¹²⁵IdUrd-induced Chromosome Fragments, Assayed by Premature Chromosome Condensation, and DNA Double-strand Breaks Have Similar Repair Kinetics in G₁-phase CHO-cells

Abstract

The effect of 125I-decay on cell lethality, and induction of chromosome and DNA damage, was studied in synchronous non-cycling, G1-phase CHO-cells. For this purpose a population of mitotic cells was allowed to divide and progress through S-phase in the presence of 125IdUrd. Cells were subsequently transferred to conditioned medium (C-med) obtained from plateau-phase cultures that allowed cells to divide and accumulate in G1-phase in a non-cycling state. To accumulate 125I-induced damage, cells were kept frozen at -80 degrees C. Freezing was carried out using a new method that optimally preserves cell integrity. After various times of cold storage, cells were thawed and assayed for survival, DNA and chromosome damage, either immediately or after various times in C-med. Neutral filter elution was used to assay repair of DNA double-strand breaks (dsbs), and premature chromosome condensation was used to assay repair of chromosome fragments and induction of ring chromosomes. The results indicate very little repair at the cell survival level (repair of PLD). At the DNA level an efficient repair of DNA dsbs was observed, with kinetics similar to those observed after exposure to X-rays. At the chromosome level a fast repair of prematurely condensed chromosome fragment was observed, with a concomitant increase in the number of ring chromosomes induced. The repair kinetics of chromosome fragments and DNA dsbs were very similar, suggesting that DNA dsbs may underlie chromosome fragmentation.

Repair of x-ray-induced DNA double-strand breaks in specific Not I restriction fragments in human fibroblasts: joining of correct and incorrect ends.

“…Despite the discrepancy in initial yields, however, there are strong correlations between measured radiation-induced dsbs and PCC breaks. The rejoining kinetics for PCC breaks and DNA dsbs are similar (10), and in addition radiosensitive mutants with defects in dsb rejoining show deficiencies in rejoining PCC breaks (11,12). Such results strongly suggest that DNA dsbs are indeed the primary initial…”

“…Since the resolution of the assay used here is much higher, on the order of 100 kbp, this difference is perhaps not surprising. However, it must also be considered in this connection that the experiments for quantitating dsb misrejoining frequencies in this study were necessarily performed at much higher doses than experiments scoring chromosomal aberrations, which are typically 10 Gy or less. The possibility that fidelity of dsb rejoining varies with dose remains, although no dose effect was detected between 80 and 160 Gy in this study, and measurements of mutation frequency have suggested a linear dose-response relationship even to very low doses (21).…”

Radiosensitivity Throughout the Cell Cycle and Repair of Potentially Lethal Damage and DNA Double-strand Breaks in an X-ray-sensitive CHO Mutant

“…Mitotic cells were routinely frozen for later use by a method that optimally preserves cell integrity (Borrelli et al . 1987, Iliakis et al 1987 . The experiments described here were performed with cells that were on subculture 41 on the day of roller bottle preparation .…”

Antimelanoma and radioprotective activity of alcoholic aqueous extract of different species ofOcimumin C57BL mice

“…The DNA DSBs induced by X-rays are repaired by the time these cells reach the first postirradiation mitosis. However, the repair is error-prone, leading to structural chromosomal aberrations (Lliakis et al, 1987). Experimental evidence suggests that DNA DSBs are among the primary lesions responsible for the induction of chromosome aberrations and cell death in mammalian cells (Bryant, 1985(Bryant, , 1988.…”