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Induction of endoreduplication in temperature-sensitive mutants of rat 3Y1 fibroblasts
Abstract:
Four temperature-sensitive mutants of rat 3Y1 fibroblasts belonging to separate complementation groups (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203) are arrested mainly with a 2C DNA content, when cells proliferating at 33.8 degrees C are shifted up to 39.8 degrees C (Ohno et al., 1984). Zaitsu and Kimura (submitted for publication) showed that 3Y1tsF121 cells synchronized in the early S phase were arrested with a 4C DNA content at 39.8 degrees C. We studied the traverse through the S and G2 phases at 39.8 degrees C in the four ts mutants synchronized at the early S phase and found that 3Y1tsG125 and 3Y1tsH203 cells were arrested with a 4C DNA content as 3Y1tsF121, while 3Y1tsD123 cells went through S and G2 phases and underwent mitosis. When 3Y1tsF121 and 3Y1tsG125 mutants arrested at 39.8 degrees C were shifted down to 33.8 degrees C, a substantial fraction of the cells with a 4C DNA content started, with a certain lag period, DNA synthesis without intervening mitosis and underwent the first mitosis with a lag period similar to that in the cells arrested with a 2C DNA content. The tetraploid cells thus generated had a proliferating ability lower than that of diploid cells.
Insights
Temperature-sensitive rat fibroblast mutants were studied for cell cycle arrest. Some mutants arrest with 4C DNA content at high temperatures, impacting proliferation.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Temperature-sensitive mutants of rat 3Y1 fibroblasts exhibit cell cycle arrest at the non-permissive temperature (39.8°C).
- Previous studies indicated arrest at a 2C DNA content for most mutants, but one mutant (3Y1tsF121) arrested at 4C DNA content when synchronized in early S phase.
Purpose of the Study:
- To investigate the cell cycle traverse through S and G2 phases at 39.8°C in four temperature-sensitive rat fibroblast mutants.
- To determine the DNA content at which these mutants arrest and to analyze the consequences of temperature shift-down on cell proliferation.
Main Methods:
- Synchronization of four temperature-sensitive rat 3Y1 fibroblast mutants (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, 3Y1tsH203) at the early S phase.
- Incubation at the non-permissive temperature (39.8°C) to induce cell cycle arrest.
- Analysis of DNA content using flow cytometry.
- Temperature shift-down to 33.8°C to observe recovery and subsequent cell division.
Main Results:
- Mutants 3Y1tsG125 and 3Y1tsH203, similar to 3Y1tsF121, arrested with a 4C DNA content at 39.8°C.
- Mutant 3Y1tsD123 progressed through S and G2 phases and underwent mitosis.
- Upon shift-down, 3Y1tsF121 and 3Y1tsG125 cells with 4C DNA content initiated DNA synthesis without intervening mitosis, followed by mitosis.
- Generated tetraploid cells exhibited reduced proliferation ability compared to diploid cells.
Conclusions:
- Two additional temperature-sensitive mutants (3Y1tsG125, 3Y1tsH203) are identified to arrest with 4C DNA content at the non-permissive temperature.
- The cell cycle arrest phenotype at high temperature is distinct among these mutants.
- Recovery from arrest and subsequent cell division can lead to the generation of tetraploid cells with compromised proliferative capacity.