A method for the synchronization of cultured cells with aphidicolin: Application to the large-scale synchronization of L1210 cells and the study of the cell cycle regulation of thymidylate synthase and dihydrofolate reductase

doi:10.1016/0003-2697(89)90605-2

Analytical Biochemistry

Volume 182, Issue 2, 1 November 1989, Pages 338-345

https://doi.org/10.1016/0003-2697(89)90605-2 Get rights and content

Abstract

The DNA polymerase α inhibitor, aphidicolin, was employed to synchronize large-scale suspension cultures (10⁹ cells) of murine L1210 leukemia cells. On the basis of the doubling time and cell cycle distribution for logarithmically growing L1210 cells, a synchronization protocol was devised involving a temporal sequence of two 12-h exposures to aphidicolin, separated by an 6-h interval in drug-free medium. After the second aphidicolin treatment, resuspension of cells into drug-free medium resulted in the rapid onset of DNA synthesis as assessed by [³H]thymidine incorporation and DNA fluorescence with flow cytometry. By 6 h after aphidicolin removal, the cells progressed into the G2-M phase and cell division was initiated. DNA synthesis was minimal during this time and remained low through 9 h when the majority of the cells were in G1 phase. Only low levels of cytotoxicity were observed when L1210 cells were treated with aphidicolin in this fashion. The levels of both thymidylate synthase and dihydrofolate reductase were relatively constant during cell cycle transit, following release from the aphidicolin blockade. Similarly, the levels of the corresponding mRNA transcripts for these enzymes, measured by Northern blot hybridizations, remained essentially unchanged through most of the cell cycle, increasing approximately twofold only as the cells entered G1 phase. Whereas intracellular dihydrofolate reductase catalytic activity was relatively unchanged throughout the cell cycle, as reflected in the metabolism of [³H]folic acid to reduced folate forms, a marked increase in in situ thymidylate synthase activity occurred during S phase that was tightly linked to the rate of DNA synthesis. These findings suggest that the regulation of thymidylate synthesis following synchronization with aphidicolin occurs primarily through effects on catalytic activity rather than enzyme synthesis or degradation. Conversely, both dihydrofolate reductase levels and catalytic activity appear to be relatively independent of DNA synthesis in this model. This simple method of synchronizing large numbers of cultured L1210 cells should be equally applicable to the study of a variety of cell cycle-dependent processes in other cultured lines.

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^☆: Supported by research Grants CA-39807, CA-09340, and HLBI-07110 from the National Institutes of Health, Grant CH 435 from the American Cancer Society, and a grant from the Children's Leukemia Foundation of Michigan. Dr. Westin is a recipient of an American Cancer Society Clinical Oncology Career Development Award.

²: Recipient of a Junior Faculty Research Award (JFRA 133) from the American Cancer Society.

³: Current address: Division of Clinical Toxicology, Department of Medicine, Medical College of Virginia, Richmond, VA 23298.

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A method for the synchronization of cultured cells with aphidicolin: Application to the large-scale synchronization of L1210 cells and the study of the cell cycle regulation of thymidylate synthase and dihydrofolate reductase☆

Abstract

Exp. Cell Res

FEBS Lett

J. Biol. Chem

J. Biol. Chem

J. Biochem. Biophys. Methods

J. Biol. Chem

Arch. Biochem. Biophys

Anal. Biochem

J. Mol. Biol

J. Biol. Chem

Biochem. Biophys. Res. Commun

J. Biol. Chem

J. Biol. Chem

Cytometry

Nucleic Acids Res

Cytometry