Gene activation by induced DNA rearrangements.

Area of Science:

• Molecular Biology
• Genetics
• Cancer Research

Background:

• DNA rearrangements can lead to gene activation and disease.
• Understanding the mechanisms of induced DNA rearrangements is crucial for cancer research.
• Existing assay systems have limitations in detecting specific types of DNA alterations.

Purpose of the Study:

• To establish and validate a murine cell line (EN/NIH) for detecting induced DNA rearrangements.
• To investigate the role of tumor promoters (12-O-tetradecanoylphorbol-13-acetate, TPA) and UV irradiation in inducing DNA rearrangements.
• To determine the effect of SV40 large T antigen on DNA rearrangement frequency and characteristics.

Main Methods:

• Utilized a retroviral vector (ZIPNeoSV(x)1) with a silenced reporter gene (neomycin phosphotransferase, neo) in EN/NIH cells.
• Exposed cells to TPA, UV irradiation plus TPA, or transfected with a temperature-sensitive SV40 T antigen mutant.
• Analyzed drug-resistant cell lines for DNA alterations using molecular techniques, including DNA cloning and hybridization.

Main Results:

• Spontaneous G418 resistance frequency was low (<10(-7)).
• TPA and UV irradiation plus TPA significantly increased drug-resistant cell emergence (5x10^6 and 67x10^6, respectively).
• SV40 T antigen exposure dose-dependently increased drug resistance frequency (9-52x10^6) and induced DNA rearrangements, including inverted duplications.

Conclusions:

• The EN/NIH cell line is a sensitive system for studying induced DNA rearrangements.
• Tumor promoters, UV light, and SV40 T antigen can all induce DNA rearrangements.
• The findings suggest SV40 T antigen may initiate rearrangements via binding to the viral replication origin.