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[DAN gene]
1Chiba Cancer Center Research Institute.
Abstract:
DAN gene has been isolated by means of differential screening method between rat fibroblast 3Y1 and Rous sarcoma virus-transformed 3Y1 (SR-Y1) cells. DAN expression was suppressed in some of the transformed cells and its re-expression reverted the various transformed phenotypes such as colony formation in soft agar and tumor formation in mice. When DAN was overexpressed in 3Y1 cells, these cell showed the retardation of the entry into the S phase. DAN cDNA encodes a 27-kD protein which consists of 178 amino acids. The deduced amino acid sequence has no homology with known proteins. The amino terminal of DAN is highly hydrophobic and DAN is indeed secreted into the culture medium. This secreted DAN, when added to the culture of SR-3Y1 cells, suppressed DNA synthesis. Human DAN is mapped to 1p36.11-p36.13, a region known to have highly significant linkage with the genesis and/or progression of human neuroblastoma. Aberrant bands on Southern blot were detected in some of the neuroblastoma cases. Rat and human genomic DANs were isolated. CAT and the electrophoretic mobility shift assays revealed the presence of possible positive and negative regulatory elements at -57 approximately +118 and -1,232 approximately -987 of rat genome, respectively.
Insights
The DAN gene, when re-expressed, reverses cancer cell phenotypes and suppresses DNA synthesis. Its human counterpart is linked to neuroblastoma, suggesting a tumor suppressor role.
Area of Science:
- Molecular Biology
- Cancer Research
- Genetics
Background:
- The DAN gene was identified through differential screening between normal rat fibroblasts and Rous sarcoma virus-transformed cells.
- DAN expression is suppressed in some transformed cells, and its re-expression reverses malignant phenotypes.
- Overexpression of DAN in fibroblasts retards cell cycle progression into the S phase.
Purpose of the Study:
- To investigate the function and regulation of the DAN gene in cellular transformation and cancer.
- To characterize the DAN protein and its role in DNA synthesis and cell proliferation.
- To explore the potential involvement of DAN in human neuroblastoma.
Main Methods:
- Differential screening for gene isolation.
- Phenotypic analysis of transformed cells (soft agar colony formation, tumor formation in mice).
- Cell cycle analysis (S phase entry).
- Protein characterization (amino acid sequence, hydrophobicity, secretion).
- DNA synthesis assays.
- Gene mapping (human DAN to chromosome 1p36.11-p36.13).
- Southern blot analysis for neuroblastoma cases.
- Genomic DNA isolation and analysis (rat and human DAN).
- Reporter assays (CAT assay) and electrophoretic mobility shift assays (EMSA) for regulatory element identification.
Main Results:
- Re-expression of DAN reversed transformed cell phenotypes, including anchorage-independent growth and tumorigenicity.
- DAN overexpression in normal cells inhibited S phase entry.
- DAN encodes a secreted 27-kD protein with no homology to known proteins.
- Secreted DAN suppressed DNA synthesis in transformed cells.
- Human DAN maps to a region strongly linked to neuroblastoma, with aberrant bands found in some cases.
- Regulatory elements (positive and negative) were identified in the rat DAN gene promoter region.
Conclusions:
- DAN functions as a tumor suppressor by inhibiting cell proliferation and reversing malignant transformation.
- The secreted nature of DAN suggests a paracrine or autocrine mechanism of action.
- The linkage of human DAN to neuroblastoma and observed aberrations highlight its potential role in this cancer.
- The identified regulatory elements provide insights into the transcriptional control of DAN expression.