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Reduction of Cripto-1 expression by a hammerhead-shaped RNA molecule results from inhibition of translation rather
1Department of Genetics & Cell Biology, Washington State University, Pullman 99164-4234, USA.
Abstract:
Cripto-1 (CR-1) is a transforming growth factor which has been associated with breast, colon, and pancreatic cancer. Overexpression of CR-1 in non-tumorigenic mouse mammary epithelial cells and fibroblasts results in an increase in anchorage-dependent and -independent growth in vitro. Reduction of CR-1 expression in human colon carcinoma or embryonal teratoma cells results in a decrease in growth in vitro. In an effort to better define the role of CR-1 in breast cancer, we have developed an underexpression vector for CR-1 to reduce CR-1 levels in a tumorigenic mouse mammary epithelial cell line (-SA). This vector specifically targets the expression of the murine homolog of CR-1 in murine cancer lines and utilizes a hammerhead ribozyme-like structure directed toward the extreme 5' end of the Cripto-1 mRNA. We dramatically reduced expression of CR-1 through the expression of this RNA. This is the first use of a ribozyme-like molecule to alter Cripto-1 expression. This ribozyme-shaped molecule appears to act principally through a block in translation. A possible mechanism for this block is described, and its implications for modifying expression of other bioactive proteins are discussed.
Insights
Cripto-1 (CR-1), a transforming growth factor linked to various cancers, was targeted using a novel ribozyme-like molecule. This approach effectively reduced CR-1 expression, impacting cancer cell growth.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Cripto-1 (CR-1) is a transforming growth factor implicated in breast, colon, and pancreatic cancers.
- CR-1 overexpression enhances anchorage-dependent and -independent growth in non-tumorigenic cells.
- CR-1 reduction decreases growth in human colon carcinoma and embryonal teratoma cells.
Purpose of the Study:
- To investigate the role of CR-1 in breast cancer by developing a method to reduce its expression.
- To create an underexpression vector targeting the murine homolog of CR-1 in a tumorigenic mouse mammary epithelial cell line (-SA).
Main Methods:
- Developed an underexpression vector utilizing a hammerhead ribozyme-like structure targeting the 5' end of Cripto-1 mRNA.
- Applied the vector to a tumorigenic mouse mammary epithelial cell line (-SA) to reduce CR-1 expression.
- Investigated the mechanism of CR-1 reduction, identifying a translational block.
Main Results:
- Successfully and dramatically reduced CR-1 expression in the targeted cell line.
- Demonstrated the first use of a ribozyme-like molecule to modulate Cripto-1 expression.
- Observed that the ribozyme-like molecule primarily acts by blocking translation.
Conclusions:
- The developed ribozyme-like molecule is effective in reducing Cripto-1 expression.
- This approach offers a novel strategy for targeting CR-1 in cancer therapy.
- The mechanism of translational blockade has implications for modifying the expression of other bioactive proteins.