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Identification of a novel NOTCH-4/INT-3 RNA species encoding an activated gene product in certain human tumor cell
1Laboratory of Tumor Immunology, National Cancer Institute, NIH, Bethesda, Maryland, MD 20892, USA.
Abstract:
Ectopic expression of the intracellular domain of NOTCH-4/INT-3 leads to tumorigenesis in the mouse mammary gland. This results from a gain-of-function mutation. To evaluate gain-of-function NOTCH-4/INT-3 activity in human cancers we have surveyed human breast, lung, and colon carcinoma tissue culture cell lines for evidence of increased NOTCH-4/INT-3 RNA expression. High levels of a 1.8 Kb NOTCH-4/INT-3 RNA species are detected in normal human testis but not in other tissues where a 6.5 Kb species is prevalent. Transformed human cancer cell lines express the 1.8 Kb NOTCH-4/INT-3 RNA species. We show that this RNA species encodes a truncated form of the NOTCH-4/INT-3 intracellular domain (ICD). This novel NOTCH-4/INT-3 protein includes the CDC10 repeats and amino acid residues C-terminal to them, but is missing the CBF-1 binding region of the NOTCH-4/INT-3 ICD. This suggests that it has a different mode of action. Furthermore, we show that a transgene which expresses the 1.8 Kb NOTCH-4/INT-3 RNA species in the 'normal' human mammary epithelial cell line MCF-10A enables these cells to grow in soft agar.
Insights
Gain-of-function NOTCH-4/INT-3 mutations drive tumorigenesis. A truncated NOTCH-4/INT-3 RNA species, found in cancer cells, promotes mammary epithelial cell growth, suggesting a role in human cancer development.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Ectopic expression of the NOTCH-4/INT-3 intracellular domain (ICD) causes mammary gland tumors in mice due to a gain-of-function mutation.
- The role of NOTCH-4/INT-3 gain-of-function in human cancers remains to be fully elucidated.
Purpose of the Study:
- To investigate gain-of-function NOTCH-4/INT-3 activity in human cancers.
- To identify and characterize novel NOTCH-4/INT-3 RNA species and their encoded proteins in human carcinoma cell lines.
Main Methods:
- Surveyed human breast, lung, and colon carcinoma cell lines for NOTCH-4/INT-3 RNA expression.
- Analyzed RNA species size and expression levels in normal and cancerous tissues.
- Characterized the protein product of the identified NOTCH-4/INT-3 RNA species.
- Transfected normal human mammary epithelial cells (MCF-10A) with the identified RNA species to assess functional changes.
Main Results:
- A specific 1.8 Kb NOTCH-4/INT-3 RNA species was detected in transformed human cancer cell lines, contrasting with the prevalent 6.5 Kb species in most normal tissues.
- This 1.8 Kb RNA encodes a truncated NOTCH-4/INT-3 intracellular domain (ICD) lacking the CBF-1 binding region.
- Expression of the 1.8 Kb NOTCH-4/INT-3 RNA species in MCF-10A cells induced anchorage-independent growth (soft agar assay), indicative of transformation.
Conclusions:
- The 1.8 Kb NOTCH-4/INT-3 RNA species represents a novel, truncated ICD with altered function, potentially contributing to human tumorigenesis.
- This truncated NOTCH-4/INT-3 protein may possess a distinct mechanism of action compared to the full-length ICD.
- The findings highlight the oncogenic potential of specific NOTCH-4/INT-3 variants in human epithelial cancers.