Effects of cold treatment on synchronous Chinese hamster cells treated in mitosis

Abstract

Chinese hamster mitotic cells (95% in mitosis with 85% in metaphase and 10% in anaphase) were shaken loose from monolayer asynchronous cultures and stored at 1°C for up to 28 hours. During this period, the mitotic index did not decrease and the cells remained cytologically normal. However, over a four‐hour period, metaphase cells located within 1.8 minutes of anaphase, 10% of the metaphase population, were able to move into anaphase; this point of 1.8 minutes corresponds in time to that reported for the spindle activation marker. When the cells were warmed to 37°C, they were delayed in entering G₁ and S (35 and 70 minutes, respectively, after a 4‐hour treatment). This delay of 70 minutes was maintained for three cell cycles, during which a high degree of synchrony was maintained. Cold treatment for 12 hours produced delays into G₁ and S of 50 and 110 minutes, respectively. A fraction of the metaphase cells (12 or 50% after treatments of 4 or 12 hours, respectively) evidenced chromosomal aggregation, were unable to complete cytokinesis, and appeared in the next division as tetraploid cells. These tetraploid cells were unable to survive and produce macroscopic colonies. It is concluded that this decrease in viability is caused by irreversible effects on the spindle and/or centriolar components in metaphase cells located prior to the spindle activation marker.