Consequences of thebleed-throughphenomenon in immunofluorescence of proteins forming radiation-induced nuclear foci

Abstract

Through this report, we would like authors to consider more carefully protein co-localizations by performing systematically, before any co-immunofluorescence, immunofluorescence of each protein separately to avoid bleed-through artifacts.

DNA double-strand break repair defects in syndromes associated with acute radiation response: At least two different assays to predict intrinsic radiosensitivity?

“…Nevertheless, the involvement of MRE11 in the NHEJ process is still controversial (Di Virgilio & Gautier 2005). Furthermore, our findings shown here are based on separate immunofluorescence and we have suggested recently that co-immunofluorescence of pH2AX and MRE11 may introduce biases due to the bleed-through fluorescence phenomenon (Rénier et al 2007). Interestingly, hyperrecombination, spontaneous breaks, abnormally large nuclei and impaired formation of radiation-induced MRE11 foci are common features of group II syndromes and ATM-mutated cells (German 1993, Savitsky et al 1995, Grompe & D'Andrea 2001, Cleaver 2005 Such common phenotype may support therefore an uncontrolled nuclease activity of MRE11 occurring in absence of ATM, FANC, BLM, and/or XPD proteins, leading to genomic instability, cancer proneness, spontaneous breaks and chromatin relaxation but to a moderate impact upon cellular response to radiation that would explain the moderate radiosensitivity of group II cells ( Figure 5).…”

“…Data provided by techniques based on the discrimination of DNA fragments by their size like PFGE, comet assay and neutral elution result in these two antagonistic phenomena: when NHEJ is deficient, DSB repair curves describe a plateau; when hyperrecombination occurs, DSB repair curves tend to the X-axis. Hence, NHEJ defects and hyperrecombination occurring concomitantly may hide DSB effectively involved in cell death (Joubert & Foray 2007). This might be notably the case of ATM-mutated cells that show yields of unrepaired DSB about 2 -4 times lower than LIG4-mutated cells when analysed with PFGE, despite similar cellular radiosensitivity (Badie et al 1995a, 1995b, Foray et al 1997a.…”

What Does the History of Research on the Repair of DNA Double-Strand Breaks Tell Us?—A Comprehensive Review of Human Radiosensitivity

“…This is notably the case for γH2AX and MRE11 [60], γH2AX and 53BP1 [61], or γH2AX and DNA-PK [12]. However, in immunofluorescence technique, the emission and/or absorption spectra of immunofluorescence dyes (often red and green) are not strictly separated, which causes biased co-localization (the bleed-through phenomenon) [60]. Hence, a number of authors who have researched the co-localization of two biomarkers have not verified whether the co-immunofluorescence data were consistent with the foci kinetics obtained from separated immunofluorescence.…”

“…The diversity of immunofluorescence biomarkers that formed nuclear foci has not facilitated the elucidation of consensual mechanisms of DSB repair: to date, nearly all the foci-making proteins were found to co-localize. This is notably the case for γH2AX and MRE11 [60], γH2AX and 53BP1 [61], or γH2AX and DNA-PK [12]. However, in immunofluorescence technique, the emission and/or absorption spectra of immunofluorescence dyes (often red and green) are not strictly separated, which causes biased co-localization (the bleed-through phenomenon) [60].…”

“…Hence, the protein partnership and the mechanistic models that are deduced from it may be biased by the bleed-through phenomenon whose occurrence is clearly independent of any confocal or non-confocal technology. Hence, particular care must be taken with co-immunofluorescence data and the resulting mechanistic models [60].…”