γ-Ray and hydrogen peroxide induction of gene amplification in hamster cells deficient in DNA double strand break repair
Introduction
A hallmark of cancer cells is genomic instability, which leads to the accumulation of genetic lesions [1]. Among these, gene amplification, that is the increase in the copy number of a portion of the genome, is a very frequent finding (reviewed in [2]). DNA amplification is a common mechanism of oncogene activation and can also be involved in the development of resistance to antiproliferative drugs.
In vitro cultured cell lines resistant to drugs because of the amplification of the target gene have been exploited to analyze the molecular mechanisms of gene amplification as well as to investigate genes and exogenous factors playing a role in the process (reviewed in [3], [4], [5], [6], [7]). Initial studies on mouse and hamster cell lines revealed that the amplified DNA can be localized either on small, acentric, autonomously replicating circular chromosomes, named double minutes (DM), or within a chromosome arm generating an expanded region with an abnormal banding pattern (ABR). Structural analysis of intra-chromosomal amplified DNA formed at early stages of the process showed that the selected gene is generally co-amplified with up to megabase long flanking sequences [8], indicating that recombination, rather than replication, based mechanisms are responsible for gene amplification. Moreover, the observation that the amplified units are frequently organized as inverted repeats led to propose intra-chromosomal bridge-breakage-fusion (BBF) cycles as the most likely mechanism driving the early phases of gene amplification [9], [10], [11], [12]. Recent evidence showing that DMs can derive from the breakage of an ABR suggested that the same initial events could be shared by intra- and extra-chromosomal amplification mechanisms [13].
Two main types of events can trigger intra-chromosomal BBF cycles: fusions between the telomeres of two sister chromatids [10] or DNA double-strand breaks (DSBs) which occur telomeric to the selected gene and are followed by the fusion of the broken ends. In both cases, dicentric sister chromatids are formed that can enter BBF cycles.
The role of DSBs in initiating gene amplification is supported by several observations. Pretreatment of the cells with agents that induce DSBs, like for example X-rays, DNA synthesis inhibitors or restriction enzymes, increases the frequency of gene amplification [14], [15], [16], [17], [18], [19], [20], [21]. Moreover, activation of fragile sites close to and telomeric to the selected gene can specifically induce gene amplification [22], [23], [24]. Finally, it has been shown that the induction of a break within an I-Sce1 endonuclease target site [25], previously introduced by transfection close to the DHFR gene of hamster cells, can initiate BBF cycles leading to intra-chromosomal amplification [26].
In mammalian cells, DSBs are mainly repaired through the non-homologous end-joining pathway (NHEJ) (reviewed in [27], [28]). Among the proteins playing a role in NHEJ, the DNA-dependent protein kinase (DNA-PK) complex, formed by the DNA-PK catalytic subunit (DNA-PKcs) and the Ku heterodimer (reviewed in [29]), is fundamental in the first steps of the process. Mutant cells deficient in one of the DNA-PK complex components are defective in DSB repair and in V(D)J recombination and are hypersensitive to DSB inducers. We have recently demonstrated a link between DSB repair and gene amplification [30] by showing an increased amplification propensity in the DNA-PKcs deficient hamster cell line V3 [31], [32] and in immortalized mouse embryo fibroblasts derived from mice in which the DNA-PKcs gene had been ablated by homologous recombination [33].
In this paper, we further investigated the relationship between DSBs, DSB repair and gene amplification by studying the induction of gene amplification by γ-rays and hydrogen peroxide (H2O2) in the V3 line. γ-Rays are well known to induce DSBs, either directly or by forming hydroxyl radicals by water radiolysis [34]. H2O2 forms a wide variety of DNA lesions among which DSBs [35]; H2O2 is generated by different cellular metabolic processes and can be a source of endogenous DNA damage (reviewed in [36], [37]). To evaluate the extent of DNA breakage induced by γ-rays and H2O2 we analyzed chromosomal abnormalities caused by the treatment with the two agents.
Section snippets
Cells and culture conditions
The radiosensitive Chinese hamster cell line V3 and its parental line AA8 were previously described [31], [38]. The cells were cultured in 10 cm petri dishes (Corning) in DMEM (Celbio) supplemented with 10% fetal calf serum, glutamine, non-essential aminoacids, and antibiotics (Hy-clone). Cells were incubated at 37 °C in 3% CO2 atmosphere.
γ-Ray irradiation
Irradiation was carried out using a γ-ray source at a dose rate of 1.3 Gy/min. Exponentially growing cells were trypsinized and resuspended in complete
Induction of gene amplification in hamster cells deficient in DNA-PKcs after treatment with γ-rays or with hydrogen peroxide
To investigate the effect of γ-rays and H2O2 pretreatment on gene amplification in V3 and AA8 parental cells, we analyzed the frequency of PALA resistant clones in cells treated with different doses of γ-rays or H2O2. In rodent cells, PALA resistance is considered a measure of gene amplification since it is mainly due to the amplification of the CAD gene [41]. In particular, in a previous paper we showed that all the PALA resistant clones isolated from AA8 and V3 cells carried amplification of
Discussion
In this paper we analyzed gene amplification after treatment with γ-rays or with H2O2 in the hamster cell lines AA8 and in its DNA-PKcs defective derivative V3.
In the V3 cells, DNA-PKcs deficiency causes an impairment in DSB repair which leads to hypersensitivity to DSB inducers. For this reason, we used doses of the two agents that allowed us to compare the frequency of amplification in the two lines both after the same dose of agent and after doses reducing survival to similar values. After
Acknowledgements
This work was supported by the European Community Grant FIGHT-CT1999-00009. We are very grateful to Prof. A. Faucitano (University of Pavia) for rendering available to us the γ-ray source and for taking care of irradiation. We thank the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute (Bethesda, MD) for the gift of PALA and Daniela Tavarnè for editorial assistance.
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