| Cell death is a fundamental biological process which plays an important role in maintaining the function and morphology of tissues.Execution of cell death needs an orchestrated interplay between some important processes: apoptosis,autophagy,necrosis and mitotic catastrophe.Apoptosis refers to a constellation of characteristic changes leading directly to cell death.Over the past decades,apoptosis has been widely studied and radiotherapy strategies targeting at apoptosis become one of the most important ways for cancer treatment.Autophagy is a lysosome-dependent self-digestion process that maintains cellular homeostasis and supplies substrates for energy generation.Autophagy can promote cell survival under some stresses,such as nutrient starvation,ROS,hypoxia,DNA damage.However when excessive cell damage is beyond the limit of repair under certain physiological conditions,autophagy turns into a programmed cell death,which we known as autophagic cell death.Other than apoptosis and autophagy,mitotic catastrophe is one of the atypical deaths,also acts as an oncosuppressive mechanism which can prevent perturbations of the mitotic machinery and genomic instability in response to DNA damage.Mitotic catastrophe occurs as a consequence of failure to complete mitosis.In that case,cells proceed into mitosis after a transient cell cycle arrest and fail to separate,leading to catastrophic cell division.Deoxycytidine kinase(d CK)is a key enzyme in the deoxyribonucleoside salvage,which is important to maintain normal DNA metabolism.d CK can phosphorylate many antiviral and anticancer nucleoside analogues.These drugs are activated only after phosphorylation,thereby inhibiting tumor growth.Our previous study found d CK can be responsible for the phosphorylation of endogenous deoxynucleosides,is also involved in DNA damage repair.d CK protein has four phosphorylation sites,Thr-3,Ser-11,Ser-15 and Ser-74.d CK activity can be increased by Ser-74 phosphorylation and Thr-3 can promote the stability of d CK.Ataxia-telangiectasia-mutated(ATM)kinase can phosphorylate d CK on Serine 74 to activate it in response to IR treatment and d CK regulates the G2/M checkpoint through interaction with cyclin-dependent kinase 1 in response to DNA damage.Besides,ATM could promote IR-induced autophagy via the Akt/m TOR/P70S6 K pathway.However,up to now,the relationship between d CK and cell death under IR treatment and the specific mechanism has not been exactly clarified.Objective: In this study,we focused on the role of d CK in radiation-induced cell death and its possible mechanism.This will provide the basis for gene targeting therapy of cancer.Methods:(1)Human cervical cancer cells Hela and human breast cancer cells SKBR3 and MDA-MB-231 were used in this study;(2)The knockdown cell models and over expression cell models of d CK were established by genetic engineering;(3)3-MA,Rapamycin,ZVAD-FMK,Necrostatin-1 and Ferrostatin-1 were used to distinguish different types of cell death;(4)CCK-8 and colony formation assays were used to detect cell viability and radiosensitivity;(5)Western blot was applied to analyze the expression of target proteins;(6)Co-immunoprecipitation was used to detect the interaction between proteins;(7)Total cell death,apoptosis,autophagy,cell cycle progression,polyploidy and mitotic cells were detected by flow cytometry;(8)X-irradiation conditions were as follows: voltage 180 k V,current 18.0 m A,filtration plate 0.25 mm Cu and 1.0 mm Al,distance between target and X source 60 cm,dose rate 0.40 Gy/min.Results: 1.Loss of d CK increased IR-induced cell deathThe d CK knock down He La cells were treated with different doses of IR.Colony formation showed that compared with the p SUPER cells,with the increase of irradiation dose,the rate of cell clone formation decreased significantly in d CK knock down cells.The D0 value was 2.35 in p SUPER cells vs 2.01 in d CK knock down cells,indicating that silencing d CK resulted in hypersensitivity to IR in He La cells.SKBR3 and MDA-MB-231 which were stable transfected with p SUPER and d CKRi plasmids were treated with 8Gy IR.Flow cytometry result showed that the mortality rate in MDA-MB-231 cells was significantly higher than that in SKBR3 cells.After IR treatment,the rate of cell death were increased by 141% and 169% in SKBR3-p SUPER and SKBR3-d CKRi cells;The rate of cell death increased by 302% and 355% in MDA-MB-231-p SUPER and MDA-MB-231-d CKRi cells,suggesting that d CK silencing increased IR-induced breast cancer cell death.Compared with SKBR3 cells,MDA-MB-231 cells show more radiosensitivity.2.d CK phosphorylation at Ser-74 site suppressed IR-induced cell deathWe transfected either empty Vector,wild-type d CK(d CK-WT),d CK-S74 A or d CK-S74 E plasmid into d CK knock-down cells and then treated with 8Gy IR.The plasmid d CK-S74 A has a Serine 74 to alanine substitution,which abrogates phosphorylation,and d CK-S74 E has a Serine 74 to glutamic acid substitution,which mimics phosphorylation.After IR treatment,the cell viability of d CK knock down He La cells reduced to 60.79%.However,IR-induced cell death can be reversed by reintroduction of d CK,the survival rate from high to low is d CK-S74E(93.58%)> d CK-WT(81.93%)> d CK-S74A(69.93%).The rate of cell death increased by 129% and 366% in d CK knock down SKBR3 and MDA-MB-231 cell models respectively afte 8Gy IR treatment.However,IR-induced cell death can be reversed by reintroduction of d CK,the cell death rate of SKBR3 cell models increased from high to low is d CK-S74A(137%)> d CK-WT(90%)> d CK-S74E(65%).The cell death rate of MDA-MB-231 cell models increased from high to low is d CK-S74A(357%)> d CK-WT(320%)> d CK-S74E(195%).These results suggested that d CK phosphorylation at Ser74 site increased radioresistance.3.d CK was involved in IR-induced different types of cell death.The stable He La models with p SUPER,d CKRi plasmids were pro-treated with 3-MA,Rapamycin,ZVAD-FMK,Necrostatin,Ferrostatin-1respectively.1 hour later,they were treated with 8Gy IR.Compared with the control group,3-MA increased IR-induced cell death significantly,which increased by 29% in p SUPER cells and increased by 25% in d CKRi cells.Rapamycin group and ZVAD-FMK group decrease IR-induced cell death.However,Necrostatin-1 and Ferrostatin-1 had no effect on IR-induced cell death.In SKBR3 cell models,3-MA had no effect on IR-induced cell death.Rapamycin increased IR-induced cell death significantly,which increased by 42% in p SUPER cells and increased by 55% in d CKRi cells.In MDA-MB-231 cell models,3-MA and ZVAD-FMK decreased IR-induced cell death obviously.Rapamycin increased IR-induced cell death significantly,which increased by 64% in p SUPER cells and increased by 40% in d CKRi cells.These results suggested d CK involved in medium dose IR-induced cell death was related to autophagy and apoptosis,but nothing to do with necrosis or ferroptosis.4.d CK Ser74 site participated in suppressing apoptosis.The stable He La models with p SUPER and d CKRi plasmids were treated with 8 Gy IR.It showed that IR could increase apoptosis significantly,and He La-d CKRi(20.92%)> He La-p SUPER(14.98%).IR could increase cleaved-caspase3/caspase3 obviously,which increased by 134% in p SUPER cells and increased by 214% in d CKRi cells.Moreover,silencing d CK and IR could decrease Bcl-2 protein level.In SKBR3 and MDA-MB-231 cell models,IR could increase cleaved-parp/parp obviously,in which SKBR3-d CKRi(1.92)>SKBR3-p SUPER(1.20),MDA-MB-231-d CKRi(3.61)> MDA-MB-231-p SUPER(1.42).These results suggested that silencing d CK increased IR-induced apoptosis.The d CK overexpression cell models of He La,SKBR3 and MDA-MB-231 were treated with 8Gy IR.In He La cell models,IR induced apoptosis increased from high to low was d CK-Vector(88%)>d CK-S74A(50%)>d CK-WT(29%)>d CK-S74E(26%);In SKBR3 cell models,IR induced apoptosis increased from high to low was d CK-S74A(107%)>d CK-Vector(101%)>d CK-WT(64%)>d CK-S74E(42%);In MDAMB-231 cell models,IR induced apoptosis increased from high to low was d CK-Vector(463%)>d CK-S74A(293%)>d CK-WT(190%)>d CK-S74E(no change).These results suggested that d CK phosphorylation at Ser74 site suppressed IR-induced apoptosis.5.d CK Ser74 site participated in promoting autophagy.He La-p SUPER and He La-d CKRi cells were treated with NH4 Cl and 8Gy IR.LC3-II increased significantly in p SUPER cells at 72 h but no obviously increase at 24 h and 48 h after IR treatment compared with sham-irradiation group,suggesting LC3-II increased in a time-dependent manner in the cells.NH4 Cl could increase LC3-II expression significantly.Compared with He La-d CKRi cells,LC3-II expression level was much higher in He Le-p SUPER cells at 72 h after IR treatment.Flow cytometry showed that IR-induced autophagy was much lower in He La-d CKRi cells(14.76%)compared with He La-p SUPER cells(25.31%).The stable SKBR3,MDA-MB-231 cell models with d CK sh RNA and control sh RNA were treated with 8Gy IR.The results showed that LC3-II increased by 52% in SKBR3-p SUPER cells but no significantly change in SKBR3-d CKRi cells after IR treatment.LC3-II increased by 91% in MDA-MB-231-p SUPER cells and only increased by 36% in MDA-MB-231-d CKRi cells after IR treatment.These results indicated that d CK could increase autophagy induced by IR.The d CK overexpression cell models of He La,SKBR3 and MDA-MB-231 were treated with 8Gy IR.The results showed that d CK overexpression increased LC3-II significantly after IR treatment.In He La cell models,IR increased LC3-II from high to low was S74E(46%)>WT(44%)> Vector(16%)>S74A(9%).In MDA-MB-231 cells,IR increased LC3-II from high to low was S74E(342%)>WT(306%)> Vector(145%)>S74A(no change).In SKBR3 cells transfected with d CK-S74 E,LC3-II induced by IR increased obviously(65%)but no significantly change in other cell models.These results suggested overexpression of d CK could increase IR-induced autophagy,and d CK phosphorylation at Ser74 site participated in the autophagy regulation.6.d CK regulated autophagy via Akt/m TOR/P70S6 K pathwayThe d CK overexpression He La and MDA-MB-231 cell models were treated with 8Gy IR.Western blot was used to test Akt/m TOR/P70S6 K and we found that IR significantly decreased levels of phospho-Akt/Akt/GAPDH by 54%,phospho-m TOR/m TOR/GAPDH by 39% and phospho-P70S6K/P70S6K/GAPDH by 31% in the He La-d CK-WT cells.IR treatment significantly decreased levels of phospho-Akt/Akt/GAPDH by 57%,phospho-m TOR/m TOR/GAPDH by 55% and phospho-P70S6K/P70S6K/GAPDH by 46% in He La-d CK-S74 E cells.However,the levels of phospho-Akt/Akt/GAPDH,phospho-m TOR/m TOR/GAPDH,phosphoP70S6K/P70S6K/GAPDH showed no significantly change in He La-d CK-S74 A cells following IR treatment.IR treatment significantly decreased levels of phospho-Akt/ GAPDH by 54%,phospho-m TOR /GAPDH by 82% and phospho-P70S6K/ GAPDH by 54% in the MDA-MB-231 cells which transfected with wild type d CK.IR treatment significantly decreased levels of phospho-Akt/ GAPDH by 57%,phospho-m TOR/ GAPDH by 87% and phospho-P70S6 K /GAPDH by 64% in MDA-MB-231 cells which transfected with d CK-S74 E.However,the levels of phospho-Akt/ GAPDH,phospho-m TOR/ GAPDH,phospho-P70S6K/ GAPDH showed only smaller decreases of 19%,38% and 7%,respectively in MDA-MB-231 cells transfected with d CK-S74 A following IR treatment.This suggested that d CK regulated IR-induced autophagy via Akt/m TOR/P70S6 K pathway7.There was a switch between autophagy and apoptosisHe Le-p SUPER and He La-d CKRi cells were pre-treated with 3-MA,ZVAD-FMK or 3-MA+ZVAD-FMK for 1h and then following 8Gy IR treatment.Western blot tested caspase-3 and cleaved-caspase3 and the result showed that 3-MA increased apoptosis and the level of apoptosis was most high in d CK knock down He La cells after IR treatment.The apoptosis level in the cells treated with 3-MA+ZVAD-FMK was much lower than that with 3-MA treatment and much higher than that with ZVAD-FMK treatment.Flow cytometry showed that 3-MA increased IR-induced apoptosis by 32% in He La-p SUPER and by 48% in He La-d CKRi cells.In d CK knock down He La cells,ZVAD-FMK inhibited apoptosis induced by IR obviously(24%),and 3-MA+ZVAD-FMK didn't change IR-induced apoptosis significantly compared with the control group,suggesting the promoting effect of 3-MA on apoptosis and the inhibition of ZVAD-FMK on apoptosis were counteracted each other.Flow cytometry showed that 3-MA inhibited IR-induced apoptosis significantly in MDA-MB-231 cell models.Rapamycin increased apoptosis induced by IR in d CK knock down cells but the effect was not obviously in p SUPER cells.Western blot tested PARP and Cleaved-PARP and the result showed that knock down d CK increased IR-induced apoptosis(17%).3-MA could inhibit apoptosis and the apoptosis induced by IR decreased most significantly in d CK knock down cells(43%).Moreover,3-MA+ZVAD-FMK inhibited apoptosis and the effect was much stronger than 3-MA or ZVAD-FMK.The results suggested that suppressing autophagy inhibited IR-induced apoptosis,and 3-MA and ZVAD-FMK had synergistic effects on cell apoptosis inhibition.8.d CK protected cells from mitotic catastrophe by regulating G2/M checkpointFlow cytometry was used to detect the related indexes of cell cycle in He La-p SUPER and He La-d CKRi cells.IR-induced G2/M phase arrest increased in a time-dependent manner,reached its peak at 12 h after 8Gy IR.At that time,the G2/M phase arrest in d CK knock-down cells was lower than that in p SUPER cell.d CK interacts with Cdk1 after IR and that the interaction inhibits Cdk1 activity.d CK phosphorylation at Ser74 site played an important role in the inhibition of Cdk1 activity.The number of mitotic control cells decreased by about 70% 12 h after IR treatment and recover a few at 24 h IR treatment.Knocking down d CK had a minimal effect on mitotic cell number after 12 h IR treatment but polyploidy increased more significantly(204%).More over,knocking down d CK increased H2 AX and reduced ERCC1 induced by IR considerably.The results demonstrate that dCK protected cells from mitotic catastrophe by regulating G2/M checkpointConclusions: 1.Loss of d CK increased IR-induced cell death and radiosensitivity.2.Phosphorylation of d CK Ser-74 site inhibits IR induced cell death,which is embodied in the inhibition of IR-induced apoptosis and the promotion of IR-induced autophagy.There is a switch between autophagy and apoptosis.3.d CK regulated IR-induced autophagy via Akt/m TOR/P70S6 K pathway 4.d CK protected cells from mitotic catastrophe by regulating G2/M checkpoint... |
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