Mutagenic effects of ionizing radiation on AL hybrid cells in different compartments of the cell cycle

Abstract

I used the AL assay to investigate mutant induction and spectra during the cell cycle. AL cells are human X hamster hybrids containing one human chromosome II and a normal complement of CHO chromosomes. On the short arm of chromosome II, at IIpI3, is the selection gene for the AL assay that codes for the CD59 surface antigen. CD59 mutants survive an antibody/complement attack that lysis wild-type cells expressing the antigen. The advantage of the AL assay is that the human chromosome provides a large non-lethal target allowing analysis of mutational spectra from a few base pairs to 1.6 X 108 bases. Centrifugal elutriation was used to collect large populations of AL cells enriched for early G1. The resultant elutriated cells were incubated and allowed to progress in phase through the cell cycle. Timing of the replication of the CHO and human chromosomes was analyzed using flow cytometry, bromodeoxyuridine (BrdU) labeling, and fluorescent in-situ hybridization (FISH). The analyses showed that S phase and DNA replication of the CHO and human chromosomes began at 5.5 hours after plating the early G1 cells and continued through 12.5 hours. Most of human chromosome II replicated between 6.5 to 8.5 hours, with the remainder replicating from 8.5 to 12.5 hours. Mutation was measured as the loss of expression of the human CD59 antigen. AL cells were irradiated in specific cell cycle compartments with 137CS-γ to determine the number and spectra of mutants produced. When irradiated, G1 cells were most susceptible to killing, followed by S phase cells. G2 cells were the most resistant to cell killing. Irradiated with 3 Gy and with background subtracted, early G1 cells produced 426 mutants/105/surviving cell (dose/LD50). In S phase, mutant induction dropped to 68 mutants/105/ surviving cell, and G2 phase produced as little as 43 mutants/105/surviving cell. Mutant induction was low when the CD59 gene was replicating. The mutant spectra were determined throughout the cell cycle. Mega-base pair deletions were much more frequent than intragenic mutations in all phases of the cycle. Mutant induction "hot-spots" were discerned on the human chromosome during different phase of the cell cycle.