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Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols
Published on: June 6, 2017
Reversible G(1) arrest by dimethyl sulfoxide as a new method to synchronize Chinese hamster cells
Mario Fiore1, Romina Zanier, Francesca Degrassi
1Centre for Evolutionary Genetics, CNR, c/o Department of Genetics and Molecular Biology, La Sapienza University, Via degli Apuli 4, 00185 Rome, Italy.
Abstract:
Dimethyl sulfoxide (DMSO), a well-known differentiation inducer in several myeloid cells, also induces a reversible G(1) arrest in many cell lines. We recently showed that DMSO induces a G(1) phase arrest in Chinese hamster ovary (CHO) cells, by restoring contact inhibition and preventing high density-dependent apoptosis. CHO cells are frequently used in cell biology and mutagenesis studies due to their good growth capacity and ease of manipulation but are very difficult to synchronize by serum starvation since they detach from monolayers when they reach confluence. In this study we investigated the possibility of using DMSO to reversibly synchronize CHO cells in the G(1) phase of the cell cycle and analysed whether toxic effects follow the arrest using growth curve, sister chromatid exchange and micronuclei assays. We carried out a kinetic analysis of the arrest by DMSO and re-entry into the cell cycle after drug release by cytofluorimetric analysis of DNA content and bromodeoxyuridine incorporation. We show that CHO cells are efficiently and reversibly arrested in G(1) by DMSO in concentrations ranging between 1 and 2%. In our experiments, >90% of cells grown for 96 h in presence of the drug were arrested in G(1) and synchronously re-entered S phase approximately 8-12 h after release. Furthermore, expression levels of p27 were down-regulated during G(1) progression and cyclin D3 and E expression patterns were similar to those observed after serum starvation. No detectable cytotoxicity or genetic damage were induced in G(1) released cells as revealed by the tests employed. Our results show that DMSO is a very powerful inducer of G(1) synchronization in CHO cells without detectable cytotoxic or genetic effects in cell populations released from G(1) arrest. DMSO synchronization represents a model system in which to analyse protein activities regulating G(1) progression and investigate the response of G(1) cells to mutagen treatments.
Insights
Dimethyl sulfoxide (DMSO) effectively synchronizes Chinese hamster ovary (CHO) cells in G(1) phase without causing toxicity. This method offers a reliable way to study cell cycle progression and responses to mutagens.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Dimethyl sulfoxide (DMSO) is a known inducer of cell differentiation and G(1) arrest.
- Chinese hamster ovary (CHO) cells are widely used but difficult to synchronize using traditional methods like serum starvation.
- Previous work demonstrated DMSO's ability to induce G(1) arrest in CHO cells, restoring contact inhibition and preventing apoptosis.
Purpose of the Study:
- To investigate the efficacy and safety of using DMSO for reversible G(1) cell cycle synchronization in CHO cells.
- To analyze the kinetics of DMSO-induced G(1) arrest and subsequent cell cycle re-entry.
- To assess potential cytotoxic and genotoxic effects of DMSO treatment in CHO cells.
Main Methods:
- Cell cycle synchronization using varying concentrations of DMSO (1-2%).
- Kinetic analysis of G(1) arrest and S phase re-entry via flow cytometry (DNA content) and bromodeoxyuridine incorporation.
- Assessment of cytotoxicity and genetic damage using growth curve, sister chromatid exchange, and micronuclei assays.
- Analysis of cell cycle regulatory protein expression (p27, cyclin D3, cyclin E).
Main Results:
- DMSO at 1-2% efficiently and reversibly arrested >90% of CHO cells in G(1) phase after 96 hours.
- Cells synchronously re-entered S phase 8-12 hours after DMSO removal.
- Down-regulation of p27 and expression patterns of cyclin D3/E mimicked serum starvation effects.
- No detectable cytotoxicity or genetic damage was observed in cells released from G(1) arrest.
Conclusions:
- DMSO is a potent and safe agent for achieving reversible G(1) synchronization in CHO cells.
- This DMSO-based synchronization method provides a valuable model for studying G(1) cell cycle regulation and responses to mutagens.
- The absence of toxic or genetic effects makes DMSO synchronization a reliable technique for various cell biology and mutagenesis studies.
