G2-checkpoint abrogation in irradiated lymphocytes: A new cytogenetic approach to assess individual radiosensitivity and predisposition to cancer

Abstract

Abstract. Increased yield of chromatid breaks, following in vitro G2-phase lymphocyte irradiation, can be a marker of individual radiosensitivity and cancer predisposing genes whose role is to respond to DNA damage. Mutations or polymorphisms of genes encoding DNA repair pathways may underlie the increased chromosomal radiosensitivity. However, genes that facilitate DNA damage recognition, using signal transduction pathways to activate cell cycle arrest and preserve genomic integrity, are perhaps the most important determinant. Based on the latter hypothesis, an individual radiosensitivity parameter (IRP) is introduced, which expresses, at individual level, the G2-checkpoint potential to facilitate DNA damage recognition and repair of radiation-induced chromosomal damage during G2 to M-phase transition. Based on this parameter a new methodology for assessment of individual radiosensitivity is proposed, which involves G2-checkpoint abrogation by caffeine to obtain the IRP values. To evaluate the proposed methodology, blood samples from 52 healthy donors were taken for inter-individual radiosensitivity analysis using both the conventional G2 chromosomal radiosensitivity assay as well as the new approach using caffeineinduced G2-checkpoint abrogation. The two assays were compared in experiments using samples from 5 hypersensitive patients, 3 AT-homozygotes, 3 AT-heterozygotes, and the GM15786, GM03188A, GM09899, HCC1937 and MCF-7 cell lines. Using the G2 chromosomal radiosensitivity assay, donors are predicted as G2 radiosensitive or normal, while according to the new approach, individuals can be classified as highly radiosensitive, radiosensitive, normal, radioresistant and highly radioresistant. Overall, the new approach provides better individual radiosensitivity discrimination and intraexperimental reproducibility. Therefore, the proposed methodology using IRP values may provide a clinically applicable predictive assay for individual radiosensitivity and predisposition to cancer.

Individual Radiosensitivity Assessment of the Families of Ataxia-Telangiectasia Patients by G2-Checkpoint Abrogation

“…11 Likewise, Terzoudi et al introduced a new approach to G2/M cell cycle checkpoint abrogation for assessing IRS using caffeine treatment; this method demonstrated better discrimination and minimal intra-individual and inter-laboratory variations compared to the conventional G2-assay. 19,20 Claes et al reported the use of a modified S-G2 micronucleus assay in which caffeine treatment formed a complementary assay for the identification of atypical A-T patients and heterozygous ATM carriers. 29 Prodosmo et al proposed a new, fast and cost-effective test to determine ATM zygosity based on p53 centrosomal localisation visualised by immunofluorescence staining.…”

“…20,22 On the other hand, some of the obligate heterozygotes may have had a range of ATM mutations; consequently, these individuals may, like A-T patients, have exhibited various degrees of radiosensitivity both in vitro and clinically. [7][8][9][10][11]19,25,28,29 Furthermore, technical issues in different laboratories can intensify such variations. 22 Finally, the assay was not specific for ATM mutations and may therefore have identified mutations in other genes which modify DNA repair system efficiency.…”

“…18 Terzoudi et al reported that caffeine treatment increased G2-assay sensitivity and reduced interlaboratory and intra-experimental variations. 19 Similarly, Pantelias et al proposed a standardised caffeine-treated G2-assay for predicting individual radiosensitivity (IRS). 20 This study aimed to evaluate the ability of a modified caffeine-treated G2 breakage assay to identify heterozygous ATM carriers in the families of A-T patients.…”

Individual Radiosensitivity in Oncological Patients: Linking Adverse Normal Tissue Reactions and Genetic Features

“…With few exceptions, chromatid aberration yields were obtained by scoring for chromatid breaks and gaps 50 metaphases per culture, under a Zeiss AxioImager Z2 microscope coupled with MSearch-AutoCapt software (Metasystems, Altlussheim Germany). Following calculation of the in vitro individual radiosensitivity index (IRS = [1-(G 2caf -G 2 )/G 2caf ] × 100%, simplified as IRS = (G 2 /G 2caf ) × 100%) patients were classified as: highly radiosensitive, HRS (IRS > 70), radiosensitive, RS (50 < IRS ≤ 70), normal, N (30 ≤ IRS ≤ 50), and radioresistant, RR (IRS < 30) (15, 29).…”

Association study of miR-22 and miR-335 expression levels and G2 assay related inherent radiosensitivity in peripheral blood of ductal carcinoma breast cancer patients

“…Enhanced CA after irradiation has been detected in ~40% of BC patients while only in ~10% of healthy individuals [9,10]. G2 assay, a widely used method for the study of radiosensitivity, is in vitro irradiation of peripheral blood lymphocytes in G2 phase of cell cycle to create DNA damage, which is often repaired during G2 to M-phase transition, residual lesions can be observed and measured at metaphase as CA [11]. The high frequencies of chromosomal aberrations expressed following G2 exposure significantly differentiate between radiosensitive and nonradiosensitive cells or individuals [12][13][14][15][16].…”