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Biomedical subjects

F E Bertrand

Publications and source records attributed to F E Bertrand.

At least 19 recordsLinked to original sources
PubMed2006

Synergy between an IGF-1R antibody and Raf/MEK/ERK and PI3K/Akt/mTOR pathway inhibitors in suppressing IGF-1R-mediated growth in hematopoietic cells.

The Insulin-like growth factor-1 receptor (IGF-1R) is overexpressed in a variety of tumors including breast, prostate and myeloma. Thus, IGF-1R and its downstream signaling effectors are good candidates for molecular-based targeted antitumor therapies. Indeed, protein inhibitors of IGF-1R signaling and IGF-1R blocking antibodies are undergoing clinical trials. Herein, the molecular basis for antibody-mediated IGF-1R signal inhibition has been investigated in a hematopoietic cell line model, FDC-P1, that has been rendered interleukin-3 independent in a ligand-dependent manner through retroviral-mediated expression of IGF-1R (FD/IGF-1R). Furthermore, the ability of an anti-IGF-1R antibody to synergize with signal-transduction pathway inhibitors and induce apoptosis was determined. The alphaIGF-1R antibody, A12, was capable of arresting IGF-1 or insulin-induced FD/IGF-1R cell proliferation in the G1 phase of the cell cycle and resulted in apoptotic induction. A12 effectiveness could be potentiated through combination treatment with small molecule inhibitors of the Ras/Raf/MEK/ERK or PI3K/Akt/mTOR pathways. These results validate the use of the FD/IGF-1R cells to evaluate the effectiveness and mechanisms of targeted IGF-1R therapeutic strategies.

Animals↗
PubMed2005

Inhibition of PI3K, mTOR and MEK signaling pathways promotes rapid apoptosis in B-lineage ALL in the presence of stromal cell support.

Bone marrow stromal cells are essential for the differentiation, survival and proliferation of normal and leukemic human B-lineage cells. Leukemic cells require stromal cell support for optimal proliferation and apoptotic resistance. Stromal cell contact can promote resistance to chemotherapeutic agents. In this study, we have made use of small molecular weight inhibitors and an established stromal cell-dependent pre-B-ALL cell line, BLIN-2, to investigate the role of the MAP kinase, PI3K/Akt, JAK/STAT and mTOR pathways in the promotion of leukemic cell growth in the presence of stromal cell support. Treatment with PI3K+JAK, PI3K+MEK, or MEK+JAK inhibitor combinations resulted in an inhibition of proliferation as measured by DNA synthesis. However, only inhibition of both PI3K and MEK or both mTOR and MEK resulted in a dramatic increase in the number of annexinV(+)/PI(+) apoptotic events within a 24 h period. Our data suggest that stromal cell-mediated apoptotic protection in B-lineage ALL is mediated by PI3K/mTOR and MEK via a synergistic mechanism(s).

Apoptosis↗
PubMed2004

JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in cell cycle progression and leukemogenesis.

The roles of the JAK/STAT, Raf/MEK/ERK and PI3K/Akt signal transduction pathways and the BCR-ABL oncoprotein in leukemogenesis and their importance in the regulation of cell cycle progression and apoptosis are discussed in this review. These pathways have evolved regulatory proteins, which serve to limit their proliferative and antiapoptotic effects. Small molecular weight cell membrane-permeable drugs that target these pathways have been developed for leukemia therapy. One such example is imatinib mesylate, which targets the BCR-ABL kinase as well as a few structurally related kinases. This drug has proven to be effective in the treatment of CML patients. However, leukemic cells have evolved mechanisms to become resistant to this drug. A means to combat drug resistance is to target other prominent signaling components involved in the pathway or to inhibit BCR-ABL by other mechanisms. Treatment of imatinib-resistant leukemia cells with drugs that target Ras (farnysyl transferase inhibitors) or with the protein destabilizer geldanamycin has proven to be a means to inhibit the growth of resistant cells. This review will tie together three important signal transduction pathways involved in the regulation of hematopoietic cell growth and indicate how their expression is dysregulated by the BCR-ABL oncoprotein.

Animals↗
PubMed2003

Treatment by design in leukemia, a meeting report, Philadelphia, Pennsylvania, December 2002.

Novel approaches have been designed to treat leukemia based on our understanding of the genetic and biochemical lesions present in different malignancies. This meeting report summarizes some of the recent advances in leukemia treatment. Based on the discoveries of cellular oncogenes, chromosomal translocations, monoclonal antibodies, multidrug resistance pumps, signal transduction pathways, genomics/proteonomic approaches to clinical diagnosis and mutations in biochemical pathways, clinicians and basic scientists have been able to identify the particular genetic mutations and signal transduction pathways involved as well as design more appropriate treatments for the leukemia patient. This meeting report discusses these exciting new therapies and the results obtained from ongoing clinical trials. Furthermore, rational approaches to treat complications of tumor lysis syndrome by administration of the recombinant urate oxidase protein, also known as rasburicase, which corrects the biochemical defect present in humans, were discussed. Clearly, over the past 25 years, molecular biology and biotechnology has provided the hematologist/oncologist novel bullets in their arsenal that will allow treatment by design in leukemia.

Antineoplastic Agents↗
PubMed2003

B-cell development in the presence of the MLL/AF4 oncoprotein proceeds in the absence of HOX A7 and HOX A9 expression.

Infant acute lymphoblastic leukemia (ALL) is frequently characterized by the t(4;11)(q21;q23) cytogenetic abnormality encoding the MLL/AF4 oncogene, increased HOX gene expression and a pro-B/monocytoid phenotype. We have previously established a novel MLL/AF4-positive cell line, B-lineage 3 (BLIN-3), which retains several features of normal B-lineage development (functional Ig gene rearrangement and apoptotic sensitivity to stromal cell withdrawal) not generally observed in infant ALL. We now use microarray analysis to identify patterns of gene expression in BLIN-3 that may modulate MLL/AF4 oncogenesis and contribute to the retention of normal B-lineage developmental characteristics. Comparison of 6815 expressed genes in BLIN-3 with published microarray data on leukemic blasts from t(4;11) patients indicated that BLIN-3 was unique in lacking the expression of certain HOX-A cluster genes. These results were validated by RT-PCR showing no expression of HOX A7 or HOX A9 in BLIN-3. A HOX C8 promoter reporter was active in BLIN-3, indicating that lack of HOX gene expression in BLIN-3 was not due to a nonfunctional MLL/AF4. Our results suggest that B-lineage development can proceed in t(4;11) leukemic blasts in the absence of HOX-A gene expression.

B-Lymphocytes↗
PubMed2001

Pro-B-cell to pre-B-cell development in B-lineage acute lymphoblastic leukemia expressing the MLL/AF4 fusion protein.

The most common chromosomal abnormality of infant acute lymphoblastic leukemia (ALL) is the t(4;11)(q21;q23) that gives rise to the MLL/AF4 fusion gene. Leukemic blasts expressing MLL/AF4 are arrested at an early progenitor stage with lymphoid or monocytoid characteristics. A novel B-lineage ALL cell line termed B-lineage-3 (BLIN-3) requiring human bone marrow (BM) stromal cell contact and interleukin-7 (IL-7) for optimal proliferation has been established. BLIN-3 cells have a CD19(+)/CD10(-) phenotype typical of infant ALL, and they harbor the t(4;11)(q21;q23) chromosomal translocation. Reverse transcription-polymerase chain reaction and Western blot analysis confirmed the presence of the MLL/AF4 fusion mRNA and protein in BLIN-3. Initial BLIN-3 cultures had a pro-B cell phenotype and did not express cytoplasmic or surface mu heavy chain. After approximately 5 months in culture on BM stromal cells plus IL-7, BLIN-3 sublines emerged expressing mu heavy chain and VpreB on the cell surfaces (ie, pre-B-cell receptor [BCR](+)). BLIN-3 cells expressing pre-BCR had the t(4;11)(q21;q23) translocation and expressed the MLL/AF4 fusion protein. Cross-linking the BLIN-3 pre-BCR led to enhanced cell proliferation, demonstrating that BLIN-3 expressed a functional pre-BCR. Increased acquisition of surface pre-BCR in BLIN-3 sublines was associated with loss of DJ rearrangements and the appearance of VDJ rearrangements. These results indicate that expression of the MLL/AF4 fusion protein is compatible with BM stromal cell and cytokine dependency, functional immunoglobulin gene segment rearrangement, and subsequent expression of a potentially diverse antigen receptor repertoire. Thus, the expression of MLL/AF4 is compatible with the normal developmental program of human B-lineage cells.

Antibodies, Monoclonal↗
PubMed2001

VH gene replacement in thymocytes.

The quasi-monoclonal (QM) mouse has a functionally rearranged H chain gene inserted into its natural position in the IgH locus. In this position, the H chain gene is subject to many of the same activities as normally arranged H chain genes, including somatic hypermutation, V(H) gene replacement, and class switch recombination. Here, we have used this mouse strain to determine some of the rules that govern the V(D)J recombination activity of the IgH locus in thymus. We focused on the requirements for V(H) gene replacement. In normal mice, thymic DJ(H) rearrangements are common, but VDJ(H) rearrangements are not. We found intermediate products of V(H) replacement in double-positive CD4(+)CD8(+) cells of the QM thymus, demonstrating that the inserted V(H) gene was accessible and ruling out the possibility that a V(H) gene per se cannot be rearranged in the thymus. We found transcripts from the knocked-in H chain gene of QM, but no mu H chain protein was detectable in thymocytes. Cloning and sequencing of these transcripts revealed that some had been generated by V(H) gene replacement. Corresponding signal joints could also be identified. These results suggest that neither a B cell-specific signal nor an Ig protein are necessary to activate V(H)-to-VDJ(H) joining in thymocytes. Possible mechanisms remaining to account for overcoming the barrier to V(H) joining in thymocytes include the insertion of a transcriptionally active gene segment and/or the inactivation of a silencer.

Animals↗
PubMed2000

Notch-1 and Notch-2 exhibit unique patterns of expression in human B-lineage cells.

The Notch genes encode a conserved family of receptors that influence developmental fate in many species. Prior studies have indicated that Notch-1 and Notch-2 signaling influence the development of hematopoietic stems cells and thymocytes, but little is known regarding Notch expression and function in B-lineage cells. We analyzed the expression of Notch receptors and Notch ligands in human B-lineage cells and bone marrow (BM) stromal cells. Notch-1 mRNA and protein is expressed throughout normal B cell development and in leukemic B-lineage cells. In contrast, Notch-2 expression is limited to pre-B cells expressing low levels of surface mu. The Notch ligand Delta is expressed in BM B-lineage cells. The Notch ligand Jagged-1 is not expressed in B-lineage cells, but is expressed in BM stromal cells. These results suggest a model wherein lateral signaling between Notch and Delta on B-lineage cells and/or Notch/Jagged-1 interactions between B-lineage cells and BM stromal cells may regulate human B cell development.

Animals↗
PubMed2000

Microenvironmental influences on human B-cell development.

Mammalian B-cell development can be viewed as a developmental performance with several acts. The acts are represented by checkpoints centered around commitment to the B-lineage and functional Ig gene rearrangement--culminating in expression of the pre-B-cell receptor (pre-BCR) and the BCR. Progression of cells through these checkpoints is profoundly influenced by the fetal liver and adult bone marrow (BM) stromal cell microenvironments. Our laboratory has developed a model of human B-cell development that utilizes freshly isolated/non-transformed human BM stromal cells as an in vitro microenvironment. Human CD34+ hematopoietic stem cells plated in this human BM stromal cell microenvironment commit to the B lineage and progress through the pre-BCR and BCR checkpoints. This human BM stromal cell microenvironment also provides survival signals that prevent apoptosis in human B-lineage cells. Human B-lineage cells exhibit differential expression of Notch receptors and human BM stromal cells express the Notch ligand Jagged-1. These results suggest a potential role for Notch in regulating B-lineage commitment and/or progression through the pre-BCR and BCR checkpoints.

Apoptosis↗
PubMed1999

IgM heavy chain complementarity-determining region 3 diversity is constrained by genetic and somatic mechanisms until two months after birth.

Due to the greater range of lengths available to the third complementarity determining region of the heavy chain (HCDR3), the Ab repertoire of normal adults includes larger Ag binding site structures than those seen in first and second trimester fetal tissues. Transition to a steady state range of HCDR3 lengths is not complete until the infant reaches 2 mo of age. Fetal constraints on length begin with a genetic predilection for use of short DH (D7-27 or DQ52) gene segments and against use of long DH (e.g., D3 or DXP) and JH (JH6) gene segments in both fetal liver and fetal bone marrow. Further control of length is achieved through DH-specific limitations in N addition, with D7-27 DJ joins including extensive N addition and D3-containing DJ joins showing a paucity of N addition. DH-specific constraints on N addition are no longer apparent in adult bone marrow. Superimposed upon these genetic mechanisms to control length is a process of somatic selection that appears to ensure expression of a restricted range of HCDR3 lengths in both fetus and adult. B cells that express Abs of an "inappropriate" length appear to be eliminated when they first display IgM on their cell surface. Control of N addition appears aberrant in X-linked agammaglobulinemia, which may exacerbate the block in B cell development seen in this disease. Restriction of the fetal repertoire appears to be an active process, forcing limits on the diversity, and hence range of Ab specificities, available to the young.

Adult↗
PubMed1998

V(H) gene replacement occurs in the spleen and bone marrow of non-autoimmune quasi-monoclonal mice.

Genes encoding the heavy chain portion of immunoglobulin molecules arise from the combinatorial association of V, D and J gene segments, which occurs during discrete stages of B lineage development in the bone marrow. Recently, V(H) replacement, a form of receptor editing, has been described, in which the variable region of an existing VDJ(H) rearrangement is replaced by another V(H) gene segment in a recombination event believed to involve an embedded heptamer within the coding region of the V(H). Studies of transgenic mice with "knocked-in" VDJ(H) genes encoding anti-DNA specificity have demonstrated that receptor editing of the heavy chain is one mechanism by which autoreactive B cell receptors can be modified. Another mouse, the "quasi-monoclonal", which encodes a "knocked-in" VDJ(H) for the hapten NP also contains B lineage cells that undergo V(H) replacement. This suggests that V(H) replacement may play a role in the normal diversification of the antibody repertoire. Using a ligation-mediated PCR assay, we have identified V(QM) double-stranded DNA breaks indicative of V(H) replacement intermediates from bone marrow and splenic B lineage cells of quasi-monoclonal mice in the absence of immunization. V(QM) to J558 recombination deletion products consistent with V(H) replacement were also detected in both the bone marrow and spleen of non-immunized quasi-monoclonal mice. Moreover, RAG-1 transcripts were detected in the spleen. These data suggest that V(H) replacement can be part of the mechanism(s) used by B lineage cells to generate diversity throughout B lineage development, including later stages occurring in secondary lymphoid tissues.

Animals↗
PubMed1998

Molecular mechanisms involved in receptor editing at the Ig heavy chain locus.

In receptor editing, a phenomenon that has recently come to light and into favor, a rearranged VDJ or VJ gene segment encoding a variable region of an Ig chain is replaced by another. In this commentary, the molecular mechanisms involved in the editing process are examined in some detail. Editing is most likely mediated by the same V(D)J recombinase activity responsible for the formation of the original VDJ or VJ segment. An embedded heptamer, which is present near the 3' end of many VH elements, is used as the recombination signal sequence at the Ig heavy chain locus. It has been postulated that the mediation of receptor editing is the evolutionary force maintaining the embedded heptamer. Some of the evidence for and against this hypothesis is discussed.

Animals↗
PubMed1998

Sequence analysis of the mouse RAG locus intergenic region.

The recombination activating genes RAG-1 and RAG-2 are highly conserved throughout evolution and are necessary and essential for the DNA rearrangement of antigen-receptor gene segments. These convergently transcribed genes are expressed primarily by developing B and T lineage cells. In addition, recent data suggest that the RAG locus can be reactivated in mouse germinal center B cells. Despite these well-defined patterns of expression, little is known about mechanism(s) regulating transcription of the RAG locus. Experiments with a mouse fibroblast line stably transfected with a genomic fragment of the RAG locus suggest that the intergenic region between RAG-1 and RAG-2 may contain information modulating RAG transcription. In order to begin testing this hypothesis, we have sequenced the 7.0-kb RAG intergenic region of the mouse. The sequence did not contain open reading frames larger than 60 amino acids. Analysis with GCG software identified several potential transcription-factor binding sequences within this region. Many of these are associated with transcriptional regulation of the Ig locus.

3' Untranslated Regions↗
PubMed1997

Ig D(H) gene segment transcription and rearrangement before surface expression of the pan-B-cell marker CD19 in normal human bone marrow.

The onset of IgH transcription and rearrangement is a defining characteristic of the progenitor population in which B-lineage commitment occurs. These features were used to better define the earliest stage of B-cell commitment in humans and to determine if these stages differ as a function of human ontogeny. Fetal and adult bone marrow mononuclear cells were sorted into B-lineage subpopulations on the basis of surface expression of the stem cell marker CD34, the pan-B-cell marker CD19, and IgM and analyzed for transcription and rearrangement of the IgH locus. The locus was found to be transcriptionally active before surface expression of CD19, as indicated by the presence of germline I mu, C mu, and D(H)Q52 transcripts in the CD34+ CD19- subpopulation. Transcripts from IgH alleles that had undergone DJC mu rearrangements were also detected in the CD34+ CD19- subpopulation. Within this subpopulation, low levels of DXP-containing DJC mu transcripts were detected in both fetal and adult cells. Although D(H)Q52 DJC mu transcripts were abundant in fetal CD34+ CD19- cells, they were not detected in cells of the same phenotype derived from adult bone marrow. In both fetus and adult, V(H)3-and V(H)6-containing VDJC mu transcripts were detected only in the CD19+ subpopulations. These data indicate that transcription of D(H)Q52-J(H) and DXP-J(H) rearrangements differs during fetal and adult B lymphopoiesis. Moreover, in both fetus and adult, transcription of unrearranged components of the IgH locus and DJ rearrangements can proceed before the surface expression of CD19.

Adult↗
PubMed1996

The J chain gene is transcribed during B and T lymphopoiesis in humans.

In mice and chickens, J chain appears to be expressed only in activated B cells and plasma cells. In humans, studies based mainly on transformed cells suggest that J chain expression may initiate during earlier stages in B lineage differentiation. In the present study, we isolated a series of hematopoietic subpopulations from human fetal and adult tissues by immunofluorescence cell sorting and examined each subpopulation for J chain expression by reverse transcriptase-PCR. In fetal and adult bone marrow, J chain transcripts were detected at all stages of B lineage differentiation, including the progenitor (CD34+/CD19-) and pro-B (CD34+/CD19+) cell subpopulations. J chain mRNA was also detected during fetal thymocyte development: double negative (CD4-/CD8-) through single positive (CD4+ or CD8+) cell subpopulations. The J chain message was not detected in peripheral CD3+ T cells, CD14+ monocytes, and CD56+ NK cells from either fetal or adult samples. The nucleotide sequence of J chain PCR products from CD34+/CD19- bone marrow progenitors and CD4+/CD8- thymocytes proved identical to the previously reported sequence of functionally spliced J chain mRNA. These results suggest that the J chain gene is transcriptionally active during early stages of both B cell and T cell differentiation, before the expression of their respective Ag receptors.

Adult↗
PubMed1995

Immunoglobulin recombinase gene activity is modulated reciprocally by interleukin 7 and CD19 in B cell progenitors.

Bone marrow stromal cells promote B cell development involving recombinase gene-directed rearrangement of the immunoglobulin genes. We observed that the stromal cell-derived cytokine interleukin 7 (IL-7) enhances the expression of CD19 molecules on progenitor B-lineage cells in human bone marrow samples and downregulates the expression of terminal deoxynucleotidyl transferase (TdT) and the recombinase-activating genes RAG-1 and RAG-2. Initiation of the TdT downregulation on the first day of treatment, CD19 upregulation during the second day, and RAG-1 and RAG-2 downmodulation during the third day implied a cascade of IL-7 effects. While CD19 ligation by divalent antibodies had no direct effect on TdT or RAG gene expression, CD19 cross-linkage complete blocked the IL-7 downregulation of RAG expression without affecting the earlier TdT response. These results suggest that signals generated through CD19 and the IL-7 receptor could modulate immunoglobulin gene rearrangement and repertoire diversification during the early stages of B cell differentiation.

Antigens, CD19↗