Effect of antisense c-raf-1 on tumorigenicity and radiation sensitivity of a human squamous carcinoma
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U Kasid1, A Pfeifer, T Brennan
1Department of Radiation Medicine, Vincent T. Lombardi Comprehensive Cancer Research Center, Georgetown University Medical Center, Washington 20007.
Abstract:
Antisense RNA-mediated inhibition of gene expression was used to investigate the biological function of the c-raf-1 gene in a radiation-resistant human squamous carcinoma cell line, SQ-20B. S1 nuclease protection assays revealed that transfection of full-length raf complementary DNA in the antisense orientation (AS) leads to a specific reduction (greater than tenfold) of steady-state levels of the endogenous c-raf-1 sense (S) transcript in SQ-20B cells. In nude mice, the malignant potential of SQ-20B cells transfected with raf (S) was significantly increased relative to that of SQ-20B cells transfected with raf (AS). SQ-20B cells containing transfected raf (S) maintained a radiation-resistant phenotype as compared to those cells harboring the AS version, which appeared to have enhanced radiation sensitivity. These data indicate that the reduced expression of endogenous c-raf-1 is sufficient to modulate the tumorigenicity and the radiation-resistant phenotype of SQ-20B cells, thus implicating c-raf-1 in a pathway important to the genesis of this type of cancer.
Insights
Investigating the c-raf-1 gene in radiation-resistant cancer cells using antisense RNA showed that reducing c-raf-1 expression decreases tumor growth and radiation resistance. This implicates c-raf-1 in cancer development.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Genetics
Background:
- The c-raf-1 gene plays a role in cell signaling pathways.
- Radiation resistance is a significant challenge in cancer treatment.
- Understanding gene function in cancer cells is crucial for developing targeted therapies.
Purpose of the Study:
- To investigate the biological function of the c-raf-1 gene in a radiation-resistant human squamous carcinoma cell line (SQ-20B).
- To determine the effect of c-raf-1 gene expression modulation on tumorigenicity and radiation sensitivity.
Main Methods:
- Antisense RNA-mediated inhibition of gene expression was employed.
- S1 nuclease protection assays were used to quantify transcript levels.
- Transfection of SQ-20B cells with sense (S) and antisense (AS) raf complementary DNA.
- In vivo studies using nude mice to assess malignant potential.
Main Results:
- Transfection with antisense raf (AS) specifically reduced endogenous c-raf-1 sense (S) transcript levels by over tenfold.
- SQ-20B cells transfected with sense raf (S) showed increased malignant potential in nude mice compared to those with antisense raf (AS).
- Cells with transfected raf (S) maintained radiation resistance, while those with raf (AS) exhibited enhanced radiation sensitivity.
Conclusions:
- Reduced expression of endogenous c-raf-1 is sufficient to modulate tumorigenicity in SQ-20B cells.
- Downregulation of c-raf-1 expression can overcome the radiation-resistant phenotype.
- The c-raf-1 gene is implicated in a critical pathway for the genesis of this type of cancer.