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Gene Activation by Induced DNA Rearrangements¹

Thorndike Memorial Laboratories at the Dana Research Institute, and Division of Medical Oncology, Department of Medicine, Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts 02215 [L. E. S., V. C.], and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts [J. S., P. J., P. A. S.]

A murine cell line (EN/NIH) containing the retroviral vector ZIPNeoSV(x)1 that was modified by deletion of the enhancer elements in the viral long terminal repeats has been used as an assay system to detect induced DNA rearrangements that result in activation of a transcriptionally silent reporter gene (neomycin phosphotransferase, neo) encoded by the viral genome. The spontaneous frequency of G418 resistance is less than 10^-7, whereas exposure to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) or the combination of UV irradiation plus TPA resulted in the emergence of drug resistant cell lines at a frequency of 5 per 10⁶ and 67 per 10⁶ cells, respectively. In several of the cell lines that were analyzed a low level of amplification of one of the two parental retroviral integrants was observed, whereas in others no alteration in the region of the viral genome was detected.

To determine the effect of the SV40 large T antigen on induced DNA rearrangements, EN/NIH cells were transfected with a temperature sensitive (ts) mutant of SV40 T. Transfectants were maintained at the permissive temperature (33°C) for varying periods of time (1–5 days) in order to vary SV40 T antigen exposure, after which they were shifted to 39.5°C for selection in G418. The frequency of emergence of drug resistant cell clones increased with duration of exposure to large T antigen (9–52 per 10⁶ cells over 1–5 days, respectively), and all cell lines analyzed demonstrated DNA rearrangements in the region of the neo gene. A novel 18-kilobase pair XbaI fragment was cloned from one cell line which revealed the presence of a 2.0-kilobase pair EcoRI segment containing an inverted duplication which hybridized to neo sequences. It is likely that the observed rearrangement was initiated by the specific binding of large T antigen to the SV40 origin of replication encoded within the viral genome.

The investigations with phorbol esters, UV light, and the SV40 large T antigen demonstrate the utility of the EN/NIH cell lines for the study of induced DNA rearrangements and support the future use of this system to investigate the mechanism by which varied stimuli or specific gene functions promote DNA rearrangements.

¹ Supported in part by American Cancer Society Grant MV-237 (L. E. S.) and National Cancer Institute Grant P01-38493 (L. E. S.).

² To whom requests for reprints should be addressed.

³ Present address: Department of Molecular Biophysics and Biochemistry, Yale University, 333 Cedar Street, New Haven, CT 06510.

⁴ Present address: Ludwig Cancer Institute in London, England.

Received 5/ 2/89. Revised 8/16/89. Accepted 9/ 7/89.