiate recipient and donor cells and are used to identify MRD when there are no other disease-specific markers. The detection of MRD does not always correlate with relapse risk. Chimerism testing may also identify normal hematopoietic cells or other cells not contributing to relapse. In this study we report our initial experience with a novel system that provides combined morphological and cytogenetical analysis on the same cells. This system allows rapid automatic scanning of a large number of cells, thus increasing the sensitivity of detection of small recipient population. The clinical significance of MRD detection is improved by identifying the morphology of recipient cells. Identification of recipient characteristics within blasts predicts overt relapse in leukemia patients and precedes it by a few weeks to months. Identification within mature hematopoietic cells may not be closely associated with relapse. The system also allows chimerism testing after sex-mismatched transplants, within cellular subsets, with no need for sorting of cells. The system merits further study in larger scale trials.

Automation↗
PubMed2001

Identification of a region in glycoprotein IIIa involved in subunit association with glycoprotein IIb: further lessons from Iraqi-Jewish Glanzmann thrombasthenia.

The most frequent mutation causing Glanzmann thrombasthenia in Iraqi-Jews (IJ-1) is an 11-bp deletion in exon 13 of the glycoprotein (GP) IIIa gene. This deletion predicts a frameshift that results in the elimination of the C406-C655 disulfide bond and a premature termination codon shortly before the transmembrane domain. To determine the contribution of each of these alterations to the thrombasthenic phenotype, Chinese hamster ovary or baby hamster kidney cells were cotransfected with normal GPIIb complementary DNA (cDNA) and the following GPIIIa cDNAs: normal, cDNA bearing IJ-1 mutation, 2011T>A mutated cDNA predicting C655S (single-letter amino acid codes) substitution, and 2019A>T mutated cDNA predicting Stop657. Elimination of the C406-C655 disulfide bond by C655S substitution did not affect GPIIb/IIIa surface expression or binding of the transfected cells to immobilized fibrinogen, whereas elimination of the transmembrane and cytoplasmic domains in IJ-1 and Stop657 mutants prevented both surface expression and binding of the transfected cells to immobilized fibrinogen. Immunohistochemical staining and immunoprecipitation demonstrated that the elimination of amino acids 657-762 in IJ-1 and Stop657 prevented intracellular GPIIb/IIIa complex formation, and differential immunofluorescence staining of GPIIIa and cellular organelles suggested that the truncated uncomplexed GPIIIa protein was retained in the endoplasmic reticulum. Because the use of GPIIIa Stop693 and normal GPIIb cDNAs yielded GPIIb/IIIa complex formation, though with lower efficiency, it is suggested that amino acids 657-692 of GPIIIa are essential for the intracellular association of GPIIb and GPIIIa. (Blood. 2001;98:1063-1069)

Animals↗
PubMed2001

Subgroup of patients with Philadelphia-positive chronic myelogenous leukemia characterized by a deletion of 9q proximal to ABL gene: expression profiling, resistance to interferon therapy, and poor prognosis.

A major deletion of the region proximal to the rearranged ABL gene on 9q was found in 14/94 (15%) of chronic myelogenous leukemia Philadelphia-positive patients by interphase fluorescent in situ hybridization with the BCR/ABL extra signal dual-color probe. Preliminary results indicated that the prognosis of the deletion 9q patients is probably worse than that of the non-deletion 9q patients. Twelve of the 14 deletion 9q patients were treated with alpha-interferon and none had a major cytogenetic response. The median duration of the chronic phase in patients not undergoing BMT was significantly shorter for the deletion 9q patients as compared to the non-deletion 9q patients (p =.0144). DNA microarray technology was performed in order to compare the gene expression patterns between the two groups of patients. A number of genes exhibiting differential expression, especially involving cell adhesion and migration, were identified. This finding may identify a sub-group of CML patients with different cell properties and a relatively poor prognosis.

Antineoplastic Agents↗
PubMed2001

Detection of unidentified chromosome abnormalities in human neuroblastoma by spectral karyotyping (SKY).

Spectral karyotyping (SKY) is a novel technique based on the simultaneous hybridization of 24 fluorescently labeled chromosome painting probes. It provides a valuable addition to the investigation of many tumors that can be difficult to define by conventional banding techniques. One such tumor is neuroblastoma, which is often characterized by poor chromosome morphology and complex karyotypes. Ten primary neuroblastoma tumor samples initially analyzed by G-banding were analyzed by SKY. In 8/10 tumors, we were able to obtain additional cytogenetic information. This included the identification of complex rearrangements and material of previously unknown origin. Structurally rearranged chromosomes can be identified even in highly condensed metaphase chromosomes. Following the SKY results, the G-banding findings were reevaluated, and the combination of the two techniques resulted in a more accurate karyotype. This combination allows identification not only of material gained and lost, but also of breakpoints and chromosomal associations. The use of SKY is therefore a powerful tool in the genetic characterization of neuroblastoma and can contribute to a better understanding of the molecular events associated with this tumor.

Child↗
PubMed2001

Reduced expression of plakoglobin correlates with adverse outcome in patients with neuroblastoma.

Plakoglobin and its homologue beta-catenin are cytoplasmic proteins that mediate adhesive functions by interacting with cadherin receptors and signaling activities by interacting with transcription factors. It has been suggested that plakoglobin can suppress tumorigenicity whereas beta-catenin can act as an oncogene. We investigated the correlation between the expression pattern of N-cadherin, beta-catenin, and plakoglobin and tumor behavior in primary tumors of 20 neuroblastoma patients of all stages and in 11 human neuroblastoma cell lines. N-cadherin and beta-catenin were detected in 9 of 11 and 11 of 11 cell lines, respectively, whereas plakoglobin was undetectable or severely reduced in 6 of 11 cell lines. Tumor cells from 16 of 20 patients expressed N-cadherin and 20 of 20 patients expressed beta-catenin at levels similar to those of normal ganglion cells. Plakoglobin was undetectable in 9 of 20 tumors. Plakoglobin deficiency in the primary tumors was significantly associated with adverse clinical outcome. Five of the patients with plakoglobin-negative tumors died whereas four patients are alive without evident disease. In contrast, all patients with plakoglobin-positive tumors are alive; 2 of 11 are alive with the disease and 9 of 11 are alive without evident disease. These results suggest that down-regulation of plakoglobin may be of prognostic value for neuroblastoma patients as predictor of poor outcome.

Blotting, Western↗
PubMed2000

Standardization criteria for the detection of BCR/ABL fusion in interphase nuclei of chronic myelogenous leukemia patients by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH), as a new clinical test, is not presently standardized. For practical reasons, each laboratory must build its own criteria. In this work, we present our standardization criteria for clinical practice, which include not only the methods for cell fixation, specimen preparation, and hybridization conditions, but mainly the definition of false-positive range and the scoring criteria of microscopic analysis. These include signal assessment, difference between individual microscopists, evaluation of specimen homogeneity, and the minimum number of scored nuclei required for a clinically reliable result. For this purpose, we analyzed by FISH 24 healthy volunteer donors, 31 patients affected by non-chronic myelogenous leukemia (CML) hematological malignancies, 47 CML patients at diagnosis, and 82 CML patients during treatment for the BCR/ABL fusion. In this article, we present several quality control and assurance methods that can be useful in providing standardization of the FISH technique.

Cell Nucleus↗
PubMed2000

Three-dimensional spectral precision distance microscopy of chromatin nanostructures after triple-colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome.

Topological analysis of the three-dimensional (3D) chromatin nanostructure and its function in intact cell nuclei implies the use of high resolution far field light microscopy, e.g. confocal laser scanning microscopy (CLSM). However, experimental evidence indicates that, in practice, under biologically relevant conditions, the spatial resolution of CLSM is limited to about 300 nm in the lateral direction and about 700 nm in the axial direction. To overcome this shortcoming, the use of a recently developed light microscopical approach, spectral precision distance microscopy (SPDM) is established. This approach is based on the precise localization of small labelling sites of a given target in spectrally differential images. By means of quantitative image analysis, the bary centres (intensity weighted centroid analogous to the centre of mass) of these independently registered labelling sites can be used as point markers for distance and angle measurements after appropriate calibration of optical aberrations (here, polychromatic shifts). In combination with specific labelling of very small chromatin target sites with dyes of different spectral signatures by fluorescence in situ hybridization (FISH), SPDM presently allows us to analyse the nuclear topology in three-dimensionally conserved nuclei with a 'resolution equivalent', many times smaller than the conventional optical resolution. Chronic myelogeneous leukaemia (CML) is genetically characterized by the fusion of parts of the BCR and ABL genes on chromosomes 22 and 9, respectively. In most cases, the fusion leads to a translocation t(9; 22) producing the Philadelphia chromosome. SPDM was applied to analyse the 3D chromatin structure of the BCR region on the intact chromosome 22 and the BCR-ABL fusion gene on the Philadelphia chromosome (Ph) by using a new triple-colour FISH protocol: two different DNA probes were used to detect the BCR region and the third DNA probe was used to identify the location of the ABL gene. Consistent 3D distance measurements down to values considerably smaller than 100 nm were performed. The angle distributions between the three labelled sites on the Philadelphia chromosome territory were compared to two state-of-the-art computer models of nuclear chromatin structure. Significant differences between measured and simulated angle distributions were obtained, indicating a complex and non-random angle distribution.

Bone Marrow Cells↗
PubMed1999

Coexistence of several unbalanced translocations in a case of neuroblastoma: the contribution of multicolor spectral karyotyping.

Spectral karyotyping (SKY) is based on the simultaneous hybridization of a set of 24 chromosome-specific DNA painting probes, each labeled with a different fluor combination. Automatic classification, based on the measurement of the spectrum for each chromosome, was applied to metaphases obtained from the affected bone marrow of a neuroblastoma case. Spectral karyotyping allowed the identification of chromosomal aberrations that could not be identified by the use of the G-banding technique, and revealed a number of gains and unbalanced translocations.

Child, Preschool↗
PubMed1998

Preleukemia in long-term plasmacytoma-regressor mice.

Our previous results have indicated that mice whose plasmacytoma regressed following curative melphalan chemotherapy manifested various persistent immunohematological abnormalities including immunosuppression, myeloproliferation, as well as excessive production of and response to growth factors. Mice not bearing plasmacytoma treated with an identical dose of melphalan chemotherapy did not exhibit such abnormalities. In the present study we show that plasmacytoma-regressor mice (PRM) contain preleukemic cells which do not progress to leukemia in these mice. However, adoptive transfer of splenocytes originating in PRM to preirradiated but otherwise untreated syngeneic recipients resulted in the development of overt leukemia in these recipients. The presence of leukemia in the primary recipient mice was ascertained by blood counts as well as by spleen histology. Furthermore, splenocytes from the irradiated primary recipients adoptively transferred to non-irradiated secondary recipients caused leukemia formation in 100% of the secondary recipients. Sex chromosome analysis of the leukemic cells in the irradiated primary recipients clearly showed that they originated in the PRM donors. Two leukemic lines were established from leukemias developing in the secondary recipients and both expressed surface markers of hematopoietic progenitor cells as well as markers of T cells. We suggest that PRM could serve as an animal model to investigate development of chemotherapy-related leukemia in humans.

Animals↗
PubMed1998

Fast-FISH detection and semi-automated image analysis of numerical chromosome aberrations in hematological malignancies.

A new fluorescence in situ hybridization (FISH) technique called Fast-FISH in combination with semi-automated image analysis was applied to detect numerical aberrations of chromosomes 8 and 12 in interphase nuclei of peripheral blood lymphocytes and bone marrow cells from patients with acute myelogenous leukemia (AML) and chronic lymphocytic leukemia (CLL). Commercially available alpha-satellite DNA probes specific for the centromere regions of chromosome 8 and chromosome 12, respectively, were used. After application of the Fast-FISH protocol, and microscopic images of the fluorescence-labelled cell nuclei were recorded by the true color CCD camera Kappa CF 15 MC and evaluated quantitatively by computer analysis on a PC. These results were compared to results obtained from the same type of specimens using the same analysis system but with a standard FISH protocol. In addition, automated spot counting after both FISH techniques was compared to visual spot counting after standard FISH. A total number of about 3,000 cell nuclei was evaluated. For quantitative brightness parameters, a good correlation between standard FISH labelling and Fast-FISH was found. Automated spot counting after Fast-FISH coincided within a few percent to automated and visual spot counting after standard FISH. The examples shown indicate the reliability and reproducibility of Fast-FISH and its potential for automatized interphase cell diagnostics of numerical chromosome aberrations. Since the Fast-FISH technique requires a hybridization time as low as 1/20 of established standard FISH techniques, omitting most of the time consuming working steps in the protocol, it may contribute considerably to clinical diagnostics. This may especially be interesting in cases where an accurate result is required within a few hours.

Aneuploidy↗
PubMed1996

In vitro proliferative advantage of bone marrow cells with tetrasomy 8 in Ewing sarcoma.

We describe a case of a 14.5-year-old boy with a clinically aggressive pelvic Ewing sarcoma. The tumor cells showed the presence of a typical t(11;22)(q24;q12) aberration and gains of chromosomes 8, 10, 14, and 21. To determine the size of the trisomy and tetrasomy 8 clones an interphase analysis by fluorescence in situ hybridization with a centromere-specific chromosome 8 probe was performed. Significant quantitative differences between metaphase and interphase data were obtained. It was shown that culturing of bone marrow cells leads to enrichment of tetrasomy 8 population that may be explained by the proliferative advantage of the tetrasomy 8 cells.

Adolescent↗
PubMed1995

Hexasomy of chromosome 8 and trisomy of chromosome 11 characterize two karyotypically independent clones in a case of acute non-lymphocytic leukemia. Conventional cytogenetic and FISH investigation.

A case of ANLL following a myelodysplastic syndrome, probably resulting from occupational exposure to ionizing irradiation, with two cytogenetically unrelated clones, hexasomy 8 and trisomy 11, was investigated by conventional cytogenetics and FISH. Significant quantitative differences between data obtained by metaphase and interphase analysis of the hexasomy 8 clone were observed. A difference in the sensitivity to chemotherapy of the two clones was found: while the hexasomy 8 clone markedly decreased in response to treatment, the trisomy 11 clone remained unchanged.

Aneuploidy↗