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Methotrexate resistance in NIH3T3 cells expressing polyoma virus oncogenes
1Department of Radiology, Ohio State University, Columbus 43210.
Abstract:
NIH3T3 cell lines expressing different polyoma virus oncogenes were found to differ by almost three orders of magnitude in frequencies of methotrexate-resistant (MTXr) cells in single-step selections. Cells expressing the polyoma small T antigen showed the highest frequencies of MTX resistance, whereas cells expressing the polyoma middle T antigen showed the lowest frequencies of MTX resistance. Amplification of the dihydrofolate reductase (dhfr) gene was not a mechanism of MTX resistance in these cell lines. Cells expressing large T antigen also showed a high frequency of MTXr colonies with only two of 20 such colonies showing evidence of marginal (less than threefold) dhfr gene amplification. Cells carrying the cloning vector without an oncogene showed intermediate frequencies of MTX resistance, and in contrast to the oncogene expressing lines, two-thirds of these MTXr colonies showed marked dhfr gene amplification. The results suggest that the plasticity of the genome as measured by frequency or extent of dhfr gene amplification is not increased by expression of either immortalizing or transforming polyoma oncogenes. These oncogenes may, however, influence MTX resistance through epigenetic mechanisms. In addition, there appears to be no relationship between frequency of methotrexate resistance and frequency of dhfr gene amplification. It is clear that mechanisms of resistance other than dhfr gene amplification can be exclusively associated with either very high or very low frequencies of resistance.
Insights
Polyoma virus oncogenes influence methotrexate resistance (MTXr) frequencies in NIH3T3 cells. Small T antigen increased MTXr, while middle T antigen decreased it, suggesting epigenetic mechanisms beyond dhfr gene amplification.
Area of Science:
- Molecular Biology
- Oncology
- Genetics
Background:
- NIH3T3 cell lines are widely used models for studying cellular transformation and oncogene function.
- Methotrexate (MTX) resistance is a common phenomenon in cancer cells, often mediated by alterations in the dihydrofolate reductase (dhfr) gene.
- Polyoma virus oncogenes are known to induce cellular immortalization and transformation.
Purpose of the Study:
- To investigate the effect of different polyoma virus oncogenes on the frequency of methotrexate resistance in NIH3T3 cells.
- To determine if dhfr gene amplification is the primary mechanism of MTX resistance in these cell lines.
- To explore potential epigenetic mechanisms underlying MTX resistance induced by polyoma oncogenes.
Main Methods:
- Single-step selection of methotrexate-resistant (MTXr) colonies in NIH3T3 cell lines expressing different polyoma virus oncogenes (small T, middle T, large T) or a cloning vector.
- Analysis of dhfr gene amplification in MTXr colonies using quantitative methods.
- Comparison of MTXr frequencies and dhfr gene amplification levels across different cell lines.
Main Results:
- Frequencies of MTXr cells varied by nearly three orders of magnitude depending on the expressed polyoma oncogene.
- Cells expressing small T antigen showed the highest MTXr frequencies, while middle T antigen expressing cells showed the lowest.
- dhfr gene amplification was not the primary mechanism for MTX resistance; instead, epigenetic mechanisms may be involved, particularly with small T and large T antigens.
Conclusions:
- Polyoma virus oncogenes do not increase genome plasticity related to dhfr gene amplification.
- Oncogenes can influence MTX resistance through epigenetic modifications, independent of dhfr gene amplification.
- Mechanisms of resistance other than dhfr gene amplification are crucial for understanding varied MTX resistance frequencies.