Related Experiment Video
Updated: Aug 4, 2026
Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression
Published on: March 5, 2013
Expression of antisense PC1 in stably transfected RIN5F cells significantly reduces CCK 8 biosynthesis
1Department of Pharmacological and Physiological Science, St. Louis University Medical Center, MO 63104, USA.
Abstract:
The subtilisin-like endoprotease PC1 (PC3) has been implicated in the processing of a number of prohormones. To evaluate whether PC1 may be important for the processing of pro CCK to CCK 8, stable cell lines expressing a portion of the PC1 cDNA in the antisense orientation were established from RIN5F cells. These cells express CCK mRNA, produce and display regulated secretion of CCK 8. One of the clones, R1E8, expresses antisense PC1 mRNA as determined by reverse transcriptase-PCR (RT-PCR) and contains a significantly reduced level of PC1 protein. As compared to both RIN5F and RIN5F control cells (transfected with the expression plasmid containing no antisense message), R1E8 contains only about 30% cell content of CCK 8. These results suggest that PC1 may be important for the processing of CCK 8 from pro CCK.
Insights
Subtilisin-like endoprotease PC1 (PC3) is crucial for processing pro-cholecystokinin (CCK) into CCK 8. Reduced PC1 levels in cell lines significantly lowered CCK 8 content, confirming PC1
Area of Science:
- Endocrinology
- Molecular Biology
- Cell Biology
Background:
- Subtilisin-like endoprotease PC1 (also known as PC3) plays a role in prohormone processing.
- Cholecystokinin (CCK) is a prohormone processed into active CCK 8.
- RIN5F cells express CCK mRNA and secrete CCK 8, making them a suitable model.
Purpose of the Study:
- To investigate the role of PC1 in the processing of pro-cholecystokinin (proCCK) to cholecystokinin 8 (CCK 8).
- To determine if PC1 is essential for CCK 8 production and secretion.
Main Methods:
- Stable cell lines (RIN5F) were engineered to express PC1 cDNA in antisense orientation.
- Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to confirm antisense PC1 mRNA expression.
- PC1 protein levels and CCK 8 content were quantified in engineered and control cell lines.
Main Results:
- An antisense PC1 clone (R1E8) showed reduced PC1 protein levels compared to controls.
- R1E8 cells contained significantly lower levels of CCK 8 (approximately 30% of control levels).
- This indicates a direct correlation between PC1 levels and CCK 8 production.
Conclusions:
- The endoprotease PC1 is important for the efficient processing of proCCK to CCK 8.
- PC1 activity is critical for achieving normal cellular CCK 8 content.
- These findings highlight PC1 as a key enzyme in the CCK processing pathway.
