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Assessing 'radiosensitivity' with kinetic profiles of ?-H2AX, 53BP1 and BRCA1 foci.

ABSTRACT:

Background and purpose

DNA repair assays to identify radiosensitive patients have had limited clinical implementation due to long turn-around times or limited specificity. This study evaluates ?-H2AX-Irradiation Induced Foci (IRIF) kinetics as a more rapid surrogate for the 'gold standard' colony survival assay (CSA) using several known DNA repair disorders as reference models.

Materials and methods

Radiosensitive cells of known and unknown etiology were studied. ?-H2AX-IRIFs were quantified over 24 h, and the curves were fitted by combining logarithmic growth and exponential decay functions. Fitted values that differed from radionormal controls were considered aberrant and compared to CSA results.

Results

We observed 87% agreement of IRIF data with the CSA for the 14 samples tested. Analysis of ?-H2AX-IRIF kinetics for known repair disorders indicated similarities between an RNF168(-/-) cell line and an RS cell of unknown etiology. These cell lines were further characterized by a reduction in BRCA1-IRIF formation and G2/M checkpoint activation.

Conclusions

?-H2AX-IRIF kinetics showed high concordance with the CSA in RS populations demonstrating its potential as a more rapid surrogate assay. This method provides a means to globally identify defective DNA repair pathways in RS cells of unknown etiology through comparison with known DNA repair defects.

SUBMITTER: Martin NT

PROVIDER: S-EPMC3202034 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Assessing 'radiosensitivity' with kinetic profiles of γ-H2AX, 53BP1 and BRCA1 foci.

Martin Nathan T NT Nahas Shareef A SA Tunuguntla Rashmi R Fike Francesca F Gatti Richard A RA

Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology 20110630 1

<h4>Background and purpose</h4>DNA repair assays to identify radiosensitive patients have had limited clinical implementation due to long turn-around times or limited specificity. This study evaluates γ-H2AX-Irradiation Induced Foci (IRIF) kinetics as a more rapid surrogate for the 'gold standard' colony survival assay (CSA) using several known DNA repair disorders as reference models.<h4>Materials and methods</h4>Radiosensitive cells of known and unknown etiology were studied. γ-H2AX-IRIFs were ...[more]

PMID: 21722985

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Project description:BackgroundIn response to DNA double-strand breaks, the histone protein H2AX becomes phosphorylated at its C-terminal serine 139 residue, referred to as ?-H2AX. Formation of ?-H2AX foci is associated with recruitment of p53-binding protein 1 (53BP1), a regulator of the cellular response to DNA double-strand breaks. ?-H2AX expression in peripheral blood mononuclear cells (PBMCs) was recently proposed as a diagnostic and disease activity marker for multiple sclerosis (MS).ObjectiveTo evaluate the significance of ?-H2AX and 53BP1 foci in PBMCs as diagnostic and disease activity markers in patients with clinically isolated syndrome (CIS) and early relapsing-remitting MS (RRMS) using automated ?-H2AX and 53BP1 foci detection.MethodsImmunocytochemistry was performed on freshly isolated PBMCs of patients with CIS/early RRMS (n = 25) and healthy controls (n = 27) with ?-H2AX and 53BP1 specific antibodies. Nuclear ?-H2AX and 53BP1 foci were determined using a fully automated reading system, assessing the numbers of ?-H2AX and 53BP1 foci per total number of cells and the percentage of cells with foci. Patients underwent contrast enhanced 3 Tesla magnetic resonance imaging (MRI) and clinical examination including expanded disability status scale (EDSS) score. ?-H2AX and 53BP1 were also compared in previously frozen PBMCs of each 10 CIS/early RRMS patients with and without contrast enhancing lesions (CEL) and 10 healthy controls.ResultsThe median (range) number of ?-H2AX (0.04 [0-0.5]) and 53BP1 (0.005 [0-0.2]) foci per cell in freshly isolated PBMCs across all study participants was low and similar to previously reported values of healthy individuals. For both, ?-H2AX and 53BP1, the cellular focus number as well as the percentage of positive cells did not differ between patients with CIS/RRMS and healthy controls. ?-H2AX and 53BP1 levels neither correlated with number nor volume of T2-weighted lesions on MRI, nor with the EDSS. Although ?-H2AX, but not 53BP1, levels were higher in previously frozen PBMCs of patients with than without CEL, ?-H2AX values of both groups overlapped and ?-H2AX did not correlate with the number or volume of CEL.Conclusion?-H2AX and 53BP1 foci do not seem to be promising diagnostic or disease activity biomarkers in patients with early MS. Lymphocytic DNA double-strand breaks are unlikely to play a major role in the pathophysiology of MS.