Mechanisms of the DNA Damage Repair Gene Work on Elevated α/β ratio after Hypofractionated Radiotherapy for Prostate Cancer

doi:10.21203/rs.3.rs-2119746/v1

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Mechanisms of the DNA Damage Repair Gene Work on Elevated α/β ratio after Hypofractionated Radiotherapy for Prostate Cancer

https://doi.org/10.21203/rs.3.rs-2119746/v1

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Purpose: Our previous study showed that the linear quadratic (LQ) model appeared to be inappropriate for high doses per fraction owing to α/β ratio tending to become higher when the dose per fraction increased. In order to verify this conclusion, we explored the mechanisms for the elevated α/β ratio after hypofractionated radiotherapy.

Materials and Methods: We selected two prostate cancer cell lines DU145 and PC3:

1) Draw the cell survival curve to calculate the α/β ratio, and then use biological effective dose (BED) formula to convert fractionated radiation dose into equivalent single hypofractionated radiation dose (calculated values) comparing with that on the survival curve (actual values).

2) Western Blot and laser confocal immunofluorescence were used to detect the expression of γ-H2AX and RAD51 after different fractionated modes of radiation at different time points.

Results:

1) When fractionated radiation dose was converted into equivalent single hypofractionated radiation dose, the ability of hypofractionated radiation was overestimated. If a larger α/β ratio is used, the discrepancy tended to become smaller.

2) Compared with fractionated radiation, the results showed that the expression of γ-H2AX was higher after 30min, 6 h and 24h after single hypofractionated radiation. Meanwhile, the expression of RAD51 lasted for 24 hours and the DNA damage still existed in single hypofractionated radiation.

3) The results also showed that the expression of γ-H2AX decreased slightly after 24 hours of fractionated radiation compared with that of 6 hours, and there was no difference in single hypofractionated radiation between 6 hours and 24 hours.

Conclusions: The results of this study suggest that after single hypofractionated radiation, the irreparable damage in cells increased (that is, α value increased), and some repairable sublethal damage (β value) was converted into irreparable damage (α value). When α value increased and β value decreased, the ratio increased.

α/β ratio

DNA damage

DNA repair

γ-H2AX

RAD51

The main application of α/β ratio in tumor radiobiology was to evaluate the sensitivity of tumors and surrounding tissues to radiation, and then to guide the selection of radiotherapy(1). According to the cell survival curve of the linear quadratic (LQ) model, where α represents irreparable lethal damage; β represents reparable sublethal damage (SLD)(2–5). Therefore, according to the definition of α/β ratio, after DNA damage, if SLD repair occur, α/β ratio decreased (β value increased), the survival curve shifted upward, and cells survival increased. When the repairment did not implement in time, the α/β ratio increased (α value increased or β value decreased), and the survival curve shifted downward, the cells survival decreased. This also explained why the α/β ratio of early reaction tissue is large, and that of late reaction tissue is small.

Our previous study indicated that the α/β ratio was not a constant and increased with the increasing of the single dose per fraction and the LQ model basing on the view that α/β ratio was a constant may not be suitable for a single high dose radiation(6). To confirm this conclusion, we conducted studies at the cytological level in vitro. The LQ model is closely related to the repairment of radiation damage. In this study, we accorded to the definition of α/β ratio (α represented irreparable damage, β represented repairable damage) and explored the mechanisms of the increased α/β ratio after hypofractionated radiation.

DNA damage after ionizing radiation leaded to DNA double-strand breaks (DSB). The expression of the phosphorylated histone H2A variant γ-H2AX, as a marker of DSB, is increased within a few minutes after irradiation and peaked within 30min. γ-H2AX has a linear relationship with the amount of DNA double-strand damage. In other words, the expression of γ-H2AX increases with the increasing of DSB and decreases with the repairment of DSB(7–9). Therefore, the expression of γ-H2AX is a marker of DNA damage.

Homologous recombination (HR) is considered to be an error-free repair mechanism because it uses undamaged DNA double strands to guide repairment of broken DNA double strands and therefore is a high-fidelity repair approach. At the start of homologous recombination repair, ataxia telangiectasis mutated gene (ATM) is activated due to DSB, which phosphorylates the breast cancer 1 gene expressed protein (BRCA1) and is then recruited to the DSB site that has bound to the MRN protein complex (MRE11-RAD50s-NBS.1). BRCA2 is then recruited by BRCA1 to DSB site, where it forms a complex with RAD51 to replace replicating protein A (RPA) to form nuclear protein filamentary. RAD51 is a key protein in homologous recombination that regulates the invasion of homologous sister chromatids and leads to the formation of Holliday junction. The Holliday junction is finally broken down into two DNA double-strand to complete homologous recombination repair(9–11).

Therefore, in this study, the expression levels of γ-H2AX (a DNA double-strand break damage marker) and RAD51 (a key protein in the homologous recombination repair pathway) were detected over time after different fractionated modes and observed the DNA damage repairment after hypofractionated radiation. In this case, according to the definition of the α/β ratio, we explore the reasons why the α/β ratio increased after hypofractionated radiation.

Prostate cancer cell lines DU145 and PC3 (granted from the Department of Nuclear Medicine, Peking University First Hospital) were cultured in RPMI1640 medium containing 10% fetal bovine serum, 1% glutamine and 1% tri-antibodies (penicillin, streptomycin and amphotericin B). Incubator temperature is set at 37℃, CO₂ concentration is 5%, and an appropriate humidity is maintained. When the cells in logarithmic growth stage were taken for experiment.

1. Instruments and devices

The CO2 incubator; Clean bench; Inverted microscope; Medical centrifuge; Microplate tester; -80℃ ultra-low temperature refrigerator; Gel image analysis system; Linear accelerator; Confocal laser microscope; Intelligent thermostatic water tank.

2. Main reagents

1640 medium; phosphate buffered solution (PBS); fetal bovine serum (FBS); glutamine; tri-antibodies (penicillin, streptomycin and amphotericin B); 0.25% EDTA-pancreatic enzymes; 4% paraformaldehyde; crystal violet solution; RIPA lysis buffer; protease inhibitor; phosphatase inhibitor; PMSF; Gel protein electrophoresis kit; Skimmed milk powder; Rabbit monoclonal antibody against γ-H2AX; Rabbit anti-RAD51 monoclonal antibody; Alexa Fluor 488 immunofluorescent antibody; α-Tubulin; HRP-goat anti-rabbit IgG (horseradish peroxidase labeled secondary antibody); HRP- Goat anti-mouse IgG (horseradish peroxidase labeled secondary antibody) ; 1×PBST; Page Ruler Prestained Protein Ladder; PVDF membrane; Anti-fluorescence quenching tablet sealing solution (including DAPI); Protein quantification BCA Kit; Tris-Glycine Buffer electrophoresis solution; transfer buffer; TSBT; 5×SDS loading buffer; 0.5% Triton X – 100; Bovine serum.

3. Clone formation experiment confirmed that the α/β ratio increased after hypofractionated radiation

DU145 and PC3 cells were seeded on 6-well plates at a certain density and 6MV X-ray radiation was given about 12h after the cells were stabilized and adhered to the plates. The irradiation doses were 0Gy, 1Gy, 2Gy, 4Gy, 6Gy, 8Gy, 12Gy and 4Gy/F×2F and 6Gy/F×2F, respectively with the dose rate was 500cGy/min. According to previous research reports, potentially lethal repair has been completed after 4h(12, 13). Considering that prostate cancer tissue is a late response tissue, the interval of radiation was extended to 6h. Cells were cultured in an incubator until 14 days after radiation. The culture medium was changed about 6 days. After 14 days, the cells were removed and rinsed by adding 1mL PBS to each well of the 6-well plates. After PBS was sucked out, 1ml 4% paraformaldehyde was added to each well for fixation; After 20min, paraformaldehyde was sucked out. After paraformaldehyde was air-dried which on the surface of cells, 1mL crystal violet solution was added to each well for staining. After 30min, the crystal violet solution was washed off by running water which on the cell surface. The clone count was performed under the microscope. More than 50 cells are generally considered to be a clone. According to the clone formation rate at different radiation doses, the survival fraction of cells was calculated. Then, used the radiation dose (0Gy, 1Gy, 2Gy, 4Gy, 6Gy, 8Gy and 12Gy) as the abscissa, the survival fraction of cells at each radiation dose was used as the ordinate to draw the cell survival curve using statistical software for curve fitting. Involved formulas were below:

$$\text{P}\text{l}\text{a}\text{t}\text{i}\text{n}\text{g} \text{e}\text{f}\text{f}\text{i}\text{c}\text{i}\text{e}\text{n}\text{c}\text{y}\left(\text{P}\text{E}\right)=\frac{\text{N}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{c}\text{o}\text{l}\text{o}\text{n}\text{i}\text{e}\text{s} \text{c}\text{o}\text{u}\text{n}\text{t}\text{e}\text{d}}{\text{N}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{c}\text{e}\text{l}\text{l}\text{s} \text{s}\text{e}\text{e}\text{d}\text{e}\text{d}}\times 100$$

$$\text{S}\text{u}\text{r}\text{v}\text{i}\text{v}\text{i}\text{n}\text{g} \text{f}\text{r}\text{a}\text{c}\text{t}\text{i}\text{o}\text{n}\left(\text{S}\text{F}\right)=\frac{\text{I}\text{r}\text{r}\text{a}\text{d}\text{i}\text{t}\text{i}\text{o}\text{n} \left(\text{P}\text{E}\right)}{\text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l} \left(\text{P}\text{E}\right)}$$

For fractionated radiation (4Gy/F×2F and 6Gy/F×2F), the cell survival fraction was also obtained according to the clone formation and the corresponding equivalent single hypofractionated radiation dose (actual value) was calculated according to the fitting curve and compared with the equivalent dose (calculated value) calculated by BED formula (n₁d₁ (1 + d₁ /α/β) = n₂d₂ (1 + d₂ /α/β)). The difference was calculated between the calculated value and the actual value, so as to prove the applicability of BED formula in single hypofractionated radiation.

4. Western blot experiment confirmed that the α/β ratio increased after single hypofractionated radiation

DU145 and PC3 cells in logarithmic growth phase were inoculated on a 10cm petri dish. When the cells grew to about 80%, they were irradiated with 8Gy/F×1F and 4Gy/F× 2F, and the interval of irradiated was 6h. Total proteins were extracted 30min, 6h and 24h after irradiation, respectively. The extracted total protein was quantified by BCA method. A protein gel electrophoresis kit was used to prepare the separating gel and stacking gel. The protein weight was 25ug to each well, and 1× loading buffer was used to fill up to 10ul for each well. Gel electrophoresis at constant voltage, stacking gel: 80V constant voltage 30min; Separating gel: 120V constant voltage 90min. After gel electrophoresis, 200mA constant current transfer for 2h was performed to transfer the proteins on the gel to the membrane. After the transfer, the membrane was removed and placed in 5% nonfat dry milk (TBST dissolved) and blocked for 1h. After blocking, the membrane was directly applied to the corresponding primary antibody (RAD51(Cell Signaling Technology, CST): 1:1000; γ-H2AX(CST): 1:1000; α-tubulin (Abcam) 1:7500) was incubated overnight at 4℃. On the second day, TBST was washed three times and the corresponding secondary antibodies (RAD51 and γ-H2AX: goat-anti-rabbit 1:1000; α-tubulin: goat-anti-mouse 1:500) were incubated for 1h, and TBST was washed three times again for chemiluminescence.

5. Laser confocal immunofluorescence experiment confirmed that the α/β ratio increased after single hypofractionated radiation

DU145 and PC3 cells in logarithmic growth phase were inoculated on special laser confocal petri dishes with 2×10⁵ cells in each dish. When the cells adhered to the wall and grew to about 80%, 8Gy/F×1F and 4Gy/F×2F were irradiated with the interval of 6h. The following operations were performed after irradiation 30min, 6h and 24h(14):

1). Rinse with PBS for 3min/f×2f; 2). Fixed: 4% paraformaldehyde (room temperature) for 20min; 3). Rinse with PBS for 5min/f×3f; 4). Permeabilize: 0.5%Triton X-100 treatment (room temperature) for 20min; 5). Rinse with PBS for 5min/f×3f; 6). Serum blocking: 1% bovine serum-PBST (containing 0.05% Tween20) was blocked at room temperature for 20min; 7). Incubated primary antibody: suck out and air-dried blocking buffers and then incubated with γH2AX (CST: 1:400) at 4°C overnight; 8). On the next day, rinse with PBST for 5min/f×3f; 9). Incubated secondary antibody: the secondary antibody is fluorescent antibody (Alexa Fluor 488(CST): 1:1000), which is incubated at room temperature and avoid light for 1h; 10). Rinse with PBST for 5min/f×3f; 11). DAPI stained and anti-fluorescence quencher blocked: each well of special laser confocal petri dishes were blocked with anti-fluorescence quencher containing DAPI, and then observed under a laser confocal microscope.

1. Clone formation experiment confirmed that the α/β ratio increased after single hypofractionated radiation

After clone formation experiment, the survival fraction $S={e}^{-\alpha \cdot d-\beta \cdot {d}^{2}}$ can be obtained and then, take the logarithm for both sides: $\text{ln}\left(s\right)=-\alpha \cdot d-\beta \cdot {d}^{2}$and the survival curves of PC3 and DU145 were drawn as Fig. 1A and 1B.

The survival curve fitting results were as follows: ① PC3: α was 0.1461(95%CI: 0.04089–0.2514), β was 0.08366(95%CI: 0.06830–0.09902), α/β = 1.75Gy, R² = 0.9913 and the fitting equation was $\text{ln}\left(s\right)=-0.15\cdot d-0.08\cdot {d}^{2}$. The survival fractions of 4Gy/F×2F and 6Gy/F×2F of fractionated radiation were corresponding to the curve and obtained corresponding single dose d_4×2=7.19Gy (actual value) and d_6×2=9.16Gy (actual value). At the same time, the α/β = 1.75Gy was substituted into the BED formula: n₁d₁ (1 + d₁/α/β) = n₂d₂ (1 + d₂/α/β). In this formula, n₁ value was 2; d₁ value were 4Gy and 6Gy respectively; n₂ value was 1 and we can calculate the equivalent single dose d_4×2=5.96Gy (calculated value) and d_6×2=8.81Gy (calculated value) according to the BED formula. (2) DU145: α was 0.1981 (95%CI: 0.1381–0.2581), β was 0.05237 (95% CI: 0.04372–0.06103), α/β = 3.78Gy, R² = 0.9949. The fitting equation was $\text{ln}\left(s\right)=-0.20\cdot d-0.05\cdot {d}^{2}$. The survival fractions of 4Gy/F× 2F and 6Gy/F× 2F of fractioned radiation were corresponding to the curve and obtained corresponding single dose d_4×2=7.93Gy (actual value) and d_6×2=10.83Gy (actual value). At the same time, the α/β = 3.78Gy was substituted into the BED formula and obtained equivalent single dose d_4×2=6.22Gy (calculated value) and d_6×2=9.11Gy (calculated value).

As shown in Table 1, for PC3 cells, when the α/β ratio was calculated according to 1.75Gy, fractionated radiation was converted into the equivalent single high dose radiation with the conventional BED formula and the calculated value was different from the actual measured value, the lowest calculated value was only 83% of the actual value. When 4Gy/F× 2F was converted into equivalent single dose, the actual value was 7.19Gy, but after BED formula calculation, it was believed that only 5.96Gy can satisfy clinical needs, which overestimated the ability of single hypofractionated radiation. If it was considered that the α/β ratio increased after single high dose radiation according to our previous research results, then the α/β ratio were 3.46Gy and 4.24Gy respectively substituted into BED formula and then recalculated, Table 2 can be obtained(6).

Table 1

The differences between the calculated values and measured values of PC3 and DU145 cells when converted the fractionated irradiation dose into equivalent single irradiation dose
			Equivalent single dose (Gy)
Cell lines	Fractionated mode	ln (survival fraction )	Calculated value	Measured value	Calculated value/ Measured value (%)
PC3	4Gy/F×2F	-5.22	5.96	7.19	83
PC3	6Gy/F×2F	-8.09	8.81	9.16	96
DU145	4Gy/F×2F	-4.73	6.22	7.93	78
DU145	6Gy/F×2F	-8.03	9.11	10.83	84

Table 2

Converted the fractionated irradiation dose into equivalent single irradiation dose and substituted the calculated values with different α/β values in PC3 cells
		Equivalent single dose (Gy)
Cell lines	Fractionated mode	Calculated value			Measured value	Calculated value/ Measured value (%)
Cell lines	Fractionated mode	α/β = 1.75	α/β = 3.46	α/β = 4.24	Measured value	α/β = 1.75	α/β = 3.46	α/β = 4.24
PC3	4Gy/F×2F	5.96	6.19	6.27	7.19	83	86	87
PC3	6Gy/F×2F	8.81	9.06	9.17	9.16	96	99	100

However, for DU145 cells, the α/β ratio was larger about 3.78Gy. When the fractionated radiation was converted to the equivalent single high dose radiation, the difference between the calculated value and the actual measured value was larger when the BED formula was used, and the lowest value was only 78% of the actual value. When 4Gy/F× 2F is converted into the equivalent single dose, the actual value was 7.93Gy, but after BED formula calculation, it was also believed that only 6.22Gy can satisfy clinical needs and the same conclusion can be drawn. After substituting into BED formula for recalculating with 6Gy and 10Gy of α/β ratio respectively, Table 3 can be obtained.

Table 3

Converted the fractionated irradiation dose into equivalent single irradiation dose and substituted the calculated values with different α/β values in DU145 cells
		Equivalent single dose (Gy)
Cell lines	Fractionated mode	Calculated value			Measured value	Calculated value/ Measured value (%)
Cell lines	Fractionated mode	α/β = 3.78	α/β = 6	α/β = 10	Measured value	α/β = 3.78	α/β = 6	α/β = 10
DU145	4Gy/F×2F	6.22	6.43	6.70	7.93	78	81	84
DU145	6Gy/F×2F	9.11	9.37	9.73	10.83	84	87	90

It can be seen from Tables 2 and 3 that when fractionated radiation dose was converted into a single high radiation dose with conventional BED formula and the actual value was closer if higher α/β ratio used.

2. Western blot experiment confirmed that the α/β ratio increased after a single hypofractionated radiation

The results were shown in Fig. 2A and 2B.

1. At 30min, 6h and 24h after a single high dose radiation of 8Gy/F, the expression of damage marker γ-H2AX was higher than the fractionated radiation of 4Gy/F×2F.

2. The expression of damage marker γ-H2AX was approximately the same after 6h and 24h radiation in the two cell lines.

3. The expression of repair protein RAD51 gradually increased after 30min of radiation and almost stabilized after 6h.

4. At 24h, the expression of RAD51 in both cell lines was nearly the same as that at 6h. After 6h, the repair protein RAD51 was continuously expressed until 24h, while the expression of damage marker γ-H2AX was not decreased, indicating that the existing DNA damage was not repaired. According to Hamasaki et al. most of the damage at this time was irreparable damage(15).

As the expression of γ-H2AX of single high dose radiation was more than that of fractionated radiation after 6h, the irreparable damage of single high dose radiation was also more than that of fractionated radiation. According to the definition of α/β ratio, α value represents irreparable damage, that is, when α value increased, α/β ratio increased.

3. Laser confocal immunofluorescence experiment confirmed that the α/β ratio increased after a single hypofractionated radiation

As shown in Fig. 3, the expression of DNA double-strand break damage marker γ-H2AX was gradually increased in the nucleus after radiation and then decreased with the progress of damage repair. For different fractionated modes, the expression of γ-H2AX in both DU145 cells and PC3 cells increased significantly after single high dose radiation compared with fractionated radiation after 30min, 6h and 24h, indicating DNA damage increased after single hypofractionated radiation.

In order to verify the damage repair after 6h, the expression of γ-H2AX was also detected 24h after radiation and the results were shown in Fig. 3C and 3F. The expression of γ-H2AX was also different 24h after radiation under different fractionated modes: 24h after single high dose radiation (8Gy/F×1F), the expression of γ-H2AX was similar to that of 6h. However, 24h after fractionated radiation (4Gy/F× 2F), the expression of γ-H2AX decreased slightly because there was still a small part of damage repair.

As shown in Fig. 4, the expression of RAD51 gradually increased with the appearance of DNA damage after radiation and decreased with the process of damage repairment. For different fractionated modes, after 6h and 24h, the expression of RAD51 of fractionated radiation was more than that of single high dose radiation; At 24h, the expression of RAD51 irradiated by different fractionated modes was basically the same as that at 6h in DU145 cells. For PC3 cells, after 24h, the expression of RAD51 irradiated by fractionated radiation was slightly lower than that after 6h due to the completion of repair, while the expression of RAD51 after single high dose radiation was basically the same as that after 6h. The survival difference between DU145 cells and PC3 cells after different fractionated modes radiation may be due to the lower α/β ratio of PC3 cells (1.75Gy) and were sensitivity to the change of fractionated dose.

Therefore, for a single high dose radiation, the repair protein RAD51 continued to express after 6h but the damage marker γ-H2AX was not reduced, indicating that the process of damage had been finished and the residual damage was irreparable damage. Meanwhile, some repairable damage may also be converted into irreparable damage. According to the definition of α/β ratio, α value represented irreparable damage, that is, α value increased. Some repairable sublethal damage (β value) was converted into irreparable damage (α value) and the ratio increased when α value increased and β value decreased.

With the rapid development of linear accelerator and the concept of precision radiotherapy being concerned by more and more doctors, the conventional fractionated radiotherapy (2Gy per fraction) has been far from satisfying clinical needs. Professor Zhang's team published a landmark study in the Lancet in 2015: Patients with operable stage I non-small cell lung cancer (NSCLC) who received stereotactic ablative radiotherapy (SABR) (54Gy/3F or 50Gy/4F) had a 3-year overall survival rate of 95%, much higher than 79% after surgery, further established the clinical position of hypofractionated radiotherapy(16). Due to its radiobiology advantages, shortening treatment time and saving hospital and patient costs, the hypofractionated radiotherapy is increasingly favored by clinical doctors. However, with the popularization of hypofractionated radiotherapy in clinical work, more and more problems are emerging. A number of in vitro and in vivo studies have shown that BED formula may not be applicable which was used for bioequivalent conversion from conventional fractionated radiotherapy to hypofractionated radiotherapy(13, 17). Moreover, in clinical practice, hypofractionated radiotherapy for prostate cancer patients did not obtain satisfactory results and only acquired that was not inferior to conventional fractionated radiotherapy(18–20). We speculated that the α/β ratio derived from the LQ model may not be a constant and our previous study using the maximum likelihood principle in mathematical statistics and clinical data confirmed that the α/β ratio of prostate cancer tend to become higher when the dose per fraction increased(6). Therefore, this study verified this conclusion in vitro and explored its potential mechanism.

There were many factors that affected α/β ratio whether internal or external factors of cells, whether physical or chemical factors, and ultimately, it influenced the repair of DNA damage caused by irradiation(5, 21). Therefore, DNA repair after damage was correlated with α/β ratio. From the definition of α/β ratio, α value represented irreparable damage and β value represented repairable sublethal damage. If irreparable damage increased after single high dose irradiation, that is, α value increased; Or part of repairable damage was converted into irreparable damage and β value decreased (α value increased), then the ratio increased.

H₂AX was the most faithful variant of histone H₂A. When DNA damage occurred, H₂AX entered the center of cell reaction to DNA damage and phosphorylated near DSB to produce γ-H₂AX. The number of γ-H₂AX foci can roughly reflect the number of DSB and was consistent with DSB at the beginning after irradiation. After a period of time, due to DNA repair, the numbers of γ-H₂AX foci reduced and the expression level decreased(8, 9, 22). Therefore, observing and counting γ-H₂AX foci in irradiated cells was a widely used method to verify the presence of DSB in DNA and to analyze its repair (23). γ-H₂AX had been shown to occur in all conditions leading to DNA double-strand break: environmental double-strand broke, metabolic broke during DNA replication, and programmed broke during meiotic recombination and V(D)J recombination(24).γ-H₂AX foci can be detected only after a few minutes of irradiation and reached its maximum value after 30min-1h, then gradually decreased(7). By 6 h, the expression level of γ-H₂AX was basically constant due to the completion of sublethal damage and most DSB detected at this time were irreparable damage(13, 15). Therefore, it can be considered that the damage after 6h was irreparable and represented the α value.

RAD51 was a recombinant enzyme that binds to BRCA2 to form presynaptic RAD51-BRCA2 nucleoprotein filaments on DNA. RAD51 was removed after sister chromatid invasion and homologous DNA sequence rearrangement, leaving a free 3'-OH terminal and enabling DNA polymerase to repair DNA in the 3'-5' direction. Once the repaired DNA was completed, these enzymes broke down the Holliday junction, and the DNA ends were joined by DNA ligase I, completing the process of homologous recombination. Therefore, RAD51 was a key protein of homologous recombination(9, 25–27).

In conclusion, the number of γ-H2AX and DSB had a linear relationship. The expression of γ-H2AX increased with the increasing of the number of DSB after irradiation and decreased with the completion of DNA repair, so it can be used as a marker of DNA damage. RAD51 as the most important repair protein in homologous recombination (HR) pathway, can be used as a marker of DNA repair. In this study, the expression levels of DNA damage marker γ-H2AX and DNA repair marker RAD51 were changed after radiation with different fractionated modes, so as to verify the influence of different fractionated modes on DNA damage and repair and to prove the change of α/β ratio after hypofractionated radiotherapy from the definition of α/β ratio.

The results of this study showed that the expression of DNA damage marker γ-H2AX was higher after 6h after single high dose radiation than fractionated radiation, and the difference lasted until 24h, indicating that single high dose radiation had more DNA damage. After 6h, the DNA repair marker RAD51 continued to be highly expressed until 24h after single high dose radiation, DNA damage still existed even with the expression of repair protein, meaning the damage was irreparable. In conclusion, irreparable damage after single high dose radiation was more than that after fractionated radiation. At the same time, according to the results of immunofluorescence, the expression of γ-H2AX decreased after 24h compared with 6h after fractionated radiation. But the expression of γ-H2AX after 24h showed no significant difference compared with 6h after single high dose radiation, indicating that single high dose radiation can transform part of repairable damage into irreparable damage. According to the definition of α/β ratio, α value represented irreparable damage, that is, the α value increased; some repairable damage (β value) was converted to irreparable damage (α value), and the ratio increased when the α value increased and the β value decreased, which was consistent with the conclusion of our previous study, confirming the view that the α/β ratio increased after the single high dose irradiation(6).

In this study, cytological experiments demonstrated that the expression of DNA damage genes increased, repair ability decreased, irreparable damage (α value) increased, and repairable damage (β value) decreased after hypofractionated radiotherapy, thus providing basic evidence for the view that α/β ratio increased after hypofractionated radiotherapy based on our previous clinical data.

LQ	Linear Quadratic
BED	Biological Effective Dose
SLD	Sublethal Damage
DSB	DNA Double-Strand Breaks
HR	Homologous Recombination
ATM	Ataxia Telangiectasis Mutated Gene
BRCA1	Breast Cancer 1 Gene Expressed Protein
MRN	MRE11-RAD50s-NBS.1
RPA	Replace Replicating Protein A
PBS	Phosphate Buffered Solution
FBS	Fetal Bovine Serum
PE	Plating Efficiency
SF	Surviving Fraction
CST	Cell Signaling Technology
NSCLC	Non-Small Cell Lung Cancer
SABR	Stereotactic Ablative Radiotherapy

Conflict of interest: none

Funding: None

Ethical Approval and Consent to participate: Not applicable.

Consent for publication: All authors have agreements on publication.

Availability of supporting data: Not applicable.

Competing interests: None.

Funding: This research was funded in part by the Japanese Ministry of Education, Culture, Sports, Science and Technology.

Authors' contributions: Corresponding author: Deyu Sun and Xian-Shu Gao contributed equally to this paper.

Acknowledgements: None

Authors' information: In the first page.

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No competing interests reported.

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Mechanisms of the DNA Damage Repair Gene Work on Elevated α/β ratio after Hypofractionated Radiotherapy for Prostate Cancer

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Abstract

Figures

Introduction

Methods And Materials

1. Instruments and devices

2. Main reagents

3. Clone formation experiment confirmed that the α/β ratio increased after hypofractionated radiation

4. Western blot experiment confirmed that the α/β ratio increased after single hypofractionated radiation

5. Laser confocal immunofluorescence experiment confirmed that the α/β ratio increased after single hypofractionated radiation

Results

1. Clone formation experiment confirmed that the α/β ratio increased after single hypofractionated radiation

2. Western blot experiment confirmed that the α/β ratio increased after a single hypofractionated radiation

3. Laser confocal immunofluorescence experiment confirmed that the α/β ratio increased after a single hypofractionated radiation

Discussion

Abbreviations

Declarations

References

Additional Declarations

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