Hemizygosity for Atm and Brca1 influence the balance between cell transformation and apoptosis

Abstract

Background: In recent years data from both mouse models and human tumors suggest that loss of one allele of genes involved in DNA repair pathways may play a central role in genomic instability and carcinogenesis. Additionally several examples in mouse models confirmed that loss of one allele of two functionally related genes may have an additive effect on tumor development. To understand some of the mechanisms involved, we examined the role of monoallelic loss or Atm and Brca1 on cell transformation and apoptosis induced by radiation.

Methods: Cell transformation and apoptosis were measured in mouse embryo fibroblasts (MEF) and thymocytes respectively. Combinations of wild type and hemizygous genotypes for ATM and BRCA1 were tested in various comparisons.

Results: Haploinsufficiency of either ATM or BRCA1 resulted in an increase in the incidence of radiation-induced transformation of MEF and a corresponding decrease in the proportion of thymocytes dying an apoptotic death, compared with cells from wild-type animals. Combined haploinsufficiency for both genes resulted in an even larger effect on apoptosis.

Conclusions: Under stress, the efficiency and capacity for DNA repair mediated by the ATM/BRCA1 cell signalling network depends on the expression levels of both proteins.

Figure 1

DNA damage measured with alkaline comet assay. Total DNA damage measured with alkaline comet assay points to the higher background DNA damage in the hemizygous genotypes. The data is from three independent experiments where total of 100 cells/genotype were scored.

Figure 2

Induction of micronuclei by graded doses of radiation. Induction of micronuclei by graded doses of radiation in mouse embryo fibroblasts having different genetic backgrounds. Data are shown as a mean and standard error from 3 independent experiments. At a dose of 3 Gy of γ-rays, there is a statistically significant difference between the double hemizygous and the other genotypes.

Figure 3

CD4⁺ CD8⁺ cell survival after γ-ray irradiation. A) CD4⁺ CD8⁺ cell survival after γ-ray irradiation. Cell survival was highest (apoptosis was lowest) in the double hemizygous background. In contrast the percent of CD4⁺ CD8⁺ cells does not depend of the genotype in nonirradiated cells. The numbers of mice used was three per genotype for the controls and five per genotype for the irradiated mice. B) Representative image of flow cytometry of the thymocytes. Top panel: Atmwt/Brca1wt genotype. CD4⁺ CD8⁺ cells appear at the upper right quadrant and are 31% of the total cell numbers. Note also the very low numbers of CD4⁺ and CD8⁺ cells which appear in the lower right and upper left quadrants. Lower panel represents Atmhz/Brca1hz genotype where 61% of the double positive CD4⁺ CD8⁺ cells survived accompanied also with high numbers of CD4⁺ and CD8⁺ cells (lower left and upper right quadrants).