Sandra Angèle

Functional consequences of ATM sequence variants for chromosomal radiosensitivity.

The ATM [for ataxia‐telangiectasia (A‐T) mutated] protein plays a key role in the detection and cellular response to DNA double‐strand breaks. Several single‐nucleotide polymorphisms (SNPs) have been described in the ATM gene; however, their association with cancer risk or radiosensitivity remains to be fully established. In this study, the functional consequences of specific ATM SNPs on in vitro radiosensitivity, as assessed by micronuclei (MN) formation, were measured in lymphoblastoid cell lines established from 10 breast cancer (BC) patients carrying different ATM missense SNPs, six A‐T patients, six A‐T heterozygotes (A‐T het), and six normal individuals. The BC, A‐T het, and A‐T cell line groups showed significantly higher mean levels of MN formation after exposure to ionizing radiation (IR) than did the group containing normal cell lines, with similar levels in the BC and A‐T het groups. Within the BC lines studied, the group composed of the six carrying the linked 2572T>C (858F>L) and 3161C>G (1054P>R) variants had a higher level of MN after IR exposure compared to that observed in the remaining four BC or in the normal cell lines. This increase was not related to the constitutive ATM mRNA level, which was similar in these BC and the normal cell lines. Our results indicate that alterations in the ATM gene, including the presence of heterozygous mutations and the 2572C and 3161G variant alleles, are associated with increased in vitro chromosomal radiosensitivity, perhaps by interfering with ATM function in a dominant‐negative manner.

ATM polymorphisms as risk factors for prostate cancer development

The risk of prostate cancer is known to be elevated in carriers of germline mutations in BRCA2, and possibly also in carriers of BRCA1 and CHEK2 mutations. These genes are components of the ATM-dependent DNA damage signalling pathways. To evaluate the hypothesis that variants in ATM itself might be associated with prostate cancer risk, we genotyped five ATM variants in DNA from 637 prostate cancer patients and 445 controls with no family history of cancer. No significant differences in the frequency of the variant alleles at 5557G>A (D1853N), 5558A>T (D1853V), ivs38-8t>c and ivs38-15g>c were found between the cases and controls. The 3161G (P1054R) variant allele was, however, significantly associated with an increased risk of developing prostate cancer (any G vs CC OR 2.13, 95% CI 1.17–3.87, P=0.016). A lymphoblastoid cell line carrying both the 3161G and the 2572C (858L) variant in the homozygote state shows a cell cycle progression profile after exposure to ionising radiation that is significantly different to that seen in cell lines carrying a wild-type ATM gene. These results provide evidence that the presence of common variants in the ATM gene, may confer an altered cellular phenotype, and that the ATM 3161C>G variant might be associated with prostate cancer risk.

The ATM gene and breast cancer: is it really a risk factor?

The genetic determinants for most breast cancer cases remain elusive. Whilst mutations in BRCA1 and BRCA2 significantly contribute to familial breast cancer risk, their contribution to sporadic breast cancer is low. In such cases genes frequently altered in the general population, such as the gene mutated in Ataxia telangiectasia (AT), ATM may be important risk factors. The initial interest in studying ATM heterozygosity in breast cancer arose from the findings of epidemiological studies of AT families in which AT heterozygote women had an increased risk of breast cancer and estimations that 1% of the population are AT heterozygotes. One of the clinical features of AT patients is extreme cellular sensitivity to ionising radiation. This observation, together with the finding that a significant proportion of breast cancer patients show an exaggerated acute or late normal tissue reactions after radiotherapy, has lead to the suggestion that AT heterozygosity plays a role in radiosensitivity and breast cancer development. Loss of heterozygosity in the region of the ATM gene on chromosome 11, has been found in about 40% of sporadic breast tumours. However, screening for ATM mutations in sporadic breast cancer cases, showing or not adverse effects to radiotherapy, has not revealed the magnitude of involvement of the ATM gene expected. Their size and the use of the protein truncation test to identify mutations limit many of these studies. This latter parameter is critical as the profile of mutations in AT patients may not be representative of the ATM mutations in other diseases. The potential role of rare sequence variants within the ATM gene, sometimes reported as polymorphisms, also needs to be fully assessed in larger cohorts of breast cancer patients and controls in order to determine whether they represent cancer and/or radiation sensitivity predisposing mutations.

Altered expression of DNA double-strand break detection and repair proteins in breast carcinomas

Aims:  To determine whether the expression of DNA damage detection and repair proteins is frequently altered in breast carcinomas.

Radiation, DNA damage and cancer

The characterization of the rare, radiation-sensitive and cancer-prone syndromes, ataxia telangiectasia and Nijmegen breakage syndrome, has demonstrated that genetic predisposition increases the risk of developing cancer after exposure to ionizing radiation (IR). Molecular analyses of these disorders provide valuable insights into the normal function of these two gene products in the cellular response to IR-induced DNA damage. Their contribution to a cellular radiosensitive phenotype and their role in sporadic cancers can now be fully assessed. For example, the gene ataxia telangiectasia mutated (ATM) has recently been shown to be a tumour suppressor gene in T-cell prolymphocytic leukaemia, and there is increasing evidence that individuals with one mutated ATM or Nijmegen breakage syndrome (NBS1) allele have an increased predisposition to cancer.

ATM protein overexpression in prostate tumors: possible role in telomere maintenance.

It has been postulated that telomere dysfunction and telomerase activation have important roles in prostate tumorigenesis. Since the ataxia-telangiectasia mutated gene product (ATM protein) is involved in maintaining telomere length and integrity, we hypothesized that its expression might be altered in prostate tumors and, thus, examined its profile in 49 tumor samples. The majority (32/49) had ATM protein levels higher than those observed in normal tissues, with only 5 of 49 tissue samples showing reduced or absent ATM levels. Three of these were from the group of 6 young-onset or sibling-pair tumors. There was a trend toward higher ATM expression in tumors with a higher Gleason score (23/32 [72%] for grade 8-10 vs 9/17 [53%] for grades 5-7), although this difference was not statistically significant. These findings support our hypothesis that the presence of the ATM protein at the same or a higher level than that in normal prostate cells might have an important role in the maintenance of the shortened telomeres commonly found in prostate cancer cells.

The molecular causes of low ATM protein expression in breast carcinoma; promoter methylation and levels of the catalytic subunit of DNA-dependent protein kinase

Aims:  To investigate whether aberrant methylation of the ATM promoter or loss of the catalytic subunit of DNA‐dependent protein kinase (DNA‐PKcs) may be the underlying causes of reduced ATM protein levels often seen in breast tumours.

Variation in radiation-induced apoptosis in ataxia telangiectasia lymphoblastoid cell lines

Purpose : To investigate and compare the ability of Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL) from healthy individuals (normals) and ataxia telangiectasia (A-T) patients to undergo apoptosis after exposure to ionizing radiation. Materials and methods : Four normal and eight A-T LCL were exposed to doses of up to 20 Gy ionizing radiation. Apoptosis induction was studied 24 h after irradiation using three different methods: measurement of caspase-3 activity, PARP-1 cleavage and estimation of the sub-G 1 cell fraction. Results : Of the eight A-T LCL tested, all harbouring truncating ATM mutations, five had a higher level of spontaneous apoptosis than the normal LCL as assessed by the sub-G 1 cell fraction. Four of the eight A-T LCLs showed a similar level of radiation-induced apoptosis after exposure to 5 Gy as the normal LCL. The other four A-T LCL showed a greater radiation-induced apoptotic response, as assessed by at least one of the three techniques. Conclusions : LCL from A-T patients can undergo ionizing radiation-induced apoptosis in spite of a defect in ATM-p53-dependent signalling pathways. However, the apoptotic response is characterized by a large degree of variability between the A-T cell lines, the causes of which remain to be established.

ATM Protein Overexpression in Prostate Tumors

It has been postulated that telomere dysfunction and telomerase activation have important roles in prostate tumorigenesis. Since the ataxia-telangiectasia mutated gene product (ATM protein) is involved in maintaining telomere length and integrity, we hypothesized that its expression might be altered in prostate tumors and, thus, examined its profile in 49 tumor samples. The majority (32/49) had ATM protein levels higher than those observed in normal tissues, with only 5 of 49 tissue samples showing reduced or absent ATM levels. Three of these were from the group of 6 young-onset or sibling-pair tumors. There was a trend toward higher ATM expression in tumors with a higher Gleason score (23/32 [72%] for grade 8-10 vs 9/17 [53%] for grades 5-7), although this difference was not statistically significant. These findings support our hypothesis that the presence of the ATM protein at the same or a higher level than that in normal prostate cells might have an important role in the maintenance of the shortened telomeres commonly found in prostate cancer cells.

Re: correspondence from Dr. Michael Swift, Disease Insight Research Foundation, concerning Gutiérrez-Enríquez S, Fernet M, Dörk T, Bremer M, Lauge A, Stoppa-Lyonnet D, Moullan N, Angèle S, Hall J, “Functional consequences of the ATM sequence variants for chromosomal radiosensitivity”

We welcome the opportunity to comment on the issues raised by Dr. Swift based on our article Gutiérrez-Enrı́quez et al. (2004), “Functional consequences of the ATM sequence variants for chromosomal radiosensitivity”. In this article, we reported on our investigation of whether certain variants in the ATM gene are associated with altered in vitro chromosomal radiosensitivity exploiting the micronucleus formation assay to provide evidence for increased sensitivity in carriers of some but not all ATM missense variants. This project is part of ongoing research in our laboratories aimed at investigating the relationship between sequence variants and cancer risk and in particular whether such genetic variants have functional consequences that may modify the cellular response to DNA damaging agents. It was our intention neither to obscure known facts about the association of ataxia-telangiectasia (A-T) mutations and breast cancer predisposition nor to claim priority for hypothesizing this association. The sentence about “our” hypothesis is far from representing a priority claim and was simply meant to designate “the” hypothesis that was the initial reason for “our” research study. As we did state in our article, several epidemiological studies on A-T families over the past 30 years have shown clear evidence that heterozygous carriers of A-T-causing mutations have an increased breast cancer risk. We are well aware of the excellent article by Athma et al. (1996), which falls into this category, and its omission in the reference list was an oversight on our part for which we apologize. We fully agree with Dr. Swift in the opinion that the epidemiological evidence for an association of A-T heterozygosity with breast cancer is strong. But we also have to recognize that an association between ATM gene mutations and breast cancer has been demonstrated in some but not all published studies outside the setting of A-T families. These breast cancer patients screened have been a heterogeneous group, including breast cancer cases with a strong family history or early onset of disease, unselected cases from the general population, and cases who show unusually high sensitivity to therapeutic radiation. As mentioned by Dr. Swift, some studies are based on small numbers of cases and controls, and with hindsight, some have used suboptimal mutation detection technology in the sense that the full spectra of ATM mutations could not have been detected. While the conclusions that can be drawn from these latter studies may be limited in terms of associations with breast cancer risk, they have allowed the frequency of truncating ATM mutations to be documented and thus make an important contribution to our understanding of the impact of this mutation type in several populations. To rank the usefulness of a particular study is a difficult task, and the categories may to some extent depend on the viewpoint. In this respect, we are intrigued by Swift’s stating that the publication by Atencio et al. (2001) can be considered both as a rigorous study and as one that uses inadequate mutation detection technology. Dr. Swift takes us to task over the introductory sentence of our discussion in which we state that “the relationship between the presence of ATM mutations or sequence variants and breast cancer predisposition remains controversial in part because of the low frequency at which such alterations have been detected in this population and the uncertainty of their biological and functional significance.” We still feel that this introductory sentence reflects the published literature at the present time on the association between common sequence variants in the ATM gene and breast cancer predisposition, but we also recognize that if misquoted and thus taken out of context, it could be misleading. One important issue that arises from a review of the published studies is the mixed use of terms such as ATM “mutations”, “polymorphisms,” and “sequence variants,” which are not interchangeable, to describe the different sequence alterations