Effect of passage number on cellular response to DNA-damaging agents: cell survival and gene expression.

Abstract

The effect of different passage numbers on plating efficiency, doubling time, cell growth, and radiation sensitivity was assessed in Syrian hamster embryo (SHE) cells. Changes in gene expression after UV or gamma-ray irradiation at different passage numbers were also examined. The SHE cells were maintained in culture medium for up to 64 passages. Cells were exposed to 60Co gamma rays or 254-nm UV radiation. Differential display of cDNAs and Northern blots were used for the study of gene expression. With increasing passage number, SHE cells demonstrated decreased doubling time, increased plating efficiency, and a decreased yield in the number of cells per plate. Between passages 41 and 48 a 'crisis' period was evident during which time cell growth in high serum (20%) was no longer optimal, and serum concentrations were reduced (to 10%) to maintain cell growth. Sensitivity to ionizing radiation was no different between early- and intermediate-passage cells. However, after UV exposure at low passages (passage 3), confluent cells were more sensitive to the killing effects of UV than were log-phase cells. At intermediate passages (passages 43, 48), confluent cells were slightly more radioresistant than were log-phase cells. By passage 64, however, both confluent and log-phase cells showed similar patterns of UV sensitivity. Expression of gamma-actin, PCNA, and p53 transcripts did not change following UV exposure. p53 mRNA was induced following gamma-ray exposure of the intermediate (passage 45) epithelial cells. The observed differences in radiation sensitivity associated with increasing passage number may be influenced by radiation-induced gene expression. We are conducting experiments to identify these genes.