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Biomedical subjects

J M Bishop

Publications and source records attributed to J M Bishop.

At least 55 records · Page 3Linked to original sources
PubMed1997

Targeted expression of MYCN causes neuroblastoma in transgenic mice.

The proto-oncogene MYCN is often amplified in human neuroblastomas. The assumption that the amplification contributes to tumorigenesis has never been tested directly. We have created transgenic mice that overexpress MYCN in neuroectodermal cells and develop neuroblastoma. Analysis of tumors by comparative genomic hybridization revealed gains and losses of at least seven chromosomal regions, all of which are syntenic with comparable abnormalities detected in human neuroblastomas. In addition, we have shown that increases in MYCN dosage or deficiencies in either of the tumor suppressor genes NF1 or RB1 can augment tumorigenesis by the transgene. Our results provide direct evidence that MYCN can contribute to the genesis of neuroblastoma, suggest that the genetic events involved in the genesis of neuroblastoma can be tumorigenic in more than one chronological sequence, and offer a model for further study of the pathogenesis and therapy of neuroblastoma.

Abdomen↗
PubMed1997

A PMLRARalpha transgene initiates murine acute promyelocytic leukemia.

The malignant cells of acute promyelocytic leukemia (APL) contain a reciprocal chromosomal translocation that fuses the promyelocytic leukemia gene (PML) with the retinoic acid receptor alpha gene (RAR alpha). To test the hypothesis that the chimera PMLRAR alpha plays a role in leukemogenesis, we expressed a PMLRAR alpha cDNA in myeloid cells of transgenic mice. PMLRAR alpha transgenic mice exhibited impaired neutrophil maturation early in life, which progressed at a low frequency over the course of several months to overt APL. Both the preleukemic state and the leukemia could be transplanted to nontransgenic mice, and the transplanted preleukemia could progress to APL. The APL recapitulated features of the human disease, including a response to retinoic acid. Retinoic acid caused the leukemic cells to differentiate in vitro and in vivo, eliciting remissions of both the preleukemic state and APL in mice. Our results demonstrate that PMLRAR alpha impairs neutrophil differentiation and initiates the development of APL. The transgenic mice described here provide an apparently accurate model for human APL that includes clear evidence of tumor progression. The model should be useful for exploring the molecular pathogenesis of APL and the mechanisms of the therapeutic response to retinoic acid, as well as for preclinical studies of therapeutic regimens.

Animals↗
PubMed1997

The DFer gene of Drosophila melanogaster encodes two membrane-associated proteins that can both transform vertebrate cells.

The vertebrate gene FER encodes two protein-tyrosine kinases with molecular weights of 51,000 and 94,000 and distinctive aminotermini. The larger kinase is expressed ubiquitously among vertebrate tissues, whereas expression of the smaller kinase appears to be limited to spermatogenic cells in the testes. Here we show that Drosophila melanogaster contains an apparent ortholog of FER (DFer) that also produces two mRNAs by separate initiation of transcription, and two proteins with molecular weights of 45,000 and 92,000. Both proteins are in part loosely associated with cytoplasmic membranes. Both can transform avian and rodent cells with roughly equal potency, when expressed from retroviral vectors. Fusing the myristoylation signal from the SRC protein-tyrosine kinase to the aminoterminus of the DFer protein increased the strength of attachment to membranes but augmented transformation only marginally. The results provide the first demonstration of neoplastic transformation by a protein-tyrosine kinase of Drosophila and by FER from any species. The products of Drosophila and vertebrate FER may be part of similar signaling pathways in the two species.

3T3 Cells↗
PubMed1997

Neoplastic transformation by truncated alleles of human NOTCH1/TAN1 and NOTCH2.

The Notch genes of Drosophila melanogaster and vertebrates encode transmembrane receptors that help determine cell fate during development. Although ligands for Notch proteins have been identified, the signaling cascade downstream of the receptors remains poorly understood. In human acute lymphoblastic T-cell leukemia, a chromosomal translocation damages the NOTCH1 gene. The damage apparently gives rise to a constitutively activated version of NOTCH protein. Here we show that a truncated version of NOTCH1 protein resembling that found in the leukemic cells can transform rat kidney cells in vitro. The transformation required cooperation with the E1A oncogene of adenovirus. The transforming version of NOTCH protein was located in the nucleus. In contrast, neither wild-type NOTCH protein nor a form of the truncated protein permanently anchored to the plasma membrane produced transformation in vitro. We conclude that constitutive activation of NOTCH similar to that found in human leukemia can contribute to neoplastic transformation. Transformation may require that the NOTCH protein be translocated to the nucleus. These results sustain a current view of how Notch transduces a signal from the surface of the cell to the nucleus.

Adenovirus E1A Proteins↗
PubMed1996

myb provides an essential function during Drosophila development.

Vertebrate c-myb encodes a transcription factor thought to play an important role in the cell cycle. To gain further insight into myb function, we have been studying a related gene in Drosophila. We found that Drosophila myb is abundantly expressed throughout development in all mitotically active tissues, at lower levels in some postmitotic tissues, but not at detectable levels in polyploid larval tissues. We performed genetic screens to isolate recessive lethal mutations in the chromosomal region that includes the myb gene. We obtained two temperature-sensitive alleles of myb, demonstrating that the gene provides an essential function. Examination of the mutant phenotype revealed that Drosophila myb is important for both embryonic and imaginal development and that myb serves a role in the development of many tissues and during oogenesis.

Animals↗
PubMed1996

Cellular adherence elicits ligand-independent activation of the Met cell-surface receptor.

Cell adhesion has a fundamental role in the proliferation and motility of normal cells and the metastasis of tumor cells. To identify signaling pathways activated by the adherence of tumor cells, we analyzed the tyrosine phosphorylation of proteins in mouse melanoma cells before and after attachment to substrata. We discovered that cellular adherence activated the protein-tyrosine kinase of the cell surface receptor Met, whose ligand is hepatocyte growth factor and scatter factor. The activation was exceedingly prompt, affected the great majority of Met in the cells, persisted so long as the cells remained adherent, and was rapidly reversed as soon as the cells were detached from substrata. Activation of Met required that cells be adherent but not that they spread on the substratum, and it occurred in the absence of any apparent ligand for the receptor. Ligand-independent activation of Met occurred in several varieties of tumor cells but not in normal endothelial cells that express the receptor. The activation of Met described here may represent a means by which cells respond to mechanical as opposed to biochemical stimuli.

Animals↗
PubMed1996

Phosphorylation of the fused protein kinase in response to signaling from hedgehog.

The hedgehog gene (hh) of Drosophila melanogaster exerts both short- and long-range effects on cell patterning during development. The product of hedgehog is a secreted protein that apparently acts by triggering an intra-cellular signaling pathway, but little is known about the details of that pathway. The Drosophila gene fused (fu) encodes a serine/threonine-protein kinase that genetic experiments have implicated in signaling initiated by hedgehog. Here we report that the fused protein is phosphorylated during the course of Drosophila embryogenesis, as a result of hedgehog activity. In cell culture, phosphorylation of fused protein occurs in response to the biologically active form of hedgehog and cannot be blocked by activation of protein kinase A, which is thought to be an antagonist of signaling from hedgehog. These results suggest that fused and protein kinase A function downstream of hedgehog but in parallel pathways that eventually converge distal to fused. The reconstruction of signaling from hedgehog in cell culture should provide further access to the mechanisms by which hedgehog acts.

Animals↗
PubMed1995

Beta PDGF receptor mutants defective for mitogenesis promote neurite outgrowth in PC12 cells.

BACKGROUND: Platelet-derived growth factor (PDGF) promotes mitogenesis in fibroblast cell lines but stimulates neurite outgrowth in PC12 cells that ectopically express the beta PDGF receptor. To determine which substrates must associate with this receptor protein-tyrosine kinase in order to promote neurite outgrowth, we introduced into PC12 pheochromocytoma cells three mutant forms of the beta PDGF receptor that no longer associate with specific substrate proteins. We then assayed the ability of these receptor mutants to affect neurite extension. RESULTS: Receptors lacking the kinase-insert domain did not associate with either phosphatidylinositol 3-kinase (PI 3-kinase) or Ras GTPase-activating protein (Ras-GAP) in PC12 cells. A carboxy-terminal truncation of the beta PDGF receptor eliminated the association of phospholipase C-gamma 1 (PLC-gamma 1) with the receptor and prevented phosphorylation of PLC-gamma 1 in PC12 cells. Finally, beta PDGF receptors that have tyrosine-to-phenylalanine point mutations at positions 708, 719, 977 and 989 did not associate with either PI 3-kinase or PLC-gamma 1. All three mutant forms of the beta PDGF receptor promoted PDGF-dependent neurite outgrowth in PC12 cells and elicited activation of mitogen-activated protein (MAP) kinases. CONCLUSIONS: PC12 cells expressing the beta PDGF receptor extend neurites in response to PDGF in the absence of signalling through PI 3-kinase, RasGAP, and PLC-gamma 1. This contrasts with the requirements for mitogenesis for epithelial and fibroblast cell lines, in which the association of PI 3-kinase with the beta PDGF receptor is essential. This receptor protein-tyrosine kinase therefore phosphorylates and activates a similar set of intracellular signalling molecules in the context of both mitogenesis and differentiation, but the importance of particular pathways for each phenotypic response is distinct.

Animals↗
PubMed1995

Myristoylation and differential palmitoylation of the HCK protein-tyrosine kinases govern their attachment to membranes and association with caveolae.

The human proto-oncogene HCK encodes two versions of a protein-tyrosine kinase, with molecular weights of 59,000 (p59hck) and 61,000 (p61hck). The two proteins arise from a single mRNA by alternative initiations of translation. In this study, we explored the functions of these proteins by determining their locations within cells and by characterizing lipid modifications required for the proteins to reach those locations. We found that p59hck is entirely associated with cellular membranes, including the organelles known as caveolae; in contrast, only a portion of p61hck is situated on membranes, and none is detectable in preparations of caveolae. These distinctions can be attributed to differential modification of the two HCK proteins with fatty acids. Both proteins are at least in part myristoylated, p59hck more so than p61hck. In addition, however, p59hck is palmitoylated on cysteine 3 in the protein. Palmitoylation of the protein requires prior myristoylation and, in turn, is required for targeting to caveolae. These findings are in accord with recent reports for other members of the SRC family of protein-tyrosine kinases. Taken together, the results suggest that HCK and several of its relatives may participate in the functions of caveolae, which apparently include the transduction of signals across the plasma membrane to the interior of the cell.

Acylation↗
PubMed1994

Genome-wide search for loss of heterozygosity in transgenic mouse tumors reveals candidate tumor suppressor genes on chromosomes 9 and 16.

A genome-wide scan for loss of heterozygosity (LOH) in tumors provides a powerful route to the identification of genes involved in tumorigenesis. This approach has not previously been applied to transgenic mice, despite the considerable advantages they afford for genetic dissection. Here, we report a genome-wide LOH analysis of insulinomas and carcinoid tumors in transgenic mice expressing the simian virus 40 large tumor oncogene. Although the overall genome-wide rate of LOH was quite low, chromosomes 9 and 16 showed high rates of allelic loss. About one-third of tumors showed partial LOH, allowing localization of the likely tumor suppressor genes to intervals of approximately 11 centimorgans. The locus on chromosome 9, named Loh-1, lies in a region with synteny conservation to human chromosomes 3q, 6q12, 15q24, and 3p21, while the locus on chromosome 16, named Loh-2, lies in a region corresponding to human chromosomes 3q and 22q. Of particular note is the synteny conservation with human 3p21, which shows frequent loss in human cancers. These regions do not encode two tumor suppressors, pRB and p53, known to interact with large tumor oncoprotein, suggesting the presence of new genes whose loss of function contributes to multistage tumorigenesis.

Alleles↗
PubMed1993

Association of pRas and pRaf-1 in a complex correlates with activation of a signal transduction pathway.

BACKGROUND: A key pathway for transduction of proliferative, developmental and oncogenic stimuli from receptors at the cell surface to transcription factors located in the nucleus involves the activation of pRas and pRaf-1. Recent publications have described a physical interaction between pRas and pRaf-1, either as ectopic proteins in yeast or as recombinant proteins added to cellular extracts. Until now, however, physical complexes that include pRas and pRaf-1 have not been identified as native structures in mammalian cells. RESULTS: We have directly identified a pRas-pRaf-1 complex in extracts of mammalian cells. Formation of the complex is augmented in neoplastically transformed cells expressing constitutively activated pRas. Moreover, the complexes form in concert with the activation of pRas during intracellular signalling through the T-cell receptor in T-leukemia cells. CONCLUSIONS: We propose that, pRas signals to pRaf-1 in vivo by means of a direct physical interaction that results in activation of the pRaf-1 protein kinase.

Journal Article↗
PubMed1993

Macrophages are required for cell death and tissue remodeling in the developing mouse eye.

To identify and characterize tissue remodeling processes mediated by macrophages, we have generated transgenic mice in which diphtheria toxin is expressed from a macrophage-specific transgene. Expression of the transgene disrupts subsets of mature macrophages in both the eye and the peritoneal cavity and results in the persistence of two normally transient ocular tissues, the hyaloid vasculature and the pupillary membrane. Furthermore, the cells comprising the pupillary membrane appear alive up to 14 days after the structure is normally remodeled, suggesting that the macrophage actively elicits target cell death. Thus, these transgenic mice provide direct evidence for the active involvement of macrophages in developmentally programmed tissue remodeling and identify the hyaloid vessels and the pupillary membrane in the eye as targets of macrophage-mediated remodeling.

Aging↗