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Unbalanced growth in mouse cells with amplified dhfr genes
1Department of Radiology, Ohio State University College of Medicine, Columbus 43210, USA.
Abstract:
When grown in the absence of methotrexate, cells carrying unstably amplified dihydrofolate reductase (dhfr) genes have a growth disadvantage that is a function of their level of gene amplification. Although this growth disadvantage is thought to drive the loss of unstably amplified dhfr genes in the absence of methotrexate, its mechanism is not understood. The present studies of murine cell lines with different levels of dhfr gene amplification demonstrate that such cells experience increased unbalanced growth (excess RNA and protein content relative to DNA content) with increased levels of dhfr gene amplification. Stathmokinetic analysis of a cell line with unstably amplified dhfr genes showed that the unbalanced growth was associated with a very low rate of G1/S transit, which suggests that amplified DNA sequences may activate a cell cycle checkpoint at the G1/S boundary. Hydroxyurea, which is known to induce rapid elimination of amplified genes at sub-cytotoxic concentrations, also inhibits the cell cycle at the G1/S transition and causes unbalanced growth. Earlier work has shown that hydroxyurea selectively targets those cells within the heterogeneous drug resistant cell populations which have the highest amplified gene dosage. The finding that unstable gene amplification and hydroxyurea have similar effects on the cell suggests that hydroxyurea may achieve this selective targeting by pushing those cells with the highest levels of gene amplification over a critical stress threshold to cause growth arrest or cell death.
Insights
Unstable gene amplification in dihydrofolate reductase (dhfr) genes causes unbalanced cell growth. This suggests a cell cycle checkpoint activation, potentially explaining how drugs like hydroxyurea eliminate cells with high gene amplification.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Unstable gene amplification of dihydrofolate reductase (dhfr) confers a growth disadvantage in the absence of methotrexate.
- The precise mechanism driving the loss of these amplified dhfr genes remains unclear.
Purpose of the Study:
- To investigate the cellular basis for the growth disadvantage associated with unstable dhfr gene amplification.
- To explore the relationship between dhfr gene amplification levels and cellular growth characteristics.
- To elucidate the mechanism by which hydroxyurea selectively eliminates cells with high gene amplification.
Main Methods:
- Analysis of murine cell lines with varying levels of dhfr gene amplification.
- Stathmokinetic analysis to assess cell cycle transit.
- Treatment with hydroxyurea at sub-cytotoxic concentrations.
Main Results:
- Increased levels of dhfr gene amplification correlated with increased unbalanced growth (excess RNA/protein relative to DNA).
- Unbalanced growth in amplified cells was linked to significantly reduced G1/S phase transit, suggesting a G1/S checkpoint activation.
- Hydroxyurea treatment induced unbalanced growth and G1/S transition inhibition, mirroring effects of gene amplification.
Conclusions:
- Unstable dhfr gene amplification activates a cell cycle checkpoint at the G1/S boundary, leading to unbalanced growth.
- Hydroxyurea's selective targeting of high-amplification cells may occur by inducing critical stress, causing cell cycle arrest or death.