Showing papers on "Cyclin-dependent kinase 8 published in 1999"

Notch signaling imposes two distinct blocks in the differentiation of c2c12 myoblasts

Abstract: Notch signal transduction regulates expression of downstream genes through the activation of the DNA-binding protein Su(H)/CBF1. In Drosophila most of Notch signaling requires Su(H); however, some Notch-dependent processes occur in the absence of Su(H) suggesting that Notch signaling does not always involve activation of this factor. Using constitutively active forms of Notch lacking CBF1-interacting sequences we identified a Notch signaling pathway that inhibits myogenic differentiation of C2C12 myoblasts in the absence of CBF1 activation. Here we show that ligand-induced Notch signaling suppresses myogenesis in C2C12 myoblasts that express a dominant negative form of CBF1, providing additional evidence for CBF1-independent Notch signal transduction. Surprisingly mutant forms of Notch deficient in CBF1 activation are unable to antagonize MyoD activity, despite the fact that they inhibit myogenesis. Moreover, Notch-induced antagonism of MyoD requires CBF1 suggesting that the CBF1-dependent pathway mediates a cell-type-specific block in the myogenic program. However, Notch signaling in the absence of CBF1 activation blocks both myogenesis and osteogenesis, indicative of a general block in cellular differentiation. Taken together our data provide evidence for two distinct Notch signaling pathways that function to block differentiation at separate steps during the process of myogenesis in C2C12 myoblasts.

Human ligands of the Notch receptor.

Abstract: During development, the Notch signaling pathway is essential for the appropriate differentiation of many cell types in organisms across the phylogenetic scale, including humans. Notch signaling is also implicated in human diseases, including a leukemia and two hereditary syndromes known as Alagille and CADASIL. To generate tools for pursuing the role of the Notch pathway in human disease and development, we have cloned and analyzed the expression of three human homologues of the Notch ligands Delta and Serrate, human Jagged1 (HJ1), human Jagged2 (HJ2), and human Delta1 (H-Delta-1), and determined their chromosomal localizations. We have also raised antibodies to HJ1, and used these antibodies in conjunction with in situ hybridization to examine the expression of these ligands in normal and cancerous cervical tissue. We find that, as reported previously for Notch, the ligands are up-regulated in certain neoplastic tissues. This observation is consistent with the notion that Notch signaling is an important element in these pathogenic conditions, raising the possibility that modulation of Notch activity could be used to influence the fate of the cells and offering a conceivable therapeutic avenue.

Rel/NF-κB can trigger the Notch signaling pathway by inducing the expression of Jagged1, a ligand for Notch receptors

Abstract: Jagged1 belongs to the DSL family of ligands for Notch receptors that control the proliferation and differentiation of various cell lineages. However, little is known about the transcription factors that regulate its expression. Here, we show that Jagged1 is a Rel/NF-kappaB-responsive gene. Both c-Rel and RelA induced jagged1 gene expression, whereas a mutant defective for transactivation did not. Importantly, jagged1 transcripts were also upregulated by endogenous NF-kappaB activation and this effect was inhibited by a dominant mutant of IkappaBalpha, a physiological inhibitor of NF-kappaB. Cell surface expression of Jagged1 in c-Rel-expressing cell monolayers led to a functional interaction with lymphocytes expressing the Notch1/TAN-1 receptor. This correlated with the initiation of signaling downstream of Notch, as evidenced by increased levels of HES-1 transcripts in co-cultivated T cells and of CD23 transcripts in co-cultivated B cells. Consistent with its Rel/NF-kappaB-dependent induction, Jagged1 was found to be highly expressed in splenic B cells where c-Rel is expressed constitutively. These results demonstrate that c-Rel can trigger the Notch signaling pathway in neighboring cells by inducing jagged1 gene expression, and suggest a role for Jagged1 in B-cell activation, differentiation or function. These findings also highlight the potential for an interplay between the Notch and NF-kappaB signaling pathways in the immune system.

The Notch 3 intracellular domain represses Notch 1-mediated activation through Hairy/Enhancer of split (HES) promoters

Abstract: The Notch signaling pathway is important for cellular differentiation. The current view is that the Notch receptor is cleaved intracellularly upon ligand activation. The intracellular Notch domain then translocates to the nucleus, binds to Suppressor of Hairless (RBP-Jk in mammals), and acts as a transactivator of Enhancer of Split (HES in mammals) gene expression. In this report we show that the Notch 3 intracellular domain (IC), in contrast to all other analysed Notch ICs, is a poor activator, and in fact acts as a repressor by blocking the ability of the Notch 1 IC to activate expression through the HES-1 and HES-5 promoters. We present a model in which Notch 3 IC interferes with Notch 1 IC-mediated activation at two levels. First, Notch 3 IC competes with Notch 1 IC for access to RBP-Jk and does not activate transcription when positioned close to a promoter. Second, Notch 3 IC appears to compete with Notch 1 IC for a common coactivator present in limiting amounts. In conclusion, this is the first example of a Notch IC that functions as a repressor in Enhancer of Split/HES upregulation, and shows that mammalian Notch receptors have acquired distinct functions during evolution.

BAP1, a candidate tumor suppressor protein that interacts with BRCA1.

Abstract: Attempts to identify a biochemical function of the Breast/Ovarian Cancer Susceptibility Gene, BRCA1, have focused on identifying its protein partners. Recent studies have yielded a novel RING finger/BRCT-domain–containing protein, BARD1, of unknown function,1 the hRAD51 protein, which is involved in DNA recombination/repair,2 DNA Polymerase II,3 and the second Breast Cancer Susceptibility Gene, BRCA2.4 We have identified a novel protein, BAP1, that binds to the wild-type BRCA1 RING finger domain but not to mutated RING fingers (i.e., those found in breast tumors from women with heritable breast cancer) or other closely related RING fingers.5 BAP1 is a novel, nuclear-localized enzyme that displays the signature motifs and has the activity of a ubiquitin carboxy-terminal hydrolase. BAP1 is a 90-kD protein (729 a.a.) that binds to BRCA1 in vitro and in vivo, cleaves ubiquitin from a model substrate (similar to other members of this family), and enhances the growth-suppressive properties of BRCA1.5 Our data suggest that the BAP1 carboxy-terminus is tethered to the BRCA1 RING finger domain, leaving the UCH catalytic domain free to interact with ubiquitinated (or ubiquitin-like) substrates. The human BAP1 locus was mapped to chromosome 3p21.3, a region of the genome that is routinely deleted or rearranged in many cancers. Indeed, we have found rearrangements, deletions, and missense mutations of BAP1 in small cell and non-small cell lung cancer cell lines and, more recently, in breast cancer tumor samples, suggesting that BAP1 may be a tumor suppressor gene.

Human mammary epithelial cells rapidly exchange empty EGFR between surface and intracellular pools.

Abstract: Binding of ligand to the epidermal growth factor receptor (EGFR) initiates a series of processes including activation of the intrinsic EGFR tyrosine kinase, receptor autophosphorylation, and the assembly of active signaling complexes at the plasma membrane. Concomitantly, receptor trafficking is initiated, and the receptor is ultimately delivered to the lysosome, where it is degraded. Virtually all studies on EGFR trafficking have used fibroblasts and transformed cells. Because EGFR exerts a potent effect on the physiology of epithelial cells, we examined the regulation of EGFR activity and trafficking in nontransformed human mammary epithelial cells (HMEC). We found that HMEC that displayed a luminal phenotype were largely unresponsive to EGF and maintained a majority of their EGFR at the cell surface. In contrast, HMEC with a basal phenotype were highly responsive to EGF and, at steady state in the absence of exogenous ligand, distributed empty EGFR into intracellular pools. Maintenance of the intracellular pools was a direct consequence of specific and rapid endocytosis of the empty EGFR. The trafficking pattern was EGFR specific, used coated pits, and did not require receptor tyrosine kinase activity. Such an mechanism redistributes EGFR signaling potential among different membrane domains and into vesicles with unique biochemical microenviroments. In addition, our data show that EGFR endocytosis can be regulated in the absence of ligand binding and receptor activation in a cell-type-specific manner. J. Cell. Physiol. 180:448–460, 1999. © 1999 Wiley-Liss, Inc.

The v-erbA oncogene (review).

Abstract: The v-erbA oncogene product is a nuclear protein and belongs to the superfamily of nuclear hormone receptors. The v-ErbA oncoprotein is involved in neoplastic transformation leading to acute erythroleukemia and sarcomas. The cellular homolog of v-ErbA oncoprotein is the thyroid hormone receptor alpha (c-erbA alpha or TRalpha). While TR has the dual role to silence gene expression in the absence of hormone and activate genes in the presence of the ligand, triiodothyronine, the v-ErbA oncoprotein has lost the ability to activate genes. The oncoprotein is thought to repress, in a constitutive manner, a certain set of genes which prevent cellular transformation. The mechanism of gene silencing is partly understood and involves the so-called corepressors. Several types of corepressors have been identified so far. Similarly, gene silencing by corepressors also plays a role in myeloid transformation by the retinoic acid receptor (RAR) which is involved in translocations, such as PML-RAR. The v-erbA oncogene was isolated from a retrovirus which contains, in addition to v-erbA, the oncogene v-erbB. The viral erbB gene encodes an EGF-receptor derivative, which is a constitutively active tyrosine kinase. Cellular transformation is enhanced when both oncoproteins are expressed. However, the mechanisms of cellular transformation by v-ErbA alone or in synergy with v-ErbB remain unclear. Novel insights into the mechanism of cellular transformation by v-ErbA, the role of corepressors and the role of the cross talk between the EGF-receptor and v-ErbA will be discussed.

Human Metalloproteinase‐1 (Collagenase‐1) Is a Tumor Suppressor Protein p53 Target Gene

Abstract: Matrix metalloproteinases (MMPs) are a family of secreted or transmembrane proteins that can degrade all the proteins of the extracellular matrix and have been implicated in many abnormal physiological conditions, including arthritis and cancer metastasis. 1,2 Rheumatoid arthritis (RA) is marked by the destruction of the extracellular matrix and the degradation of native collagen, which are initiated mainly by the action of one type of metalloproteinase, the collagenases: collagenase-1 (MMP-1), neutrophil collagenase (MMP-8), and a recently discovered one, collagenase-3 (MMP-13) 1,3 The p53 tumor suppressor gene has been implicated in the malignant progression of cancers, and the mutational inactivation of p53 is the most frequent genetic alteration in human cancers. Moreover, recent studies have linked this powerful tumor suppressor to RA. 4–6 It was demonstrated that p53 protein was overexpressed in RA synovium, 4 and that mutant p53 transcripts are present. 5,6 Because p53 protein can function directly as a transcriptional regulator, we speculated that the overexpression of MMP-1 seen in cancer or RA might be associated with the inactivation of p53. We tested this hypothesis directly by cotransfecting Saos-2 cells with a fulllength hMMP1 promoter/luciferase construct 7 (hMMP-1/luci) plus wild-type (wt) and mutant p53 expression plasmids. Transcription assays revealed that wt-p53 downregulated the promoter activity of hMMP-1 in a dose-dependent fashion (F IG . 1, top). Clearly, if this repression activity was crucial to the tumor suppressor activity of wt-p53, naturally occurring p53 mutants might be defective in this function. As expected, most of tumor-derived p53 mutants examined lost this repression activity (F IG . 1, top). Northern blot assays give similar results (F IG . 1, bottom). Two lines of evidence suggest that the repression of hMMP-1 by wt-p53 is a physiologically relevant response. First of all, serum-stimulated expression of endogenous hMMP1 messenger was completely repressed by wt-p53, but not mutant p53 (F IG . 1, bottom). Second, hMMP-1/luci reporter activities were stimulated by etoposide only

P15—A new tumor suppressor gene

Abstract: In addition to the tumor suppressor genes such asRb andp53, it has been found that some molecules of the same class named CKI (cyclin-dependent kinase inhibitor) also play an important role in the inhibition of tumorigenesis and the tumor progression. In the KIP and INK4 families of CKls,p15 shares extensive homology withp16. Findings in many tumors and their cell lines show that the inactivation ofp15 (deletion, mutation, rearrangement, etc.) is very frequent, and inactivep15 is involved in the progress of some tumors. These studies provide evidence that thep15 is a new tumor suppressor gene. Furthermore, the research on the molecular mechanism ofp15 in regulation of cell proliferation shows thatp15 can inhibit the growth of some kinds of tumor cells, andp15 is the mediator of TGF-β-induced cell arrest. Investigations onp15 in cell differentiation suggest that increasedp15 is related to the change of malignant phenotype. These results supply clues for further interpretation about the molecular mechnism of cell cycle control and cell tumorigenesis. And they may provide theoretical and experimental basis for application ofp15 to clinical therapy of tumors.

DNA sequence encoding a tumor suppressor gene

Abstract: The invention provides novel tumor suppressor genes, methods for making and using these and related tumor suppressor genes and proteins and peptides, and nucleic acids encoding these and related tumor suppressor proteins and peptides.

PPP2R1B is a tumor suppressor

Abstract: The present invention identifies the PPP2R1B gene, as a human tumor suppressor gene. Sequencing of the PPP2R1B revealed that the gene is located on human chromosome 11q22-24 and that gene were mutated in tumors and tumor cell lines, leading to the classification of this gene as a tumor suppressor. Further analyses have demonstrated the presence of a number of mutations in the gene in lung, colon, breast and cervical cancer cells. Methods for diagnosing and treating cancers related to this tumor suppressor also are disclosed.