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Biomedical subjects

N Heisterkamp

Publications and source records attributed to N Heisterkamp.

At least 37 records · Page 2Linked to original sources
PubMed1998

Bcr/Abl associated leukemogenesis in bcr null mutant mice.

The BCR gene contributes to Philadelphia-positive leukemogenesis via a number of discrete mechanisms, one of which may be through interaction of its normal gene product with the Bcr/Abl oncoprotein. In the current study this hypothesis was tested in vivo by introducing a Bcr/Abl P190 transgene into mice lacking endogenous bcr protein. Our finding, that the P190 BCR/ABL oncogene is still capable of producing leukemia in these mice with indistinguishable latency and clinical pattern as in genetically matched counterparts, rules out any significant or major contribution of the bcr protein as a whole to leukemia development in these mice.

Animals↗
PubMed1998

Progressive neurodegeneration in aspartylglycosaminuria mice.

Aspartylglycosaminuria (AGU) is one of the most common lysosomal storage disorders in humans. A mouse model for AGU has been recently generated through targeted disruption of the glycosylasparaginase gene, and at a young age the glycosyl asparaginase-deficient mice demonstrated many pathological changes found in human AGU patients (Kaartinen V, Mononen I, Voncken J-W, Gonzalez-Gomez I, Heisterkamp N, Groffen J: A mouse model for aspartylglycosaminuria. Nat Med 1996, 2:1375-1378). Our current findings demonstrate that after the age of 10 months, the general condition of null mutant mice gradually deteriorated. They suffered from a progressive motoric impairment and impaired bladder function and died prematurely. A widespread lysosomal hypertrophy in the central nervous system was detected. This neuronal vacuolation was particularly severe in the lateral thalamic nuclei, medullary reticular nuclei, vestibular nuclei, inferior olivary complex, and deep cerebellar nuclei. The oldest animals (20 months old) displayed a clear neuronal loss and gliosis, particularly in those regions, where the most severe vacuolation was found. The severe ataxic gait of the older mice was likely due to the dramatic loss of Purkinje cells, intensive astrogliosis and vacuolation of neurons in the deep cerebellar nuclei, and the severe vacuolation of the cells in vestibular and cochlear nuclei. The impaired bladder function and subsequent hydronephrosis were secondary to involvement of the central nervous system. These findings demonstrate that the glycosylasparaginase-deficient mice share many neuropathological features with human AGU patients, providing a suitable animal model to test therapeutic strategies in the treatment of the central nervous system effects in AGU.

Acetylglucosamine↗
PubMed1998

Philadelphia-negative chronic myelogenous leukemia in a patient with a unique complex translocation: 46,XY,t(9;12;15)(q34;12;q21).

Chronic myelogenous leukemia (CML) is associated with an acquired karyotypic abnormality, the Philadelphia (Ph) chromosome, in 95% of cases. The Ph chromosome is the product of a balanced translocation that results in a hybrid gene that is considered essential for the pathogenesis of this disease. We have found a complex translocation involving chromosomes 9, 12, and 15 in a 42-year-old Haitian male with the clinical findings of CML. Complex translocations have been shown to result in the masking of the Ph chromosome. We used a mixture of two BCR-specific DNA probes for Southern blot analysis in order to test this hypothesis in our patient. High-molecular weight DNA was digested with the restriction enzymes BglII, BamHI and HindIII. The BglII digestion revealed the presence of two abnormal fragments of 3.9 and 3.0 kb and the BamHI digestion an abnormal 15-kb fragment. These data suggest there is a breakpoint in region 2 of M-bcr. The identification of this breakpoint confirms our hypothesis that a rearrangement involving 22q11 has occurred in the leukemic cells of our patient. A secondary translocation involving chromosomes 12 and 15 has hidden the effects of this translocation. Combined cytogenetic and molecular analysis establishes the karyotype of our patient as 46,XY,t(9;12;15;22)(q34;q12;q21;q11).

Adult↗
PubMed1998

Involvement of the tyrosine kinase fer in cell adhesion.

The Fer protein belongs to the fes/fps family of nontransmembrane receptor tyrosine kinases. Lack of success in attempts to establish a permanent cell line overexpressing it at significant levels suggested a strong negative selection against too much Fer protein and pointed to a critical cellular function for Fer. Using a tetracycline-regulatable expression system, overexpression of Fer in embryonic fibroblasts was shown to evoke a massive rounding up, and the subsequent detachment of the cells from the substratum, which eventually led to cell death. Induction of Fer expression coincided with increased complex formation between Fer and the cadherin/src-associated substrate p120(cas) and elevated tyrosine phosphorylation of p120(cas). beta-Catenin also exhibited clearly increased phosphotyrosine levels, and Fer and beta-catenin were found to be in complex. Significantly, although the levels of alpha-catenin, beta-catenin, and E-cadherin were unaffected by Fer overexpression, decreased amounts of alpha-catenin and beta-catenin were coimmunoprecipitated with E-cadherin, demonstrating a dissolution of adherens junction complexes. A concomitant decrease in levels of phosphotyrosine in the focal adhesion-associated protein p130 was also observed. Together, these results provide a mechanism for explaining the phenotype of cells overexpressing Fer and indicate that the Fer tyrosine kinase has a function in the regulation of cell-cell adhesion.

Animals↗
PubMed1998

Identification of a novel Rac3-interacting protein C1D.

Rac3 is a small GTPase of the Rho family, members of which have been implicated in tumorigenesis, cell growth/death and organization of the actin cytoskeleton. Many Rac-interacting proteins or effectors identified to date have a role in cytoskeletal organization. Using the yeast two-hybrid system, we have isolated a novel Rac3-interacting protein from a human placenta cDNA library which has no homology to other previously identified Rac-interacting proteins. Sequence analysis revealed that this protein is C1D, the human homolog of the murine SUN-CoR protein which acts as a corepressor for the thyroid hormone receptor. In yeast cells, C1D binds to constitutively activated but not to GDP-bound Rac3. When coexpressed with Rac3 in COS cells, C1D complexed with constitutively active Rac3 but not with wild-type Rac3, demonstrating that C1D-Rac3 interactions take place in vivo in mammalian cells and that C1D appears to be an effector of Rac3. The C1D gene was mapped to human chromosome 2, which frequently shows deletions in human follicular thyroid carcinomas.

Amino Acid Sequence↗
PubMed1998

Abnormal stress response and increased fighting behavior in mice lacking the bcr gene product.

The in vivo function of proteins which regulate activity of the GTPase Rac is largely unknown. Here we establish that mice lacking bcr, a known GTPase activating protein for Rac, exhibit a defect in the regulation of both hormonal and behavioral stress responses. Bcr null mutants demonstrate prolonged elevation of plasma glucocorticoids and increased fighting in males in response to physiological and social stress, respectively. Combined biochemical and behavioral data indicate that bcr is involved in mediating the cellular effects of glucocorticoids, specifically down-regulation of the stress-activated hippocampal hypothalamic-pituitary-adrenal axis.

Aggression↗
PubMed1997

BCR/ABL-induced leukemogenesis causes phosphorylation of Hef1 and its association with Crkl.

BCR/ABL is considered responsible for the development of Philadelphia chromosome-positive leukemia. Experimental animal models, such as transgenic mice, have demonstrated unambiguously that Bcr/Abl is capable of inducing leukemogenesis. The adaptor molecule Crkl is a major in vivo substrate of the deregulated Bcr/Abl tyrosine kinase and functions as a molecular link with other signaling proteins. While associated in vivo with Bcr/Abl through its SH3 domain, Crkl can interact simultaneously via its SH2 domain with other tyrosine-phosphorylated proteins. Here we report the identification of prominently tyrosine-phosphorylated proteins with a molecular mass of approximately 110 kDa, which bind specifically to the Crkl SH2 domain in leukemic tissues of P190BCR/ABL transgenic mice. We demonstrate that these proteins are identical to Hef1/Cas-L, which is related to p130(Cas). The proto-oncoprotein p120(Cbl) and Hef1, but not p130(Cas), were detectably phosphorylated on tyrosine in P190Bcr/Abl-expressing leukemic cells and were found in complex with Crkl, showing the existence of protein complexes in P190Bcr/Abl leukemic cells, consisting of P190Bcr/Abl, Crkl, and Hef1 or p120(Cbl). This supports a model in which Crkl acts as mediator between Bcr/Abl and downstream effectors. Since Hef1 is involved in the beta1-integrin signaling pathway, our study demonstrates that Bcr/Abl could specifically interfere with normal beta1-integrin signaling.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Characterization of RAC3, a novel member of the Rho family.

The small GTP-binding proteins Rac1 and Rac2 are critically important in regulating multiple signal transduction pathways in eukaryotic cells. Here we report the isolation of a novel third Rac family member, Rac3. Rac3 differs from Rac1/2 at its carboxyl-terminal end, a domain associated with subcellular localization and binding to specific cellular regulators. RAC3 mRNA expression patterns differ from those of RAC2, which is hematopoietic specific and also from those of RAC1. The RAC3 gene was mapped to chromosome 17q23-25, a region frequently deleted in breast cancer. Rac3 protein levels are not affected by organization of the actin cytoskeleton but remarkably, are serum-inducible. Rac3 is an active GTPase, and this activity is regulated by Bcr. When constitutively activated, Rac3 is able to stimulate efficiently the c-Jun amino-terminal kinase signaling pathway. These findings support a role for Rac3 in intracellular signaling.

Amino Acid Sequence↗
PubMed1997

Tyrosine 207 in CRKL is the BCR/ABL phosphorylation site.

BCR/ABL has a causal role in Philadelphia (Ph)-chromosome positive leukemia. The SH2/SH3 adapter protein CRKL is a major substrate of the deregulated BCR/ABL tyrosine kinase and is aberrantly tyrosine-phosphorylated in Ph-positive leukemia cells. In this study, experiments were pursued to identify and analyse the CRKL phosphorylation site(s). In an in vitro kinase assay, CRKL phosphorylation by the abl kinase was limited to a small region between the two CRKL SH3 domains. Within this region, mutation of tyrosine residue 207 yielded a mutant CRKL which could not be phosphorylated by BCR/ABL. Stable overexpression of CRKL or CRKL-Y207F did not transform NIH3T3 cells, while the Y207F mutation eliminated tyrosine-phosphorylation of CRKL. These studies indicate that Y207 in CRKL represents the major in vivo phosphorylation site. Phosphorylation of Y207 provides a binding site for the CRKL SH2 domain and potentially for other SH2-containing proteins. The Y207F mutation in CRKL did not enhance or decrease association with various target signalling proteins, including SOS or C3G, which interact specifically with the CRKL N-SH3 domain. These findings suggest that complex formation with cellular targets is not modulated by CRKL tyrosine-phosphorylation.

3T3 Cells↗
PubMed1997

Deletion within the D17S34 locus in a primitive neuroectodermal tumor.

Loss of heterozygosity on chromosome 17p13.3 is frequently observed in solid tumors, and the presence of a tumor suppressor gene has been predicted in this region of chromosome 17. We have analyzed a primitive neuroectodermal tumor sample exhibiting loss of heterozygosity at the D17S34 locus, a commonly used telomeric marker on the short arm of chromosome 17. The remaining allele showed a rearrangement. Cosmids spanning the D17S34 locus and probes from that region were used to demonstrate a 9-kb deletion within the D17S34 locus and were found to contain evolutionary, conserved sequences. Genetic alterations in this region may also affect expression of immediately adjacent genes, such as ABR, and could be a common mechanism in the causation of primitive neuroectodermal tumors.

Chromosomes, Human, Pair 17↗
PubMed1997

Characterization of the fate of midline epithelial cells during the fusion of mandibular prominences in vivo.

The fusion of the mandibular prominences along the midline is achieved with the absence of medial epithelial cells at the fusion site. Failure of fusion of the mandibular prominences results in median cleft of the lower lip and mandible. Cellular and molecular events controlling mandibular fusion were examined during the fusion process in mouse embryogenesis. Cell lineage analyses at the fusion site revealed that epithelial cells migrated to the surface and oral epithelia. DiI-labeled epithelial cells were not observed within the mandibular mesenchyme at any state of fusion. Examination of the midline region did not reveal cells with ultrastructural changes characteristic of apoptotic cell death. An increase in lysosomal enzymes in the midline epithelial cells, which would be correlated with programmed cell death, was not observed. Mice lacking TGF-beta 3 did not have cleft mandible, but had clefting of the secondary palate as a feature of null mutation phenotype. We interpret our comparisons between wild type and homozygous TGF-beta 3 (-/-) mice to suggest that different developmental processes control palatal vs. mandibular fusion. We hypothesize that medical epithelial cells at the fusion site of mandibular prominences migrate to the surface epithelium during the fusion process and neither transdifferentiate into mesenchyme nor express apoptosis.

Animals↗
PubMed1997

Transforming growth factor-beta3 regulates transdifferentiation of medial edge epithelium during palatal fusion and associated degradation of the basement membrane.

Studies on transforming growth factor beta3 (TGF-beta3) deficient mice have shown that TGF-beta3 plays a critical role in palatogenesis. These null mutant mice have clefting of the secondary palate, caused by a defect in the process of fusion of the palatal shelves. A critical step in mammalian palatal fusion is removal of the medial edge epithelial cells from the midline seam and formation of continuous mesenchyme. To determine in more detail the role of TGF-beta3 in palatogenesis, we cultured TGF-beta3 null mutant and wild-type control palatal shelves in an organ culture system. The fate of the medial edge epithelial cells was studied in vitro using vital cell labeling and immunohistochemical techniques. Despite clear adherence, the null mutant palatal shelves did not fuse in vitro, but instead the medial edge epithelial cells survived at the midline position, and the basement membrane was resistant towards degradation. Supplementation of the culture medium with the mature form of TGF-beta3 was able to fully correct the defective fusion in the null mutant specimens. Our results demonstrate that the reason for the defective palatal fusion in TGF-beta3 (-/-) samples is not impaired adhesion. Our data define a specific role for TGF-beta3 in the events that control transdifferentiation of the medial edge epithelial cells including degradation of the underlying basement membrane.

Animals↗
PubMed1997

The chimeric BCR-ABL gene.

The 1982 discovery that in chronic myeloid leukaemia (CML) the ABL proto-oncogene is translocated to the BCR gene located on chromosome 22 initiated many studies on the structural organization and function of these genes. The nucleotide sequence of the entire BCR and major parts of the ABL gene has now been determined. However, the actual cause of the fusion of BCR with ABL remains essentially unknown. Mouse models have been helpful to unravel the normal cellular function of BCR and ABL, as well the activity of BCR-ABL, although a single mechanism explaining the transforming activity of the latter has not been discovered. The cause of progression of the disease remains unknown, and no single genetic abnormality has been linked to the blast phase of CML. Much has been learned concerning the molecular biology of CML, but answers to the fundamental questions above may be expected in the coming years in parallel to increasing knowledge of genome structure, signal transduction and cell cycle control.

Animals↗
PubMed1996

A mouse model for the human lysosomal disease aspartylglycosaminuria.

Aspartylglycosaminuria (AGU), the most common disorder of glycoprotein degradation in humans, is caused by mutations in the gene encoding the lysosomal enzyme glycosylasparaginase (Aga). The resulting enzyme deficiency allows aspartylglucosamine (GlcNAc-Asn) and other glycoasparagines to accumulate in tissues and body fluids, from early fetal life onward. The clinical course is characterized by normal early development, slowly progressing to severe mental and motor retardation in early adulthood. The exact pathogenesis of AGU in humans is unknown and neither therapy nor an animal model for this debilitating and ultimately fatal disease exists. Through targeted disruption of the mouse Aga gene in embryonic stem cells, we generated mice that completely lack Aga activity. At the age of 5-10 months a massive accumulation of GlcNAc-Asn was detected along with lysosomal vacuolization, axonal swelling in the gracile nucleus and impaired neuromotor coordination. A significant number of older male mice had massively swollen bladders, which was not caused by obstruction, but most likely related to the impaired function of the nervous system. These findings are consistent with the pathogenesis of AGU and provide further data explaining the impaired neurological function in AGU patients.

Acetylglucosamine↗
PubMed1996

Inhibition of Bcr serine kinase by tyrosine phosphorylation.

The first exon of the BCR gene encodes a new serine/threonine protein kinase. Abnormal fusion of the BCR and ABL genes, resulting from the formation of the Philadelphia chromosome (Ph), is the hallmark of Ph-positive leukemia. We have previously demonstrated that the Bcr protein is tyrosine phosphorylated within first-exon sequences by the Bcr-Abl oncoprotein. Here we report that in addition to tyrose 177 (Y-177), Y-360 and Y283 are phosphorylated in Bcr-Abl proteins in vitro. Moreover, Bcr tyrosine 360 is phosphorylated in vivo within both Bcr-Abl and Bcr. Bcr mutant Y177F had a greatly reduced ability to transphosphorylate casein and histone H1, whereas Bcr mutants Y177F and Y283F had wild-type activities. In contrast, the Y360F mutation had little effect on Bcr's autophosphorylation activity. Tyrosine-phosphorylated Bcr, phosphorylated in vitro by Bcr-Abl, was greatly inhibited in its serine/threonine kinase activity, impairing both auto- and transkinase activities of Bcr. Similarly, the isolation of Bcr from cells expressing Bcr-Abl under conditions that preserve phosphotyrosine residues also reduced Bcr's kinase activity. These results indicate that tyrosine 360 of Bcr is critical for the transphosphorylation activity of Bcr and that in Ph-positive leukemia, Bcr serine/threonine kinase activity is seriously impaired.

Animals↗
PubMed1995

BCR/ABL P210 and P190 cause distinct leukemia in transgenic mice.

DNA constructs encoding BCR/ABL P210 have been introduced into the mouse germ line using microinjection of one-cell fertilized eggs. Kinetics of BCR/ABL P210 expression in transgenic mice were very similar to those of BCR/ABL P190 constructs in transgenic mice. mRNA transcripts were detectable early in embryonic development and also in hematopoietic tissue of adult animals. Expression of BCR/ABL in peripheral blood preceded development of overt disease. P210 founder and progeny transgenic animals, when becoming ill, developed leukemia of B, T-lymphoid, or myeloid origin after a relatively long latency period. In contrast, P190-transgenic mice exclusively developed leukemia of B-cell origin, with a relatively short period of latency. The observed dissimilarities are most likely due to intrinsically different properties of the P190 and P210 oncoproteins and may also involve sequences that control transgene expression. The delayed progression of BCR/ABL P210-associated disease in the transgenic mice is consistent with the apparent indolence of human chronic myeloid leukemia during the chronic phase. We conclude that, in transgenic models, comparable expression of BCR/ABL P210 and BCR/ABL P190 results in clinically distinct conditions.

Animals↗
PubMed1995

CRKL links p210BCR/ABL with paxillin in chronic myelogenous leukemia cells.

The Philadelphia chromosome translocation generates a chimeric oncogene, BCR/ABL, which causes chronic myelogenous leukemia (CML). In primary neutrophils from patients with CML, the major novel tyrosine-phosphorylated protein is CRKL, an SH2-SH3-SH3 linker protein which has an overall homology of 60% to CRK, the human homologue of the v-crk oncogene product. Anti-CRKL immunoprecipitates from CML cells, but not normal cells, were found to contain p210BCR/ABL and c-ABL. Several other phosphoproteins were also detected in anti-CRKL immunoprecipitates, one of which has been identified as paxillin, a 68-kDa focal adhesion protein which we have previously shown to be phosphorylated by p210BCR/ABL. Using GST-CRKL fusion proteins, the SH3 domains of CRKL were found to bind c-ABL and p210BCR/ABL, while the SH2 domain of CRKL bound to paxillin, suggesting that CRKL could physically link p210BCR/ABL to paxillin. Paxillin contains three tyrosines in Tyr-X-X-Pro (Y-X-X-P) motifs consistent with amino acid sequences predicted to be optimal for binding to the CRKL-SH2 domain (at positions Tyr-31, Tyr-118, and Tyr-181). Each of these tyrosine residues was mutated to a phenylalanine residue, and in vitro binding assays indicated that paxillin tyrosines at positions 31 and 118, but not 181, are likely to be involved in CRKL-SH2 binding. These results suggest that the p210BCR/ABL oncogene may be physically linked to the focal adhesion-associated protein paxillin in hematopoietic cells by CRKL. This interaction could contribute to the known adhesive defects of CML cells.

Adaptor Proteins, Signal Transducing↗