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J Groffen

Publications and source records attributed to J Groffen.

At least 73 records · Page 4Linked to original sources
PubMed1994

Negative cooperativity in the insulin-like growth factor-I receptor and a chimeric IGF-I/insulin receptor.

Insulin and insulin-like growth factor-I (IGF-I) share a spectrum of metabolic and growth-promoting effects, mediated through homologous receptors that belong to the tyrosine kinase family. The dissociation rate of insulin from its receptor is affected by negative cooperativity, i.e. accelerates with increased receptor occupancy. The dose-response curve for the acceleration of tracer dissociation by unlabeled insulin has a distinct bell-shaped curve, with a progressive slowing down at insulin concentrations greater than 100 nM. The kinetics of the IGF-I interaction with its receptor has not been studied in such detail. In the present work, we report that while the IGF-I receptor exhibits negative cooperativity like the insulin receptor, the concentration dependence of the dissociation kinetics is distinct from that of native human insulin by not being bell-shaped, but monophasic like that of insulin analogues mutated at the hexamer-forming surface; it is changed to an insulin-type curve by substitution of IGF-I receptor's sequence including residues 382-565 with the homologous insulin receptor domain. The data suggest that like insulin, IGF-I has a bivalent binding mode and crosslinks two distinct areas of the two alpha subunits that are close, but distinct from the equivalent insulin receptor binding sites.

Arteries↗
PubMed1993

Human ABR encodes a protein with GAPrac activity and homology to the DBL nucleotide exchange factor domain.

We have previously cloned a segment of a gene, ABR, homologous to the BCR gene, which encodes a protein consisting of three distinct functional domains. In the present study, genomic ABR sequences were used to isolate human ABR cDNAs. Surprisingly, the two types of ABR cDNAs identified differed only in their most 5' coding sequences. These are predicted to encode proteins of 93.5 and 92.3 kDa molecular mass. ABR showed a differential expression pattern in various mouse tissues, analogous to that of BCR, and the highest level was found in brain. Similar to BCR, ABR contains a region with homology to DBL, vav, and CDC24, which are likely to or have been shown to encode GTP exchange factors. A domain of ABR with similarity to GAPrho was expressed as a fusion protein in Escherichia coli and was shown to have GAP activity toward rac. Although both ABR and BCR have GAP activity, ABR lacks homology to the serine/threonine kinase domain of BCR. Therefore, ABR is likely to have cellular functions overlapping with but also distinct from those of BCR.

Amino Acid Sequence↗
PubMed1993

Tyrosine phosphorylation of P160 BCR by P210 BCR-ABL.

It is well established that the chimeric BCR-ABL gene formed by joining parts of the BCR and ABL genes plays a key role in the pathogenesis of Philadelphia (Ph) chromosome-positive leukemias. We report that simultaneous expression of P210 BCR-ABL and P160 BCR in simian COS-1 cells yielded stable complexes of these two proteins, and induced phosphorylation of P160 BCR on tyrosine residues in vivo. Tyrosine phosphorylation of a deletion mutant encoding 553 amino acids of BCR N-terminal sequences was also detected when it was coexpressed with P210 BCR-ABL. We propose that tyrosine phosphorylation of P160 BCR by P210 BCR-ABL and their stable physical interaction may perturb normal BCR functions and that these alterations are directly involved in the pathologic processes found in Ph chromosome-associated leukemias.

Amino Acid Sequence↗
PubMed1993

Comparison of baculovirus-expressed c-Abl and BCR/ABL protein tyrosine kinases.

Mouse c-Abl type IV and human BCR/ABL proteins have been expressed in insect cells using the baculovirus system. The proteins were expressed as full-length polypeptides as judged by electrophoresis in denaturing gels. They were identified by immunoprecipitation and immunoblotting with antibodies against ABL peptides and, for BCR/ABL, against a BCR peptide. In these immunoprecipitates both proteins gave autophosphorylation principally on tyrosine. Both proteins were active tyrosine kinases, phosphorylating a variety of tyrosine-containing substrates. In fresh extracts both proteins contained phosphotyrosine as shown by Western blots with antiphosphotyrosine antibodies. Partial purification could be achieved readily using ion exchange columns, and the BCR/ABL protein, p210BCR/ABL, could be further purified to near-homogeneity using an antiphosphotyrosine column. Both enzymes required a divalent metal ion for activity. At low concentrations of ATP (2 microM) and with angiotensin II as substrate both enzymes were activated by Mn2+ or by Mg2+. No major differences in catalytic properties were found between the two isolated enzymes in solution. The oncogenic properties of p210BCR/ABL may be due to its different subcellular location, or to the presence of an intracellular inhibitor of c-Abl that does not inhibit BCR/ABL, or to altered substrate-specificity such that it can phosphorylate a unique substrate which is not recognised by c-Abl.

Amino Acid Sequence↗
PubMed1993

Localization of a gamma-glutamyl-transferase-related gene family on chromosome 22.

A gene family encompassing a minimum of four genes or pseudogenes for gamma-glutamyl transferase (GGT; EC 2.3.2.2) is present on chromosome 22q11. We have previously isolated a cDNA related to GGT but clearly not belonging to its gene family. The chromosomal location of this related gene, GGTLA1, has been determined by both isotopic and fluorescence in situ hybridization to metaphase cells and by Southern blot analysis of somatic cell hybrid DNAs. We show that GGTLA1 is part of a distinct gene family, which has at least four members (GGTLA1, GGTLA2, GGTLA3, GGTLA4). At least two loci are located on chromosome 22 within band q11 and proximal to the chronic myelogenous leukemia (CML) breakpoint in BCR (breakpoint cluster region gene). At least one other member is located more distally between the breakpoints found in Ewings sarcoma and CML. Some of the GGT and GGTLA family members are located on NotI restriction enzyme fragments of a similar size. Combined results indicate that a segment of human chromosome 22q11 has undergone large-scale amplification events relatively recently in evolution.

Blotting, Southern↗
PubMed1993

Isolation of NotI sites from chromosome 22q11.

Chromosome 22q11 contains a large number of interesting loci, including genes associated with cancer and developmental defects. The region is also the site of the lambda immunoglobulin variable and constant regions and the BCR, gamma-glutamyl transpeptidase, and GGT-like activity multigene families. Because of the complexities associated with mapping highly related gene families, we have examined the utility of mapping large areas of DNA using a defined approach. A total of 21 complete NotI sites from band q11 were cloned and ordered into six noncontiguous clusters of sites using a combination of somatic cell hybrid panels, NotI jumping and linking libraries, and fluorescence in situ hybridization. The largest cluster spanned an estimated 2 Mb of NotI fragments, the smallest 115 kb. Approximately 3.5 Mb of band q11 could be examined for rearrangements in NotI restriction enzyme fragments. A number of conserved sequences, two genes, and a minimum of two families of related sequences were identified adjacent to NotI sites.

Animals↗
PubMed1993

Ph-positive leukemia: a transgenic mouse model.

The presence of the BCR/ABL chimeric gene is the hallmark of defined types of human leukemia. To increase our knowledge of the oncogenic processes and to develop a model for this type of leukemia we generated a BCR/ABL (P190) transgenic mouse line. Over 95% of mice of this line die of leukemia or leukemia/lymphoma within 35-200 days of age. Karyotypically visible genetic alterations were absent from the early stages of BCR/ABL generated leukemia. A high frequency of aneuploidy was found in advanced leukemia indicating a primary and pivotal role for BCR/ABL in leukemogenesis. Moreover, the data suggest that BCR/ABL has a destabilizing effect on the regulation of the cell cycle. BCR/ABL expression was also found in tissues other than hematopoietic cells. However, this did not result in the development of solid tumors, strongly suggesting that the oncogenicity of BCR/ABL is limited to the hematopoietic lineage.

Animals↗
PubMed1993

Isolation and chromosomal localization of CRKL, a human crk-like gene.

We have identified and partially characterized a gene located on chromosome 22, band q11, centromeric of the chronic myelogenous leukemia breakpoint region. A number of overlapping cDNAs were isolated from this locus and the largest of 1.8 kb was sequenced. Its deduced amino acid sequence shows homology to the SH2 domains of protein tyrosine kinases such as FER, and is strikingly similar to the cellular part of the v-crk oncogene product. We identified one SH2 and two SH3 domains within the 303 amino acid open reading frame of this crk-like gene, CRKL. The CRKL gene product is predicted to have a molecular mass of 36 kDa. In addition, we demonstrate that this gene does not represent the human homolog of v-crk but rather a novel gene potentially capable of mediating the transduction of intracellular signals.

Adaptor Proteins, Signal Transducing↗
PubMed1993

CRK proto-oncogene maps to human chromosome band 17p13.

A genomic DNA fragment, isolated from a human phage library using a chicken crk cDNA probe, was shown to derive from the human CRK locus. We have used fluorescent in situ hybridization (FISH) to map CRK distal in chromosome band 17p13, a region which demonstrates frequent deletion or loss of heterozygosity in a wide range of human cancers.

Amino Acid Sequence↗
PubMed1992

Restricted oncogenicity of BCR/ABL p190 in transgenic mice.

A chimeric BCR/ABL oncogene encoding the p190 protein has been introduced into the mouse germline using microinjection of one-cell fertilized eggs. Founder and progeny transgenic animals, when becoming ill, were found to develop lymphoblastic leukemia/lymphoma which was transplantable to compatible recipients. Lymphoblasts were arrested at the pre-B stage of development. Expression of BCR/ABL was not detected in peripheral blood during the early stages of leukemia but became evident as the disease progressed. However, the transgene was expressed early in development in bone marrow and was also transcribed in nonhematopoietic tissues although this did not result in tumorigenesis. These results strongly suggest that the oncogenicity of BCR/ABL is limited to hematopoietic cells, including pre-B cells or their progenitors.

Animals↗
PubMed1992

Clonal development and karyotype evolution during leukemogenesis of BCR/ABL transgenic mice.

The Philadelphia (Ph) translocation is responsible for the generation of the chimeric BCR/ABL oncogene. The Ph chromosome constitutes the earliest detectable chromosome abnormality in chronic myelogenous leukemia and is also found in acute lymphoblastic leukemia. Mice transgenic for a P190 BCR/ABL-producing DNA construct develop lymphoblastic leukemia/lymphoma and provide an opportunity to study early stages of the disease as well as progression. In this study, we have karyotyped the bone marrow of 10 19-day-old BCR/ABL P190 transgenic mice from a line that reproducibly develops leukemia/lymphoma. Leukemic cells from 17 terminally ill transgenic founders and progeny were also karyotyped as well as bone marrow transplant recipients of leukemic donor marrow. Karyotypically visible aberrations were absent from the early stages of BCR/ABL P190-generated leukemia and normal metaphases could be found even in the terminal stages of the disease. A high frequency of aneuploidy was found in advanced leukemia, with a marked preference for the gain of mouse chromosomes 12, 14, or 17. These results point to a primary role for BCR/ABL in leukemogenesis and suggest a destabilizing effect of the BCR/ABL gene on the regulation of cell division.

Aneuploidy↗
PubMed1992

Aspartylglycosaminuria in a non-Finnish patient caused by a donor splice mutation in the glycoasparaginase gene.

Aspartylglycosaminuria is a lysosomal storage disease caused by deficient activity of glycoasparaginase (EC 3.5.1.26), and it occurs with a high frequency among Finns. We have recently shown that the molecular defect in all Finnish aspartylglycosaminuria patients examined to date consists of two single base changes in the heavy chain of glycoasparaginase (Mononen, I., Heisterkamp, N., Kaartinen, V., Williams, J. C., Yates, J. R., III, Griffin, P. R., Hood, L. E., and Groffen, J. (1991) Proc. Natl. Acad. Sci U.S.A. 88, 2941-2945). This is the first report on the identification of the molecular defect causing aspartylglycosaminuria in a patient of non-Finnish origin. Total RNA from fibroblasts of a black American aspartylglycosaminuria patient was isolated, first-strand cDNA was synthesized, and the cDNA encoding glycoasparaginase was amplified by the polymerase chain reaction. The patient's mRNA nucleotide sequence was different from the normal sequence by a deletion of 134 nucleotides at positions 807-940. Nucleotide sequence analysis of the normal glycoasparaginase gene demonstrated that the deletion corresponded precisely to a 134-base pair exon. Moreover, analysis of the splice sites demonstrated a single base change, G to T, that altered the donor splice site of the exon deleted in the patient's mRNA. This change led to an exon-skipping event resulting in a frame shift and generation of a stop codon.

Amino Acid Sequence↗
PubMed1992

Chromosomal localization of the human glycoasparaginase gene to 4q32-q33.

Glycoasparaginase cleaves the N-glycosidic linkage between asparagine and N-acetylglucosamine in the degradation of glycoproteins. In humans, a deficient activity of glycoasparaginase results in accumulation of glycoasparagines, causing the lysosomal storage disease aspartylglycosaminuria. Recombinant plasmid containing the cDNA insert encoding human glycoasparaginase was used to localize the enzyme to chromosome 4q32-q33 by in situ hybridization to metaphase chromosomes prepared from normal human lymphocytes.

Aspartylglucosylaminase↗
PubMed1992

Transfection with gamma-glutamyl transpeptidase enhances recovery from glutathione depletion using extracellular glutathione.

Glutathione (L-gamma-glutamyl-L-cysteinylglycine) is an important constituent of the antioxidant and detoxifying mechanisms of cells. The plasma membrane bound enzyme, gamma-glutamyl transpeptidase (GGT), catalyzes the first step in the degradation of extracellular glutathione, the components of which are then used for de novo glutathione synthesis. We tested the hypothesis that an increase in GGT activity would enhance the utilization of extracellular glutathione by cells challenged with a glutathione-depleting agent. A eukaryotic system stably overexpressing GGT (nearly 200-fold) was developed by transfection of NIH-3T3 fibroblasts with a human placental GGT cDNA. These cells and controls were incubated for 30 min with 1 mM diethyl maleate, which caused approximately 80% intracellular glutathione depletion. Glutathione was added to the medium and cells were allowed to resynthesize intracellular glutathione. The transfected cells used extracellular glutathione much more efficiently than controls in terms of both the concentration dependence and the rate of glutathione resynthesis. Serine-borate, a competitive inhibitor of GGT, blocked the restoration of intracellular glutathione. The results support the hypothesis that the increase in GGT activity that occurs in some toxicologic or pathologic conditions could provide protection against glutathione depletion.

Animals↗
PubMed1992

Clonal characteristics of acute lymphoblastic cells derived from BCR/ABL p190 transgenic mice.

The clonal and immunophenotypic characteristics of blood leukemic cells from BCR/ABL p190 transgenic mice were investigated. All cell populations evaluated in vivo and in vitro had B-lymphocyte progenitor immunophenotypes. Immunoglobulin (JH) rearrangement patterns provided evidence for clonal diversification at different sites in vivo. Multiple clones were established in vitro from two of these mice (nos. 730 and 753). These cells expressed BCR/ABL p190 protein tyrosine kinase (PTK) and were highly malignant on transfer to secondary recipients. Cells independently cloned in vitro shared identical immunophenotypes and clonal IgH rearrangements, but these were distinct from those of the dominant clones in the mouse from which they were derived. Nevertheless, in vitro clones from mouse no. 753 had an abnormal karyotype (chromosome 14 trisomy) in common with the dominant clone in blood, providing evidence for a hierarchy or clonal selection in vivo and in vitro. Two sets of in vitro clones proliferated independently of exogenous growth factors and stroma and released autocrine interleukin 7 growth factor activity. These data provide evidence for rapid divergent clonal evolution and selection of B-cell progenitors initiated by BCR/ABL p190, followed by other, secondary genetic events mirroring similar changes in the equivalent, highly malignant human leukemia Philadelphia (Ph)-positive/B-precursor acute lymphoblastic leukemia (ALL).

Animals↗
PubMed1991

Entire ABL gene is joined with 5'-BCR in some patients with Philadelphia-positive leukemia.

In four patients, the chromosome 9 breakpoint of the t(9; 22)(q34;q11) had occurred at different sites within an 8.25-kilobase (kb) region situated 5' of ABL exon 1B. Chromosome in situ hybridization and field inversion gel electrophoresis (FIGE) studies showed that ABL exons 1A and 1B were present on the Ph chromosome. Yet this large fusion gene produced an mRNA conventional for chronic myelogenous leukemia (CML). Splicing from BCR exon 3 to ABL exon 2 crossed more than 200 kb and deleted exons 1A and 1B. This breakpoint site may occur in about 10% of all CML patients. Three of our patients have pronounced thrombocytosis, and two had been diagnosed as having Ph-positive essential thrombocythemia. The platelet count of the other patient was not available.

Blotting, Southern↗