Dissertation committee:
Δοντά Αικατερίνη, Αναπλ. Καθηγήτρια, Τμήμα Οδοντιατρικής, Σχολή Επιστημών Υγείας, ΕΚΠΑ.
Βοργιάς Κωνσταντίνος, Καθηγητής, Τμήμα Βιοχημείας, Σχολή Θετικών Επιστημών, ΕΚΠΑ.
Τσιχλάκης Κώστας, Καθηγητής, Τμήμα Οδοντιατρικής, Σχολή Επιστημών Υγείας, ΕΚΠΑ.
Μαργαρίτης Λουκάς, Ομότιμος Καθηγητής, Τμήμα Βιολογίας, Σχολή Θετικών Επιστημών, ΕΚΠΑ.
Γκρίτζαλης Παναγιώτης, Επίκουρος Καθηγητής, Τμήμα Οδοντιατρικής, Σχολή Επιστημών Υγείας, ΕΚΠΑ.
Παπαδάκης Ευάγγελος, Επίκουρος Καθηγητής, Τμήμα Οδοντιατρικής, Σχολή Επιστημών Υγείας, ΕΚΠΑ.
Πιπέρη Ευαγγελία, Επίκουρη Καθηγήτρια, Τμήμα Οδοντιατρικής, Σχολή Επιστημών Υγείας, ΕΚΠΑ.
Summary:
Purpose
The aim of this thesis is to study specific focal points related to the response and repair of DNA after ionizing radiation in a human cellular range and in conditions similar to radiation conditions and dose received by the epithelial cells of the oral epithelium of the patients examined. Molecular markers are related to:
• The cellular cycle due to the possible induction of the P21WAF1 protein, which is an indicator of inhibition of cell proliferation in the G1 phase of the cell cycle,
• The viability of irradiated cells as determined by using the MTT assay from 30 minutes up to 48 hours after irradiation,
• The detection of DNA double helix fractures through the detection of the phosphoryated form of the histone γH2Ax
• Activation of the genome repair high-fidelity mechanism with an homologous recombination triggered by the protein BRCA1 and the participation of the RAD51 protein
• The induction of cellular apoptosis as detected by the analysis of the induction of the tumor suppressor gene p53 and
• The levels of movement activity induced by the stress of cells, e.g. such as CK1 kinases, regulating the functions of p53, BRCA1 and Rad51 through phosphoryosis in special positions.
Materials and methods
For the irradiation of cells, a plexiglass (phantom) device was designed and constructed to simulate the actual irradiation conditions of the patient.
The above construction simulating the human head consists of two concentric cylinders between which there is deionized water. In the phantom there is a socket to accept up to three plates 8 cm in diameter or a cell culture box, in which the cells are placed for irradiation, according to the proposed radiation doses of the CBCT. Prior to irradiation of the cells, the phantom device was calibrated with thermoluminesccal dosimeters (TLD). Radiation was measured by dosimeters (TLD) which were placed on both the inner and outer surface of the cylinder and at different points of the phantom. Irradiation was carried out under similar conditions to the patient's irradiation conditions, the dosimeters was collected and the absorbed dose was measured in the Dosimetry Laboratory of the C.E.O. Democritus. The proposed irradiation conditions correspond to doses taken by an adult person of average physique.
The irradiations of a large number of cells were performed at least at two independent times, in triple samples, and the biological material was used to study the various parameters. The irradiation of the cells was done in the Come Beam Dental Computed Tomography Newtom 9000 QR Verona Italy 110KV, located in the Dental School of the University of Athens, at the maximum FOV (field of view) 15cm diameter with elements 3.2 mA and 17sec irradiation time. Also, to have a positive witness for the experiments, our experimental material was irradiated at doses of 2 Gy (Gray) per minute. The irradiation of the cells was carried out with a source of cobalt-60 (60Co) GammaCell 220 Irradiator (Atomic Energy of Canada Ltd, Ottawa, Canada, January 1974), located at the premises of the E.K.E.F.E. "Democritus".
In total there were 50 radiations in the CBCT that were needed for all the molecular methods we chose but also to ensure repeatability in our results. GammaCell 220 Irradiator performed 10 such radiations for western blotting and immunofluorescence methods.
After irradiations the cells were incubated for different periods of time depending on the molecular method we chose. The hek 293 cell line was used as an experimental material for this study, which was developed in a simple layer (2D culture) in glass covers. . The cells are allowed to grow until they cover 70-90% of the surface of the cover so that there is no inhibition of cell proliferation and modification of their metabolic processes due to lack of space (contact inhibition) and then irradiated. Each dish contained cells at a concentration of 1-2x105 cells/mL. Non-irradiated cells were used as controls.
The experimental part of the PhD thesis was conducted at the Biochemistry and Molecular Biology Laboratory of the Biology Department of the University of Athens (supervisor Prof. K. E. Vorgias). The laboratory equipment was used for the performance of the experiments, as well as all the necessary materials. More specifically, a combination of cytological and molecular techniques was used (MTT method, Immunofluorescence, Real Time PCR, Western blotting, casein kinase 1 activity study).It should be pointed out that the main part of the study was the detection of short-term lesions and the measurement of direct response of cellular repair mechanisms to complete cell retrieval or the onset of rebate procedures.
Results
• With the MTT method for possible induction of cytotoxicity into the irradiated HEK293 cell line, the results show that cell survival levels after irradiation did not differ significantly from cells not irradiated (control).
• With the Western blotting method for protein expression, Rad51, BRCA1, p21 and p53 in irradiated cells, the results showed an increase in BRCA1 in cells irradiated with 2 Gy at both 20 minutes and 4 hours, while no changes were observed in protein expression in both cells irradiated in CBCT and control cells at all intervals. RAD51 participates in the repair of DNA damage through an homologous recombination. There was a significant increase in the fluorescence intensity of Rad51 0, 5h after irradiation while the fluorescence intensity decreased 24h after irradiation.
• With the Western blotting method for the expression of P-γH2Ax in irradiated cells as well as in controls, the results showed an increase in P-γΗ2Ax in cells irradiated in CBCT in the first half hour compared to cells control. At 6 hours the signal decreases while at 48 hours there are no findings.
• The results of immunofluorescence for intracellular detection of proteins Rad51, BRCA1 and p53, 0.5h and 24h after irradiation in CBCT showed a slight increase in the Rad 51 signal 0,5h after irradiation. 24 hours later it returns to normal. No changes for BRCA1 and P53.
• To detect the creation of fragments of the double strand of DNA by immunofluorescence of the phosphoryled form of γ-H2AX was made in each cell an automated count of the number of foci/nuclear at different intervals. The results showed an increase in the number of foci/nuclear in both the first 20 minutes and 240 minutes while 48h after irradiation in the CBCT no traces of fragment residues of the double strand of DNA were detected, since γ-H2Ax was almost undetectable. The test was carried out for the first time on such a large number of cells (12,000).
• After irradiation of HEK293 cells, total RNA was isolated and seemed to remain intact.
• Using the Real Time PCR method for quantifying the expression of the BRCA1, P21, Rad51 and p53 genes in irradiated cells, the results showed a slight increase in Rad51 half an hour after irradiation in cells irradiated in the CBCT in relation to the control cells. The other genes showed no substantial changes. A decrease in p21 expression was also observed at all intervals after irradiation.
• The study of the activity of casein kinase 1 (CK1) with in vitro phosphorylation of its substrates in cell extracts after irradiation showed that cells irradiated with an increased dose of radiation (2Gy) exhibit increased kinase activity CK1 of both 20min and 240min after irradiation. In contrast, in the cells irradiated in the dental computational tomography, the activity of CK1 kinase is very low.
Conclusions
1) No inhibition of cell proliferation was observed as found with the constant value of p21 levels at all intervals studied after irradiation
2) An increase in the expression of the Rad51 protein gene was observed at 0.5h after irradiation, at 24h by the immunofluorescence method and at 48h western blotting the expression of the Rad51 gene is significantly reduced in irradiated cells by all methods used.
3) The activity of CK1 kinase (which regulates the functions of p53, BRCA1 and Rad51 through phosphorylation in specific locations) was very low in the cells irradiated in the dental computational tomography. In particular, it was lower than the non-irradiated cells, but they had suffered the stress that remained outside the furnace for at least 20 min and as is known the activity of kinase CK1 increases when there is cellular stress.The viability of irradiated cells, as estimated by the use of the MTT test after kinetic analysis over a period of 30 minutes up to 24 hours after irradiation, did not have significant differences in relation to cells not were irradiated (control).
4) The viability of irradiated cells, as assessed using the MTT assay after kinetic analysis over a period of 30 minutes up to 48 hours after irradiation, had no significant differences from the cells not irradiated.
5) No fragment residue signals of DNA DSB 48h after irradiation were detected, since γ-H2Ax was almost undetectable. The test was carried out both through western blotting and through immunofluorescence for the first time in such a large number of cells.
6) No changes in the levels of the BRCA1 protein involved in activating the high-fidelity genome repair mechanism via a recombination bond were observed. The test was carried out via RT-PCR, through immunological quantification as well as through cell immunofluorescence.
7) P53 known as the focal buffer of the cell cycle and cell apoptosis showed no change in the intensity of its fluorescence of both 0.5h and 24h after irradiation, by all methods used.
8) Under the described experimental conditions and using multiple cytological and genetic biomarkers we conclude that the irradiation of epithelial cells for the duration required for the dental examination has created a limited number of double-stranded DNA fragments witch the cells repaired within a reasonable period of time that is less than the cell cycle time. Therefore no significant irreversible harmful lesions were created.
9) It does not appear to take place during the specific irradiation conditions, activation of the high-fidelity repair mechanism as shown by the absence of changes in BRCA levels (analysis with RT-PCR).
