Rad52 inactivation is synthetically lethal with BRCA2 deficiency.

Synthetic lethality is a powerful approach to study selective cell killing based on genotype. We show that loss of Rad52 function is synthetically lethal with breast cancer 2, early onset (BRCA2) deficiency, whereas there was no impact on cell growth and viability in BRCA2-complemented cells. The frequency of both spontaneous and double-strand break-induced homologous recombination and ionizing radiation-induced Rad51 foci decreased by 2-10 times when Rad52 was depleted in BRCA2-deficient cells, with little to no effect in BRCA2-complemented cells. The absence of both Rad52 and BRCA2 resulted in extensive chromosome aberrations, especially chromatid-type aberrations. Ionizing radiation-induced and S phase-associated Rad52-Rad51 foci form equally well in the presence or absence of BRCA2, indicating that Rad52 can respond to DNA double-strand breaks and replication stalling independently of BRCA2. Rad52 thus is an independent and alternative repair pathway of homologous recombination and a target for therapy in BRCA2-deficient cells.

Mice heterozygous for mutation in Atm, the gene involved in ataxia-telangiectasia, have heightened susceptibility to cancer.

Ataxia-telangiectasia is characterized by radiosensitivity, genome instability and predisposition to cancer. Heterozygous carriers of ATM, the gene defective in ataxia-telangiectasia, have a higher than normal risk of developing breast and other cancers. We demonstrate here that Atm 'knock-in' (Atm-Delta SRI) heterozygous mice harboring an in-frame deletion corresponding to the human 7636del9 mutation show an increased susceptibility to developing tumors. In contrast, no tumors are observed in Atm knockout (Atm(+/-)) heterozygous mice. In parallel, we report the appearance of tumors in 6 humans from 12 families who are heterozygous for the 7636del9 mutation. Expression of ATM cDNA containing the 7636del9 mutation had a dominant-negative effect in control cells, inhibiting radiation-induced ATM kinase activity in vivo and in vitro. This reduces the survival of these cells after radiation exposure and enhances the level of radiation-induced chromosomal aberrations. These results show for the first time that mouse carriers of a mutated Atm that are capable of expressing Atm have a higher risk of cancer. This finding provides further support for cancer predisposition in human ataxia-telangiectasia carriers.

Cancer stem cells - from initiation to elimination, how far have we reached? (Review).

Cancer stem cells (CSCs) are rare tumor cells that have the potential to proliferate, self-renew and induce tumorigenesis. Over the past few years, CSCs have been isolated from several different tumors and when implanted into immune-deficient mice, generate tumors that are identical to the parental tumors. In this review, we summarize the current literature on CSCs, which suggests that since these cells have the ability to drive tumor formation, specifically targeting them may lead to more effective therapies against tumors.

One-step site-directed mutagenesis of ATM cDNA in large (20kb) plasmid constructs.

In vitro mutagenesis of large genes has proven to be difficult for a number of reasons, including the number of steps involved and the instability of large inserts. We describe here a single-step PCR method to introduce mutations into an ataxia-telangiectasia mutated (ATM) gene cDNA construct (20 kb). This involved several modifications of the Stratagene QuikChange Site-Directed Mutagenesis Kit. Four sites implicated in the function of ATM were mutagenised in a 20 kb plasmid, improving upon existing methods capable of mutagenesis in DNA constructs up to 13 kb, while maintaining a high efficiency of mutagenesis (85-100%). This approach makes it possible to introduce multiple mutations into large cDNA for structural/functional studies.

Analyzing the regulation and function of ATM.

We describe here the cloning of full-length ataxia-telangiectasia mutated (ATM) cDNA and characterization of its activity. Full-length ATM cDNA is cloned into an inducible EBV-based vector (pMEP4) and its expression analyzed in a stably transfected cell line. ATM protein induction is monitored by immunoblotting with antibodies against both ATM and a FLAG sequence tag in the recombinant protein. Extracts from irradiated cells are immunoprecipitated with anti-ATM antibodies, and protein kinase activity is measured using p53(1-44)-specific substrate or by immunoblotting extracts with an anti-phosphoserine 15 p53-specific antibody. Missense mutations affecting ATM kinase activity are detected using in vitro mutagenesis of ATM cDNA followed by the procedures outlined above.

Disassembly of MDC1 foci is controlled by ubiquitin-proteasome-dependent degradation.

The orderly recruitment, retention, and disassembly of DNA damage response proteins at sites of damaged DNA is a conserved process throughout eukaryotic evolution. The recruitment and retention of DNA repair factors in foci is mediated by a complex network of protein-protein interactions; however, the mechanisms of focus disassembly remain to be defined. Mediator of DNA damage checkpoint protein 1 (MDC1) is an early and key component of the genome surveillance network activated by DNA double-strand breaks (DSBs). Here, we investigated the disassembly of MDC1 foci. First, we show that ubiquitylation directs the MDC1 protein for proteasome-dependent degradation. Ubiquitylated MDC1 associates with chromatin before and after exposure of cells to ionizing radiation (IR). In addition, increased MDC1 ubiquitylation in the chromatin fraction is observed in response to IR, which is correlated with a reduction in total MDC1 protein levels. We demonstrate that blocking MDC1 degradation by proteasome inhibitors leads to a persistence of MDC1 foci. Consistent with this observation, chromatin immunoprecipitation experiments reveal increased MDC1 protein at site-specific DSBs. Interestingly, we show that the persistence of MDC1 foci is associated with an abrogated recruitment of the downstream factor BRCA1 in a manner that is RNF8 independent. Collectively, the evidence presented here supports a novel mechanism for the disassembly of MDC1 foci via ubiquitin-proteasome dependent degradation, which appears to be a key step for the efficient assembly of BRCA1 foci.

The cellular control of DNA double-strand breaks.

DNA double-strand breaks (DSBs) are the most hazardous lesions arising in the genome of eukaryotic organisms, and yet occur normally during DNA replication, meiosis, and immune system development. The efficient repair of DSBs is crucial in maintaining genomic integrity, cellular viability, and the prevention of tumorigenesis. As a consequence, eukaryotic cells have evolved efficient mechanisms that sense and respond to DSBs and ultimately repair the break. The swiftness of the DNA DSB response has paved to the identification of sensors and transducers which allowed to generate a hierarchical signaling paradigm depicting the transduction of the damage signal to numerous downstream effectors (Fig. 1). The function of such effectors involve posttranslational modifications through phosphorylation, acetylation, and methylation of the substrates. This review will address the control of DSBs in damaged eukaryotic cells, the physiological processes that require the introduction of a DSB into the genome, and the maintenance of DSBs in non-damaged cells.

Missense mutations but not allelic variants alter the function of ATM by dominant interference in patients with breast cancer.

The human genetic disorder ataxia-telangiectasia (A-T) is characterized by hypersensitivity to ionizing radiation and an elevated risk of malignancy. Epidemiological data support an increased risk for breast and other cancers in A-T heterozygotes. However, screening breast cancer cases for truncating mutations in the ATM (A-T mutated) gene has failed largely to reveal an increased incidence in these patients. It has been hypothesized that ATM missense mutations are implicated in breast cancer, and there is some evidence to support this. The presence of a large variety of rare missense variants in addition to common polymorphisms in ATM makes it difficult to establish such a relationship by association studies. To investigate the functional significance of these changes we have introduced missense substitutions, identified in either A-T or breast cancer patients, into ATM cDNA before establishing stable cell lines to determine their effect on ATM function. Pathogenic missense mutations and neutral missense variants were distinguished initially by their capacity to correct the radiosensitive phenotype in A-T cells. Furthermore missense mutations abolished the radiation-induced kinase activity of ATM in normal control cells, caused chromosome instability, and reduced cell viability in irradiated control cells, whereas neutral variants failed to do so. Mutant ATM was expressed at the same level as endogenous protein, and interference with normal ATM function seemed to be by multimerization. This approach represents a means of identifying genuine ATM mutations and addressing the significance of missense changes in the ATM gene in a variety of cancers including breast cancer.

Quantitative analysis of the expression of ACAT genes in human tissues by real-time PCR.

ACAT (also called sterol o-acyltransferase) catalyzes the esterification of cholesterol by reaction with long-chain acyl-CoA derivatives and plays a pivotal role in the regulation of cholesterol homeostasis. Although two human ACAT genes termed ACAT-1 and ACAT-2 have been reported, prior research on differential tissue expression is qualitative and incomplete. We have developed a quantitative multiplex assay for each ACAT isoform after RT treatment of total RNA using TaqMan real-time quantitative PCR normalized to beta-actin in the same reaction tube. This enabled us to calculate the relative abundance of transcripts in several human tissues as an ACAT-2/ACAT-1 ratio. In liver (n = 17), ACAT-1 transcripts were on average 9-fold (range, 1.7- to 167-fold) more abundant than ACAT-2, whereas in duodenal samples (n = 10), ACAT-2 transcripts were on average 3-fold (range, 0.39- to 12.2-fold) more abundant than ACAT-1. ACAT-2 was detected for the first time in peripheral blood mononuclear cells. Interesting differences in ACAT-2 mRNA expression were evident in subgroup analysis of samples from different sources. These results demonstrate quantitatively that ACAT-1 transcripts predominate in human liver and ACAT-2 transcripts predominate in human duodenum and support the notion that ACAT-2 has an important regulatory role in liver and intestine.