Study of aberrations in the DNA of cancer cells (MCF7), following irradiation and use of the inhibitor NU7026

82 σ.Οι βλάβες στο DNA των κυττάρων διακρίνονται από ποικιλία και κυμαινόμενη πολυπλοκότητα. Οι πιο σημαντικές από άποψη δυσκολίας επιδιόρθωσης είναι οι διπλές θραύσεις της έλικας (double strand breaks, DSBs) και τα συμπλέγματα βλαβών που δεν περιλαμβάνουν τις διπλές θραύσεις, ή αλλιώς ‘οξειδωτικώς επαγόμενες ομαδοποιημένες βλάβες DNA’ (non-DSB oxidatively-induced clustered DNA lesions, OCDLs). Λόγω της πολυπλοκότητάς τους, αυτού του είδους οι ανωμαλίες στο DNA είναι εν δυνάμει προοίμια καρκίνου, αλλά παραδόξως χρησιμοποιούνται και στην αντιμετώπισή του, για την καταστροφή καρκινικών κυττάρων. Η επιδιόρθωση των DSBs συμβαίνει κατά κύριο λόγο μέσω του μηχανισμού της μη ομόλογης επιδιόρθωσης τελικής σύνδεσης (non homologous end joining, NHEJ). Θέση-κλειδί στη διαδικασία κρατάει η DNA-εξαρτώμενη πρωτεϊνική κινάση (DNA-dependent protein kinase). Για να μελετηθεί ο ρόλος αυτής της πρωτεΐνης στην επιδιόρθωση των βλαβών σε ανθρώπινα καρκινικά κύτταρα του μαστού MCF7, χρησιμοποιήθηκε, σε συνδυασμό με ιονίζουσα ακτινοβολία (ακτίνες γ, 1 Gy), ο DNA-PK αναστολέας NU7026, ο οποίος αδρανοποιεί χημικά την DNA-PK. Η δράση του αναστολέα μελετήθηκε σε δύο επίπεδα, στο DNA με χρήση της φωσφορυλιωμένης ιστόνης γΗ2ΑΧ ως σηματοδότη και στα χρωμοσώματα των κυττάρων σε συνδυασμό με την καφεΐνη, γνωστή ουσία για την κατάργηση του σημείου ελέγχου της G2/M φάσης. Για την ανίχνευση και την ποσοτικοποίηση των βλαβών χρησιμοποιήθηκε το ανοσοϊστοχημικό πρωτόκολλο σηματοδότησης της γΗ2ΑΧ και κυτταρογενετική ανάλυση στο οπτικό μικροσκόπιο, αντίστοιχα. Σε κάθε περίπτωση, η παρεμπόδιση του επιδιορθωτικού μηχανισμού NHEJ μέσω της αδρανοποίησης της βασικής εμπλεκόμενης πρωτεΐνης DNA-PK, έδειξε συσσώρευση σημαντικού αριθμού βλαβών 24 ώρες μετά την ακτινοβόληση και συνδυασμένη, προσθετική δράση αναστολέα και καφεΐνης, αντίστοιχα. Οι πληροφορίες που προέκυψαν είναι σημαντικές για την κατανόηση της δράσης της DNA-PK και κατά συνέπεια του επιδιορθωτικού μονοπατιού NHEJ, τον ρόλο τους στην ανάπτυξη καρκίνου του μαστού, αλλά και στην βελτιστοποίηση της ραδιοθεραπείας και εξέλιξη νέων αντικαρκινικών φαρμάκων.The damage accumulated in the genome of cells is diverse and varies in complexity. From a repair difficulty point of view, the most complex types of lesions are double strand breaks (DSBs) and non-DSB oxidatively-induced clustered DNA lesions (OCDLs). Because of their complexity, these types of aberrations in DNA are potentially cancer preludes, while paradoxically they are being used in the fight against it at the same time, to kill cancer cells. The DSB repair is mediated primarily through the non-homologous end joining pathway (NHEJ). A key protein, orchestrating this repair mechanism, is the DNA-dependent protein kinase (DNA-PK). To study the role of this particular key protein in repairing complex DNA damage induced in human breast cancer cells MCF7, we used 1 Gy of γ-irradiation combined with the action of a novel DNA-PK inhibitor, NU7026, which chemically deactivates the kinase activity of DNA-PK. The effect of the inhibitor was studied at two levels, in DNA using the phosphorylated histone γ-Η2ΑΧ as a damage marker, and in chromosomes in combination with caffeine, a well-documented abrogator of the G2/M checkpoint of the cell cycle. For the detection and the evaluation of the damage we used the immunohistochemistry protocol of γΗ2ΑΧ staining and cytogenetic analysis in the optical microscope, respectively. In any case, the NHEJ pathway blocking through the inactivation of its key protein regulator DNA-PK, showed a significant accumulation of damage 24 hrs post-irradiation, and an additive effect when combined with caffeine, respectively. The information resulting from the experiments bear great importance regarding the understanding of the DNA-PK function and consequently the NHEJ repair pathway, their implication in cancer development, but also the monitoring of radiotherapy and development of new anti-cancer drugs.Δανάη-Βασιλική Α. Λασκαράτο

Key mechanisms involved in ionizing radiation-induced systemic effects. A current review

International audienceOrganisms respond to physical, chemical and biological threats by a potent inflammatory response, aimed at preserving tissue integrity and restoring tissue homeostasis and function. Systemic effects in an organism refer to an effect or phenomenon which originates at a specific point and can spread throughout the body affecting a group of organs or tissues. Ionizing radiation (IR)-induced systemic effects arise usually from a local exposure of an organ or part of the body. This stress induces a variety of responses in the irradiated cells/tissues, initiated by the DNA damage response and DNA repair (DDR/R), apoptosis or immune response, including inflammation. Activation of this IR-response (IRR) system, especially at the organism level, consists of several subsystems and exerts a variety of targeted and non-targeted effects. Based on the above, we believe that in order to understand this complex response system better one should follow a 'holistic' approach including all possible mechanisms and at all organization levels. In this review, we describe the current status of knowledge on the topic, as well as the key molecules and main mechanisms involved in the 'spreading' of the message throughout the body or cells. Last but not least, we discuss the danger-signal mediated systemic immune effects of radiotherapy for the clinical setup

Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET)

Detrimental effects of ionizing radiation (IR) are correlated to the varying efficiency of IR to induce complex DNA damage. A double strand break (DSB) can be considered the simpler form of complex DNA damage. These types of damage can consist of DSBs, single strand breaks (SSBs) and/or non-DSB lesions such as base damages and apurinic/apyrimidinic (AP; abasic) sites in different combinations. Enthralling theoretical (Monte Carlo simulations) and experimental evidence suggests an increase in the complexity of DNA damage and therefore repair resistance with linear energy transfer (LET). In this study, we have measured the induction and processing of DSB and non-DSB oxidative clusters using adaptations of immunofluorescence. Specifically, we applied foci colocalization approaches as the most current methodologies for the in situ detection of clustered DNA lesions in a variety of human normal (FEP18-11-T1) and cancerous cell lines of varying repair efficiency (MCF7, HepG2, A549, MO59K/J) and radiation qualities of increasing LET, that is γ-, X-rays 0.3–1 keV/μm, α-particles 116 keV/μm and 36Ar ions 270 keV/μm. Using γ-H2AX or 53BP1 foci staining as DSB probes, we calculated a DSB apparent rate of 5–16 DSBs/cell/Gy decreasing with LET. A similar trend was measured for non-DSB oxidized base lesions detected using antibodies against the human repair enzymes 8-oxoguanine-DNA glycosylase (OGG1) or AP endonuclease (APE1), that is damage foci as probes for oxidized purines or abasic sites, respectively. In addition, using colocalization parameters previously introduced by our groups, we detected an increasing clustering of damage for DSBs and non-DSBs. We also make correlations of damage complexity with the repair efficiency of each cell line and we discuss the biological importance of these new findings with regard to the severity of IR due to the complex nature of its DNA damage

Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET)

<p>Detrimental effects of ionizing radiation (IR) are correlated to the varying efficiency of IR to induce complex DNA damage. A double strand break (DSB) can be considered the simpler form of complex DNA damage. These types of damage can consist of DSBs, single strand breaks (SSBs) and/or non-DSB lesions such as base damages and apurinic/apyrimidinic (AP; abasic) sites in different combinations. Enthralling theoretical (Monte Carlo simulations) and experimental evidence suggests an increase in the complexity of DNA damage and therefore repair resistance with linear energy transfer (LET). In this study, we have measured the induction and processing of DSB and non-DSB oxidative clusters using adaptations of immunofluorescence. Specifically, we applied foci colocalization approaches as the most current methodologies for the <i>in situ</i> detection of clustered DNA lesions in a variety of human normal (FEP18-11-T1) and cancerous cell lines of varying repair efficiency (MCF7, HepG2, A549, MO59K/J) and radiation qualities of increasing LET, that is γ-, X-rays 0.3–1 keV/μm, α-particles 116 keV/μm and ³⁶Ar ions 270 keV/μm. Using γ-H2AX or 53BP1 foci staining as DSB probes, we calculated a DSB apparent rate of 5–16 DSBs/cell/Gy decreasing with LET. A similar trend was measured for non-DSB oxidized base lesions detected using antibodies against the human repair enzymes 8-oxoguanine-DNA glycosylase (OGG1) or AP endonuclease (APE1), that is damage foci as probes for oxidized purines or abasic sites, respectively. In addition, using colocalization parameters previously introduced by our groups, we detected an increasing clustering of damage for DSBs and non-DSBs. We also make correlations of damage complexity with the repair efficiency of each cell line and we discuss the biological importance of these new findings with regard to the severity of IR due to the complex nature of its DNA damage.</p