FP7-PEOPLE-2011-CIG MC-CIG - Career Integration Grants - GRANT 303514


We have created a model based on extensive experimental and bioinformatical work , for the systemic DNA damage especially in the case of repair deficient organisms. More details for this extensive work can be found in the published paper  http://www.ncbi.nlm.nih.gov/pubmed/26873647 :

 Fig. 1  A schematic putative model of proposed stress mediators regulating radiation-induced bystander effects (RIBE) and systemic responses as summarized in this review. These effects are expected to be high in the case of repair deficient cells!

 Exposure of cells to ionizing radiation induces high levels of complex DNA damage thus necessitating the induction of DNA damage response and repair (DDR/R) at levels exceeding the standard background levels due to endogenous damage. ROS and RNS produced by directly irradiated cells may transmit to bystander cells either through gap junction intercellular communication (GJIC) or they are released extracellularly in a nitric oxide (NO)-dependent way. Additionally, the inflammatory cytokines released by irradiated cells can bind to bystander cells through their corresponding membrane receptors, initiating specific signaling pathways (JAK2-STAT3, NF-κB, MAPK) subsequently leading to the induction of cytokines, COX2 and NOSs expression. Moreover, irradiated cells that undergo apoptosis due to extensive DNA damage may emit ‘danger’ signals to bystander cell that seem to play a significant role in the transmission of this stress system-wide and at distant sites (systemic response). In this case, an innate immune response is required. Radiation toxicity and effects will result as the integration of targeted (irradiated cells) and non-targeted effects in non-irradiated cells and tissues. The possible synergistic effect from other types of stresses like DNA replication or oncogene-induced stress will further enhance the pathway to genomic instability and cancer development through the activation of various molecular switches like Cdc6 and others.


 Fig. 2 DNA damage and apoptosis asymmetry as regulators of NTE: a putative model.

A. Description of our biophysical hypothesis where the DNA damage (DD) and apoptosis (AP) changes with distance from irradiation point (area: center 1). This DNA damage and apoptosis gradient probably plays one of the most important roles in bystander and systemic signaling.

B. A depth-dose distribution of a 200 MeV proton therapy beam (Fig. 5) where the systemic responses are evaluated calculating the DNA damage changes with distance from the center of the beam and the so called Bragg Peak (decreasing dose from ~2 → ~0.1 Gy).


In addition towards the better understanding of the processing of clustered DNA lesions in the human cell we have developed a new methodology for the detection of non-DSB lesion in situ as shown below and can be found in the recently accepter paper  http://www.ncbi.nlm.nih.gov/pubmed/26082923

And also regarding the critical role of DSB repair inhibitors in http://www.ncbi.nlm.nih.gov/pubmed/26306465


Figure 5: A) Principles of colocalization between foci of dissimilar sizeColocalization exists if the ratio of B foci number per A nucleus area is greater than the ratio of B foci number per cell nucleus area.  B) Realistic example of colocalization between APE1 and γ-H2AX foci in human HepG2 cells irradiated with high-LET Ar heavy ions (Ar-36, LET 269.4 keV/µm). In our case γ-H2Ax forms large and bright red foci with clear boundaries, while APE1 gives a punctuate and diffused staining, forming numerous small “foci”, even in case of non-irradiated cells. In an attempt of using a freely available software, we have performed the analysis using the Jcount software (courtesy of Dr. Pavel Lobachevsky group, Peter McCallum Institute, Australia). As expected a significant increase in the colocalization of APE1 foci with the DSB focus is observed for all irradiated samples compared to non-irradiated (0 Gy). (A)-(D): 0 Gy 1h. (E)-(I): 1 Gy 1h. (A)&(E) γ-H2AX foci (per cell). (B)&(F) APE1 foci (per cell). (C)&(G) complex γ-H2AX foci that would serve as the area for estimation APE1 foci per complex γ-H2AX focus in (D)&(I).

Link to publications


Proposal acronym: DSBR and clusters

Duration (months): 48

Proposal title: Processing of oxidatively induced clustered DNA lesions under a double strand break repair deficiency in human tumor cells


Although a number of risk factors associated with cancer have been well established for many years, there is an emerging need for delineation of the role of DNA repair factors relative to cancer risk, and especially the discovery and quantification of risks associated with gene mutations in double strand break (DSB) repair factors (e.g. BRCA1, DNA-PK, Lig4, XRCC4). In addition, higher levels of oxidative stress (reactive oxygen species, ROS), DNA damage and/or defective DNA repair have been reported in different malignancies and tumors. The long term objective of this project is to identify the role(s) of BRCA1 (homologous recombination-HR) and DNA-PK (non-homologous end joining-NHEJ) two key double strand break repair (DSBR) proteins in the processing of non-DSB oxidatively induced single and clustered DNA lesions (OCDLs) in human gamma-irradiated tumor cells. In addition, chromosomal instability and apoptosis will be measured in each case.

There are very limited and only fragmentary data on this specific field. Based also on preliminary data, our central hypothesis is that a compromised DSB repair pathway will also lead to deficient OCDL repair and accumulation of oxidatively-generated DNA lesions when DSBR deficient cells are challenged by various oxidizing agents like ionizing radiation. These studies are expected to provide meaningful mechanistic insights into DNA repair pathways involved in the processing of non-DSB clusters and therefore advance the field. They will also contribute to cancer etiology associated with complex DNA damage since accumulation of OCDLs is associated with increased mutation rate and chromosomal instability.

Fingerprinting the molecular identity of these DNA modifications can be utilized in designing more efficient cancer therapeutic strategies based on the concept of selective apoptotic activation in malignant cells after exposure to therapeutic sources of oxidative stress like radiation and chemotherapy drugs.

Actions so far:

We have already worked in the following aims 1 and 2. (download A) (download B)

We have already worked in following aims 1-3. (download A) (download B)


(links in file)

Repair of Clustered DNA lesions in Human Breast cancer cells under a DSB repair deficiency<!--[if !mso]> <![endif]--><!--[if gte mso 10]> <![endif]-->



MCF-7 human breast cancer  cells exposed to 1 Gy of γ-rays (IR) with or without the specific DNA-PK inhibitor NU7026. The cells were allowed to repair damage at 37 oC for the indicated times 0-24 hrs.    

Double strand breaks were followed by means of γ-H2AX foci. In the case of cells exposed to radiation with the inhibitor (NU+IR) we can see an accumulation of foci in much higher levels than without the inhibitor (only IR). Ctrl: Non-irradiated cells; NU7026 : non-irradiated just with the inhibitor; IR: Irradiated without the inhibitor and NU+IR: Irradiated with the inhibitor during also repair.