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Synchronization of replicons in Ehrlich ascites cells.

V Gekeler¹, H Probst

¹Physiologisch-Chemisches Institut der Universität Tübingen, Federal Republic of Germany.

Experimental Cell Research

|March 1, 1988

Summary

This study developed a protocol to synchronize Ehrlich ascites cells for DNA replication studies. Hypoxia and cycloheximide effectively suppressed and synchronized DNA replication initiation and growth.

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Area of Science:

Cell Biology
Molecular Biology
Biochemistry

Background:

Synchronizing cell populations is crucial for studying DNA replication dynamics.
Previous methods for cell cycle synchronization have limitations in maintaining high synchrony.

Purpose of the Study:

To establish a reliable protocol for obtaining highly synchronous Ehrlich ascites cells at the beginning of S phase.
To investigate the effects of hypoxia and cycloheximide on DNA replication initiation and elongation.

Main Methods:

Zonal centrifugation for G1 cell selection.
Controlled hypoxia and reaeration.
Cycloheximide treatment.
Monitoring thymidine incorporation, labeling indices, and mitotic indices.
DNA fiber autoradiography and alkaline sucrose gradient analysis.

Main Results:

A 12-hour hypoxia followed by reaeration and immediate cycloheximide addition yielded highly synchronous cell cycles.
Both hypoxia and cycloheximide suppressed DNA replication (replicon) initiation without affecting DNA chain elongation or maturation.
Cycloheximide's suppression of initiation was less reversible than hypoxia's.

Conclusions:

The developed protocol effectively synchronizes Ehrlich ascites cells for S phase studies.
Hypoxia and cycloheximide are potent inhibitors of DNA replication initiation, offering tools to study replication control mechanisms.

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