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Oncogenes result in genomic alterations that activate a transcriptionally silent, dominantly selectable reporter gene
R E Drews1, V T Chan, L E Schnipper
1Charles A. Dana Research Institute, Beth Israel Hospital, Boston, Massachusetts 02215.
Abstract:
Although oncogenes and tumor suppressor genes have been implicated in carcinogenesis and tumor progression, their relationship to the development of genomic instability has not been elucidated. To examine this role, we transfected oncogenes (polyomavirus middle [Py] and large T [MT and LT]) and adenovirus serotype 5 E1A) into two NIH 3T3-derived cell lines, EN/NIH 2-4 and EN/NIH 2-20. Both cell lines contain two stable integrants of a variant of the retrovirus vector pZipNeoSV(x)1 that has been modified by deletion of the enhancer elements from the long terminal repeats. DNA rearrangements activating the silent neomycin phosphotransferase gene (neo) present in these integrants were identified by selection of cells in the antibiotic G418. Whereas control-transfected EN/NIH cell lines do not yield G418-resistant subclones (GRSs), a fraction of oncogene-transfected EN/NIH 2-4 (8 of 19 Py MT, 5 of 17 Py LT, and 11 of 19 E1A) and 2-20 (7 of 15 Py MT) cell lines gave rise to GRSs at differing frequencies (0.33 x 10(-6) to 46 x 10(-6) for line 2-4 versus 0.11 x 10(-6) to 1.3 x 10(-6) for line 2-20) independent of cell generation time. In contrast, a distinctly smaller fraction of mutant Py MT-transfected EN/NIH cell lines (1 of 10 MT23, 1 of 10 MT1015, and 0 of 10 MT59b) resulted in GRSs. Southern analysis of DNA from selected oncogene-transfected GRSs demonstrated genomic rearrangements of neo-containing cellular DNA that varied in type (amplification and/or novel fragments) and frequency depending on the specific oncogene and EN/NIH cell line used in transfection. Furthermore, only one of the two neo-containing genomic loci present in both EN/NIH cell lines appeared to be involved in these genomic events. In addition to effects related to the genomic locus, these observations support a role for oncogenes in the development of genetic changes associated with tumor progression.
Insights
Oncogenes, such as polyomavirus middle T antigen and adenovirus E1A, can induce genomic instability and DNA rearrangements in NIH 3T3 cells. This study reveals oncogenes
Area of Science:
- Molecular Biology
- Cancer Research
- Genetics
Background:
- Oncogenes and tumor suppressor genes are known to play roles in cancer development.
- The specific contribution of oncogenes to genomic instability has remained unclear.
- Genomic instability is a hallmark of cancer, driving tumor progression.
Purpose of the Study:
- To investigate the role of specific oncogenes in inducing genomic instability.
- To examine how oncogenes affect DNA rearrangements in mouse fibroblast cell lines.
- To elucidate the relationship between oncogene expression and the development of genetic alterations.
Main Methods:
- Transfection of NIH 3T3-derived cell lines (EN/NIH 2-4 and EN/NIH 2-20) with oncogenes: polyomavirus middle T antigen (Py MT), polyomavirus large T antigen (Py LT), and adenovirus serotype 5 E1A.
- Selection of G418-resistant subclones (GRSs) to identify DNA rearrangements activating the neomycin phosphotransferase gene (neo).
- Southern blot analysis of DNA from GRSs to characterize the nature and frequency of genomic rearrangements.
Main Results:
- Oncogene transfection, particularly with Py MT, Py LT, and E1A, significantly increased the frequency of GRSs compared to control cells.
- Specific oncogenes and cell lines exhibited varying frequencies and types of genomic rearrangements (amplification, novel fragments) in the neo locus.
- Mutant Py MT oncogenes showed a markedly reduced ability to induce GRSs, suggesting oncogene-specific effects.
Conclusions:
- Oncogenes can directly promote the development of genomic instability.
- The observed DNA rearrangements are dependent on the specific oncogene and the cellular genomic context.
- These findings support a role for oncogenes in driving the genetic alterations crucial for tumor progression.