The Preliminary Study Of DNA Double Strands Breaks Forming Iingeringly And Its Mechanism In H1299 Cells After Thermal Damage

1、Background&ObjectiveThermotherapy of tumor refers to apply various heating mode to elevate the temperature of whole body and (or) the local tumor tissue only, take the advantage of thermal effect and its secondary effect to treat malignant tumor.It has a history of hundreds of years for heat treatment was applied in tumor treatment, the tumor thermotherapy study based on modern science began in the early 1960s, thereafter many breakthroughs have been achieved. A large number of experimental and clinical data showed that tumor thermotherapy although can’t replace surgery, chemotherapy or radiotherapy as an independent cancer treatment, but it’s has obvious synergetic and complementary effect on other cancer treatments, such as chemotherapy, radiotherapy and surgery. Because of this, tumor hyperthermia has been developing rapidly in recent years, become an important means for cancer therapy after surgery, radiotherapy, chemotherapy and biological treatment. After the German academics Gordon proposed a new technology:magnetic fluid hyperthermia(MFH) in 1996, heat treatment become a hot research topic in the field of tumor once again.Along with the deepening of the research on mechanism of heat treatment of the tumor, people could realize the heat effect on cancer cells’ structure, function and the impact on growth metabolism gradually from the level of cell and molecular biology, then make a scientific explanation of the heat effect on tumor. Tumor thermotherapy is divided into mild heat (40-42 ℃), hyperthermia (43-47 ℃), thermal ablation therapy (>47 ℃). Mild heat usually needs long time to work, but patients can not bear the long time it needs, so the clinical application of mild heat is limited;Thermal ablation is highly depend on tumor lesion’ size and position,what’s more, the thermal ablation can cause coagulation necrosis with no difference between tumor tissue and the surrounding normal tissues, its clinical application is limited too; therefore, hyperthermia is the major method for the current clinical application and experimental study, due to the difference of microcirculation between tumor and normal tissue, hyperthermia can kill tumor cells specifically without harming normal cells; Its function mechanism mainly include the following points:1, direct killing effect, its theory is based on the experiments which found that various biological membrane of tumor cells and its cytoskeleton can be damaged after heat treatment and affects its fluidity and permeability in turn; heat treatment can also destroy the intracellular proteins and then affects some cell activities such as DNA replication, transcription and expression, eventually leading to cell death.2, induction of apoptosis, studies found that caspase-3 and so on which are cell apoptosis-promotion related gene express more and survivin and so on which are apoptosis-inhibition related genes express less after thermal injury, finally lead to cancer cell apoptosis through the mitochondrial pathway or death receptor pathway.3, the inhibitory effect of invasion, metastasis, the protein hydrolysis enzyme which is responsible for the degradation of extracellular matrix significantly reduced in tumor tissues after heat, and E-calcium which is an intercellular adhesion molecule increased obviously, this two can weaken the migration ability of tumor cells eventually.4, improve the body’s immune function, tumor’s occurrence and development is closely related to the body’s immune status, literature reported that many humoral immune factors such as IL-6, CD4, CD57 and so on have a wide distribution in tumor lesions after heat treatment, and mononuclear macrophages and T cells、 B cells which produce such factors also gathered here too, these results confirmed that heat can improve the body’s immunologic function to cure tumor.It is worth noting that:the research found that the thermal damage is cell cycle specificity, namely, the S-phase cells are higher heat sensitive than cells in other phases; so thermal therapy was often applied combined with radidtherapy to kill S-phase cells which are radiation resistant. In addition, the heat also can inhibit the repair of DNA double strands break (DSB) that induced by radiotherapy to enhance the curative effect of radiotherapy. Due to the faster proliferation of tumor cells than normal cells, the tumor has the characteristics of fast development and poor prognosis;Therefore, making clear of the mechanism that why S-phase cells are heat sensitive is of great significance for the treatment of tumor. Some researchers suspect that whether the heat treatment can also lead to DSB, especially the S-phase cells, and then did the further research about the conjecture. But the current research results has not yet form a unified point of view on whether the thermal damage leads to the formation of DSB and the DSB formation depend on cell cycle or not are controversial. This study emphasize on the DSB formation in the S-phase cells after thermal damage, then analysis the possible mechanisms of DSB formation after thermal damage combined with the results of DNA replication and DNA damage repair;It is aimed to provide more theoretical basis for the clinical application of heat, and promote the wide application of heat treatment in the clinical.2、Method(1) The exploration of heating time of H1299 cells in 45 ℃ incubator and S-phase synchronous conditionThis study choose the hyperthermia treatment of 45 ℃ as the heating temperature, heating time is decided by clone forming assay:make use of the HI 299 cells in logarithmic phase to plank again and cultivate 24h then conduct heat treatment in 45 ℃ preheating incubator by group,the heat time are Oh,0.5h, lh, 1.5h,2h,2.5h respectively,the cells return to 14 days’ normal cultivation after heat treatment,and then continue dyeing、 counting analysising the results of the experiment in the final. When the clone formation rate is too high may stand for the thermal damage did not come into being, and when the clone formation rate is too low can not guarantee the experiments in the late progress smoothly, so take the heating time which is corresponding to about 50% clone formation rate as the heat treatment time for this research. To explore the reason why S-phase cells are heat sensitive, our study adopt the serum starvation method to synchronize H1299 cells to S-phase, the starvation time refer to 28h which is reported in one research, and then confirm how much time still needed to regain the normal cultivation after serum-free culture by flow cytometry to maximize the S-phase synchronization effect.(2) The DNA double strand breaks formation in S-phase H1299 cells and its influence on cell survival after thermal damageAcquire the S-phase synchronized H1299 cells according to the above results and then conduct the heat treatment, collects each group’s cells to detect "comet tail" dynamically of different time points after thermal damage by using neutral single cell gel electrophoresis (SCGE), the CASP software was used to analysis DNA double strands rupture degree. Lastly, trypan blue exclusion assay was applied to detect the survival rate of S-phase H1299 cells after thermal damage, analysis the link of DNA double strands with cell death by the results of neutral single cell gel electrophoresis experiment and trypan blue exclusion assay.(3) The possible mechanisms of the DNA double strands break formation in H1299 cells after heat treatmentBased on current research results, we speculated that the possible mechanisms of the DNA double strands break formation in S-phase H1299 cells after heat treatment is that some unrepaired DNA damage developed in thermal therapy continue directing replication when cells are blocked in S phase. So we use western blot (WB) to detect the expression of ub-PCNA and Rad18 which are involved in post replication repair and determine whether the DNA damage repair is inhibited, what’s more,we also apply flow cytometry、p-ATM expression and EdU incorporation assay to analysis if the DNA replication is still ongoing after thermal damage.3、 Results(1) The exploration of heating time of H1299 cells in 45 ℃ incubator and S-phase synchronous conditionThe clone formation rate of H1299 cells that have underwent 45 ℃ heat treatment for 0h,0.5h,1.5h,2h,2.5h were 100%,67.87%,43.73%,67.87%,21.29%, 7.79% respectively, thus the 45 ℃ heating for 1h is a better heat treatment condition. Put H1299 cells in serum-free culture 28h then return to 10% fetal bovine serum to continue cultivating, and collect the cells from different time points to detect the cell cycle distribution by using flow cytometry, the results show that when the starvated cells keep on cultivating in 10% fetal bovine serum 18h can effectively synchronize the cells to S phase, the proportion of S-synchronized cells can reach 69.27 ±0.81%; but the S-phase proportion of non-starved cell maintained at the low level of 27.24 ±1.75%.(2) The DNA double strand breaks formation in S-phase H1299 cells and its influence on cell survival after thermal damageAcquire the S-phase synchronized H1299 cells according to the above cell cycle synchronization conditions and then process thermal damage(45℃x1h), apply neutral single cell gel electrophoresis experiment to dynamically detect the DNA double strands breaks of 37℃ normal condition developed cells after heat treatment at different time points, the detecting results suggest that obvious "comet tail" phenomenon in S-phase cells could not be seen at 2.5h post 45℃ thermal damage, the Olive Tail Moment value(OTM) of heat group had no statistical difference compared with the control group; the obvious "comet tail" began to appear at 5h after recovering to normal culture tail", (the OTM value is statistically different with the control group, P<0.05) and the curve reaches a plateau at 7.5h post heat treatment (OTM value=29.54±4.78). The results of trypan blue exclusion assay which is applied to analysis the mortality of S-phase synchronized H1299 cells after thermal injury show that the mortality rate remain low before 7.5h in normal culture after heat treatment (there is no statistical significance with the control group), then the dead cells increased sharply after 7.5h, the mortality rate reached 78.72% ±2.07% at 12.5h in normal culture after heat.(3) The possible mechanisms of the DNA double strands break formation in H1299 cells after heat treatmentThe western blot (WB) results show that the DNA binding protein Rad18 and ub-PCNA which involved in post replication repair pathway (PRR) in heat group cells was significantly reduced compared to control group, and as the the extension of incubation time in normal culture after thermal damage, the Rad18 in heat group showed a trend of gradual decrease. Although the ub-PCNA was increased gradually,it was lower than the control group. The p-ATM protein which is responsible for the S-phase arrest signal has no expression in non-heat group cells, but its expression level in heat group cells increased gradually as the extension of incubation time in normal culture after thermal damage,and until 5h post heat, its expression up to a maximum quantity, and then drop to disappear. Conduct the heat treatment according to the heating condition (45℃x1h) and followed by recovering to normal cultivate condition for multiple time points, then collect cells to detect each group’s cell cycle distribution dynamically by flow cytometry, the testing results show that the proportion of S-phase cells rise first along with the extension of incubation time in normal condition, and reach the peak at 7.5h post heat treatment (51.63%±0.27%, are statistically significant compared with non-heating cells, P< 0.01)and then the S-phase proportion of heat group gradually decline after 7.5h. The heat group’s ratio of G0/G1, G2/M phase rises and falls with the change of S-phase proportion, the non-heat group’s proportion of each cell cycle changes slightly. EdU incorporating experiments showed that the DNA replication of non-synchronized H1299 cells increased at first and then decreased after thermal damage, it reached the peak at 7.5h post heat (44.48% ±1.23%, and it is significantly different with the control group, P<0.01); the DNA replication rate started to reduce after 7.5h.4、 Conclusions(1) Non-small cell lung cancer H1299 cells can occur S phase block and form DNA double strands breaks lingeringly after thermal injury of 45 ℃ x 1h.(2) Under this study’s condition of thermal damage can inhibit Rad18 and ub-PCNA which are responsible for post replication repair binding to DNA.(3) The DSBs possible forming mechanism in S-phase H1299 cells after 45℃ xlh thermal injury is DNA damage repair inhibited but the replication keep on-going at the same time, then the base damage convert to DSB.5、 The innovation of this study(1) This study found that thermal damage can lead to DNA double strands breaks lingeringly in the cells for the first time.(2) This study found that the thermal damage can make Rad18 and ub-PCNA binding to DNA less then inhibit translesion synthesis pathway for the first time.(3) This study explored the possible mechanisms of DSB forming in the S-phase cells after thermal damage for the first time and then it establishes some certain theoretical basis for the independent clinical application of tumor thermal therapy.