Oncogene database

4855

NOTCH4

notch 4;NOTCH4;notch 4

Notch homolog 4|neurogenic locus notch homolog protein 4

6p21.3

protein-coding

The protein encoded by the Notch4 gene is a member of the Notch/lin-12 family of transmembrane receptor proteins, which have been shown to control cell fate determination and cell differentiation in a wide variety of organisms. expression of Notch4(int-3), a truncated form of Notch4 having most of its extracellular domain deleted, as a transgene in mice induces the formation of poorly differentiated mammary carcinomas. To establish whether Notch4(int-3) has the capacity of subverting normal epithelial architecture, we assessed the effect of Notch4(int-3) expression on the in vitro morphogenetic properties of TAC-2 mammary epithelial cells. When grown in three-dimensional collagen gels in the presence of hydrocortisone, both wild-type and LacZ-transfected TAC-2 cells formed alveolar-like structures composed of polarized epithelial cells surrounding a central lumen. In contrast, TAC-2 cells programmed to express Notch4(int-3) formed compact cell aggregates devoid of tissue-specific organization. In addition, when grown on the surface of a collagen gel, Notch4(int-3)-expressing TAC-2 cells invaded the underlying matrix, whereas TAC-2 LacZ cells remained strictly confined to the gel surface. expression of Notch4(int-3) in TAC-2 cells also disrupted contact-inhibition of cell proliferation, resulting in cell multilayering. Our results suggest that the ability of Notch4(int-3) to subvert normal epithelial morphogenesis and to promote invasion of the extracellular matrix contributes significantly to its tumorigenic potential.

NIH3T3 cells transformed by mouse Int-2/Fgf-3 cDNA express a series of Int-2-related products representing discrete stages of processing and glycosylation. We confirm that in at least two highly transformed clonal lines, Int-2 products acquire further modifications and are efficiently secreted into the culture medium. Secreted proteins become associated with the cell surface and extracellular matrix and can be displaced by addition of soluble glycosaminoglycans, specifically heparin, heparan sulfate, and dermatan sulfate. Increasing concentrations of heparin not only compete for Int-2 binding in a dose-dependent manner but also inhibit the growth of these cells and revert the transformed phenotype. These findings reaffirm the notion that extracellular or surface-bound Int-2 protein is instrumental in the morphological transformation of these cells.

In a previous study, activated rat c-raf was detected by an NIH 3T3 cell transfection assay, and a rearrangement was demonstrated in the 5 half of the sequence of the gene. In the present study, the cDNAs of normal and activated rat c-raf were analyzed. Results showed that the activated c-raf gene is transcribed to produce a fused mRNA, in which the 5 half of the sequence is replaced by an unknown rat sequence. This mRNA codes a fused c-raf protein. The normal and activated c-raf cDNAs were each connected to the long terminal repeat of Rous sarcoma virus and transfected into NIH 3T3 cells. Only the activated form had transforming activity. We conclude that the rearrangement is responsible for the activation of c-raf.

The int-3 oncogene was identified as a frequent target in Mouse Mammary tumor Virus (MMTV)-induced mammary carcinomas and encodes the intracellular domain of a novel mouse Notch gene. To investigate the role of the int-3 proto-oncogene in mouse development and carcinogenesis, we isolated cDNA clones corresponding to the entire coding potential of the int-3 proto-oncogene. We propose to name this gene Notch4 and reserve the int-3 nomenclature for references to the oncogenic form. The deduced amino acid sequence of Notch4 contains conserved motifs found in Notch proteins; however Notch4 has fewer epidermal growth factor (EGF)-like repeats and a shorter intracellular domain than other mouse Notch homologues. Comparison of the coding potential of the int-3 gene to that of Notch4 suggests that loss of the extracellular domain of Notch4 leads to constitutive activation of this murine Notch protein. In situ hybridization revealed that Notch4 transcripts are primarily restricted to endothelial cells in embryonic and adult life. Truncated Notch4 transcripts were detected in post-meiotic male germ cells. The distinct Notch4 protein features and its restricted expression pattern suggests a specific role for Notch4 during development of vertebrate endothelium.

The cDNA and genomic clones for the human counterpart of the mouse mammary tumor gene Int3 were isolated and sequenced. We designated this human major histocompatibility complex (MHC) class III gene as NOTCH4, since very recently, by sequencing cDNA clones, the complete form of the mouse proto-oncogene Int3 has been clarified and named Notch4. The present human NOTCH4 sequence is the first example of the genomic sequence for the extracellular portion of the mammalian Notch4, and by comparing it with the mouse Notch4 cDNA sequence, the exon/intron organization was clarified. The comparison of the predicted amino acid sequence of human NOTCH4 with those of other Notch homologues of a wide range of species revealed four subfamilies for mammalian Notch. In the protein coding region of human NOTCH4, we found (CTG)n repeats showing a variable number tandem repeat (VNTR) polymorphism for different human leukocyte antigen (HLA) haplotypes. Ten genes mapped on 6p21.3, including NOTCH4, were found to have counterparts structurally and functionally similar to those mostly mapped on 9q33-q34, indicating segmental chromosome duplication during the course of evolution. Similarity of genes on chromosomes 1, 6, 9 and 19 was also discussed.

BACKGROUND: Notch and its homologues are key regulatory receptors of the cell fate decision in various developmental processes. The int-3 oncogene was originally identified as a frequent target in Mouse Mammary tumour Virus (MMTV)-induced mammary tumours and has been regarded as a Notch homologue, based on its similarity to the intracellular domain of Notch. Studies with int-3 transgenic mice have suggested that the int-3 transgene affects the differentiation capacity of stem cells and leads to neoplastic proliferation in epithelial cells. However, the exact nature and the in vivo expression pattern of the int-3 proto-oncogene are unknown. The function of gene products in embryogenesis is also not clear. RESULTS: We isolated cDNA clones corresponding to the proto-oncogene of int-3 and analysed its overall structure. The predicted amino acid sequence of the int-3 proto-oncogene contains the conserved motif found in Notch family receptors. Therefore, we name Notch-4 for the int-3 proto-oncogene. However, Notch-4 has fewer EGF repeats and shows less similarity to Notch, compared with other mammalian Notch homologues. In embryogenesis, the expression of Notch-4 was detected in endothelial cells of blood vessels forming tissues such as the dorsal aorta, intersegmental vessels, yolk sac vessels, cephalic vessels, heart, vessels in branchial arches, and capillary plexuses. In these tissues, Notch-4 expression coincided with flk-1, the major regulatory gene of vasculogenesis and angiogenesis. We also found that Notch-4 expression was up-regulated in vitro during the differentiation of endothelial cells from embryonic stem cells (ES cells). CONCLUSION: The endothelial cell specific expression pattern of Notch-4, as well as its structural similarity of Notch, suggest that Notch-4 is an endothelial cell specific homologue of Notch and it may play a crucial role in vasculogenesis and angiogenesis.