Maintenance of genome integrity is essential for homeostasis and survival as impaired DNA damage response (DDR) may predispose to grave pathologies such as neurodegenerative and immunodeficiency syndromes, cancer and premature aging. Therefore, accurate assessment of DNA damage caused by environmental or metabolic genotoxic insults is critical for contemporary biomedicine. The available physical, flow cytometry and sophisticated scanning approaches to DNA damage estimation each have some drawbacks such as insufficient sensitivity, limitation to analysis of cells in suspension, or high costs and demand for trained personnel. Here we present an option how to transform a regular fluorescence microscope and personal computer with common software into a functional alternative to high-throughput screening devices. In two detailed protocols we introduce a new semi-automatic procedure allowing for very sensitive, quantitative, rapid and simple fluorescence image analysis in thousands of adherent cells per day. Sensitive DNA breakage estimation through analysis of phosphorylated histone H2AX (gamma-H2AX), and homologous recombination (HR) assessed by a new RPA/Rad51 dual-marker approach illustrate the advantages and applicability of this technique. Our present data on assessment of low radiation doses, repair kinetics, spontaneous DNA damage in cancer cells, as well as constitutive and replication stress-induced HR events and their dependence on upstream factors within the DDR machinery document the versatility of the method. We believe this affordable approach may facilitate mechanistic insights into the role of low-dose DNA damage in human diseases, and generally promote both basic and translational research in many areas of biomedicine where suitable fluorescence markers are available.
The MRE11, RAD50, and NBS1 genes encode proteins of the MRE11-RAD50-NBS1 (MRN) complex critical for proper maintenance of genomic integrity and tumour suppression; however, the extent and impact of their cancer-predisposing defects, and potential clinical value remain to be determined. Here, we report that among a large series of approximately 1000 breast carcinomas, around 3%, 7% and 10% tumours showed aberrantly reduced protein expression for RAD50, MRE11 and NBS1, respectively. Such defects were more frequent among the ER/PR/ERBB2 triple-negative and higher-grade tumours, among familial (especially BRCA1/BRCA2-associated) rather than sporadic cases, and the NBS1 defects correlated with shorter patients' survival. The BRCA1-associated and ER/PR/ERBB2 triple-negative tumours also showed high incidence of constitutively active DNA damage signalling (gammaH2AX) and p53 aberrations. Sequencing the RAD50, MRE11 and NBS1 genes of 8 patients from non-BRCA1/2 breast cancer families whose tumours showed concomitant reduction/loss of all three MRN-complex proteins revealed two germline mutations in MRE11: a missense mutation R202G and a truncating mutation R633STOP (R633X). Gene transfer and protein analysis of cell culture models with mutant MRE11 implicated various destabilization patterns among the MRN complex proteins including NBS1, the abundance of which was restored by re-expression of wild-type MRE11. We propose that germline mutations qualify MRE11 as a novel candidate breast cancer susceptibility gene in a subset of non-BRCA1/2 families. Our data have implications for the concept of the DNA damage response as an intrinsic anti-cancer barrier, various components of which become inactivated during cancer progression and also represent the bulk of breast cancer susceptibility genes discovered to date.
- MeSH
- DNA vazebné proteiny * analýza genetika MeSH
- enzymy opravy DNA * analýza genetika MeSH
- genetická predispozice k nemoci MeSH
- imunohistochemie MeSH
- lidé MeSH
- mutační analýza DNA MeSH
- nádory prsu * etiologie genetika patologie MeSH
- poškození DNA * genetika MeSH
- protein BRCA1 analýza MeSH
- protein BRCA2 analýza MeSH
- proteiny buněčného cyklu * analýza genetika MeSH
- zárodečné mutace * MeSH
- zdraví rodiny MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
Cíl studie: Analýza erytrocytárních nukleotidů je využívána k diagnostice defektů záchranných cest purinového metabolismu. Cílem této práce je objasnit, zda je toto vyšetření použitelné také v diagnostice defektů druhé poloviny purinové de novo syntézy (PDNS). Ke studiu membránového transportu a následné biotransformace byly použity defosforylované meziprodukty druhé poloviny PDNS. Materiál a metody: Patofyziologická situace u pacientů trpících deficity PDNS byla simulována inkubací nativních erytrocytů, erytrocytárních membrán a lyzovaných erytrocytů s defosforylovanými meziprodukty druhé poloviny PDNS: 5-aminoimidazolribosid (AIr), 5-amino-4-imidazolkarboxribosid (CAIr), 5-amino-4-imidazolsukcinokarboxamidribosid (SAICAr), 5-formamido-4-karboxamidribosid (FAICAr) a 5-amino-4-imidazolkarboxamidribosid (AICAr). Tyto sloučeniny byly chemicky syntetizovány a použity jako standardní látky. Inkubační směsi byly analyzovány pomocí kapilární elektroforézy s UV detekcí pomocí tří separačních systémů, které dovolují analyzovat ribosidy i ribotidy. Výsledky: Jen dva ze studovaných ribosidů (AICAr, FAICAr) prostoupily membránou erytrocytu v detekovatelném množství. Pouze AICAr je vhodným substrátem erytrocytárních kináz a je konvertován na odpovídající mono-, di- a trifosfáty. Závěr: Výsledky ukazují, že analýza erytrocytů je použitelná pouze pro diagnostiku defektu bifunkčního enzymu AICAR- -transformylázy/IMP-cyklohydrolázy.
Objective: Analysis of erythrocyte nucleotides is useful for diagnosing defects in purine salvage pathways. The aim of this work was to elucidate whether the investigation could be used in diagnosing defects of a second part purine de novo synthesis (PDNS). Dephosphorylated intermediates of the second part of PDNS (aminoimidazoleribosides) were used for membrane transport and biotransformation study. Material and Methods: Pathophysiological situation in patients suffering from defects of PDNS was simulated by incubation of native erythrocytes, erythrocyte membranes and erythrocyte lysates with dephosphorylated intermediates of second part of PDNS: 5-amino-4-imidazoleriboside (AIr), 5-amino-4-imidazolecarboxyriboside (CAIr), 5-amino-4- -imidazolesuccinocarboxamideriboside (SAICAr), 5-formylamino-4-imidazolecarboxamideriboside (FAICAr) a 5-amino- -4-imidazolecarboxamideriboside (AICAr). The compounds were synthesized and taken as standard compounds. Incubation mixtures were analyzed by capillary electrophoresis using three separation systems allowing analysis of both ribosides and ribotides. Results: Two of all studied ribosides (AICAr a FAICAr) were able to permeate through the erythrocyte membrane in detectable amounts. AICAr is an acceptable substrate for erythrocyte kinases and is converted to mono-, di- and triphosphates. Conclusions: The results suggest that erythrocytes are only useful for diagnosing AICAR-transformylase/IMPcyclohydrolase deficiency.
