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

Larry H. Matherly, John D. Schuetz, Eric Westin, I. David Goldman

Research output: Contribution to journalArticle

47 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)338-345
Number of pages8
JournalAnalytical Biochemistry
Volume182
Issue number2
DOIs
StatePublished - Nov 1 1989
Externally publishedYes

Fingerprint

Aphidicolin
Cultured Cells
Cell Cycle
Synchronization
Cells
DNA
Catalyst activity
Tetrahydrofolate Dehydrogenase
G1 Phase
Folic Acid
Cell Division
Nucleic Acid Synthesis Inhibitors
Leukemia L1210
Thymidylate Synthase
Flow cytometry
G2 Phase
Enzymes
Cytotoxicity
thymidylate synthase-dihydrofolate reductase
S Phase

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

@article{3d037b59c3a04dd1beeedb3e1a580748,
title = "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 = "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.",
author = "Matherly, {Larry H.} and Schuetz, {John D.} and Eric Westin and Goldman, {I. David}",
year = "1989",
month = "11",
day = "1",
doi = "10.1016/0003-2697(89)90605-2",
language = "English (US)",
volume = "182",
pages = "338--345",
journal = "Analytical Biochemistry",
issn = "0003-2697",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - A method for the synchronization of cultured cells with aphidicolin

T2 - Application to the large-scale synchronization of L1210 cells and the study of the cell cycle regulation of thymidylate synthase and dihydrofolate reductase

AU - Matherly, Larry H.

AU - Schuetz, John D.

AU - Westin, Eric

AU - Goldman, I. David

PY - 1989/11/1

Y1 - 1989/11/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0024462244&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024462244&partnerID=8YFLogxK

U2 - 10.1016/0003-2697(89)90605-2

DO - 10.1016/0003-2697(89)90605-2

M3 - Article

C2 - 2514611

AN - SCOPUS:0024462244

VL - 182

SP - 338

EP - 345

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 2

ER -