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The colon anion transporter, down-regulated in adenoma, induces growth suppression that is abrogated by E1A
Jeannie M Chapman1, Stewart M Knoepp, Mee Kyeong Byeon
1Laboratory of Cancer Genomics, Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29403, USA.
Abstract:
The down-regulated in adenoma (DRA) gene is significantly down-regulated in adenomas and adenocarcinomas of the colon as well as in colon cancer cell lines. It is also mutated in the disease congenital chloride diarrhea, which is characterized by loss of chloride transport and diarrhea. We now show a second function for DRA relevant to colon tumorigenesis, i.e., growth suppression. Transfection of full-length DRA into various cell lines (DLD-1, HT-29, HCT-15, SW837, SW480, MCF-7, NIH3T3, CaSki, and HeLa) that lack endogenous DRA expression results in a reduced number of drug-resistant colonies compared with vector control, suggesting growth suppression by DRA. In addition, expression of DRA under the control of an inducible promoter reduced the growth rate of DLD-1 cells compared with cells not expressing DRA. The COOH-terminal cytoplasmic domain of DRA is required for growth suppression, but an in-frame deletion (DeltaVal317) that causes congenital chloride diarrhea and results in a loss of anion transport had no effect on growth suppression, indicating that anion transport and growth suppression are independent functions of DRA. One cell line, adenovirus-transformed HEK293, exhibited significant resistance to DRA-induced growth suppression, whereas the human papillomavirus-transformed cell lines, CaSki and HeLa, did not. E1A is an adenoviral protein required to transform HEK293 cells. DLD-1 cells that stably express 12S E1A are resistant to growth suppression by DRA, similar to HEK293 cells.
Insights
The down-regulated in adenoma (DRA) gene suppresses tumor growth in colon cancer cells. Its growth suppression function is separate from its role in chloride transport, but can be blocked by adenoviral E1A protein.
Area of Science:
- Molecular Biology
- Oncology
- Genetics
Background:
- The down-regulated in adenoma (DRA) gene is frequently lost in colon tumors and linked to congenital chloride diarrhea.
- Previous research indicated DRA's role in colon adenomas and adenocarcinomas.
- The gene's function beyond chloride transport in tumorigenesis remained unclear.
Purpose of the Study:
- To investigate the potential growth-suppressive role of the DRA gene in colon cancer.
- To determine if DRA's anion transport function is linked to its growth-suppressive activity.
- To identify factors that may confer resistance to DRA-mediated growth suppression.
Main Methods:
- Transfection of DRA into various cancer and non-cancer cell lines lacking endogenous DRA expression.
- Inducible expression of DRA to monitor cell growth rates.
- Site-directed mutagenesis to assess the role of the COOH-terminal domain and specific mutations (DeltaVal317).
- Co-expression studies with adenoviral E1A protein in relevant cell lines.
Main Results:
- DRA transfection significantly reduced drug-resistant colony formation in multiple cell lines, indicating growth suppression.
- Inducible DRA expression slowed the growth rate of DLD-1 colon cancer cells.
- The COOH-terminal cytoplasmic domain of DRA is essential for growth suppression, but the DeltaVal317 mutation, which impairs anion transport, did not affect this function.
- Adenovirus-transformed HEK293 cells and DLD-1 cells expressing E1A showed resistance to DRA-induced growth suppression.
Conclusions:
- The DRA gene possesses a distinct growth-suppressive function relevant to colon tumorigenesis.
- DRA's growth suppression activity is independent of its anion transport function.
- Adenoviral E1A protein can interfere with DRA's growth-suppressive effects, suggesting a potential mechanism for viral oncogenesis or tumor progression.