Tumor Suppressor Caliban Antagonizes E2F1 Activity To Regulate Cell Cycle Progression

Background:Deoxyribonucleotides(DNA),the main genetic material of living organisms,are constantly attacked by a variety of exogenous and endogenous damage factors,which could cause various forms of damage in composition and structure,threatening the stability and integrity of the genome.In order to maintain the stability and integrity of the genome,organisms have formed complex DNA damage response(DDR)mechanisms during the long evolutionary process to repair damaged DNA.If damaged DNA can not be effectively repaired,there would generate gene mutations,a few of which are beneficial and finally lead to evolution of organisms,but most of which are harmful and can lead to abnormal cell function and disease.The occurrence and development of tumors are the results of the accumulation of harmful gene mutations.Therefore,DNA damage response plays a very important role in the long-term maintenance of cell function and organ health.On the other hand,DNA damage response can also make cancer to be resistant to treatment such as chemotherapy and radiotherapy.DNA damage response is a double-edged sword in the formation and treatment of cancers.Iin addition,mutation,inactivation or dysfunction of genes related to DNA danage response could prevent DNA damage from being repaired in a timely and effective manner,which could lead to tumors and other diseases.Therefore,in-depth study on DNA damage response mechanism is of multiple value in the exploration of tumor pathogenesis,prevention,diagnosis and treatment.Different forms of DNA damage were generated under attack of some different damage factors,which include single base conversion or transversion caused by DNA replication errors,DNA replication fork arrest,DNA single strand breaks(SSBs)or double strand breaks(DSBs)and so on.Cells repair damaged DNA through DNA damage repair mechanisms and clear damaged DNA through apoptosis.Different forms of DNA damage can activate different repair mechanisms or apoptosis pathways.Cell cycle checkpoint block cell cycle progress to gain sufficient time for DNA damage response after DNA damage.Cell cycle checkpoint is an important mechanism to regulate cell cycle.In the process of cell growth and division,cells undergo four phases in turn:G1,S,G2 and M.The whole cell cycle process is driven by cyclins and cyclin-dependent kinases(CDKs)together.For example,in mammals,cyclin D-CDK4/6 and cyclin E-CDK2 complexes drive the Gl-to-S phase progression of the cell cycle,and cyclin A-CDK2 complexes drive the S-to-G2 phase progression,and cyclin B-CDK1 complexes drive the G2-to-M phase progression.Cell cycle checkpoints regulate cyclins and CDKs through some si.gnaling pathways to block cell cycle.G1-S checkpoint,S phase checkpoint and G2-M checkpoint are three main cell cycle checkpoints.When DNA damage occurs at Gl phase,G1-S checkpoint starts to prevent cells from entering S phase.After DNA damage 1s founded by ATM and other sensor proteins,p5 3 is activated via mediator and transducer protein,and interacts with CDK-interacting protein/kinase inhibitor protein(CIP/KIP)and INK4a/ARF families,which play negative regulatory roles in cell cycle.Among them,CIP/KIP family members p21,p27 and p57 can inhibit the activities of cyclins/CDKs complexes to cause cell cycle arrest.On the other hand,Retinoblastoma inhibition protein(Rb)which is one of tumor suppressors also plays a negative regulatory role in cell cycle.Rb binds to the key factor E2F1 which regulates cell cycle and inhibits the role of E2F1.E2F1 can promote the transcription expression of cyclin D,cyclin E,Cdc25 A and other protein driving cell cycle,and E2F1 also can promote the transcription expression of DNA synthesis genes.There is interaction between E2F1 and some other proteins.P53 has physical and functional interaction with E2F1 and DPI which is an E2F1 molecular chaperone.The mechanism of S phase checkpoint 1s rich and important.When DNA damage or replication arrest appears in S phase,different S phase checkpoint mechanisms are activated to repair different damage and prevent damage from affecting the accuracy of DNA replication.The S phase checkpoint is replication fork-dependent or replication fork-independent.When the replication fork is blocked,the checkpoint mechanism which depends on the replication fork is activated.Many proteins participate in this mechanism.Activated ATR phosphorylates Chkl and Chkl phosphorylates Cdc25A,which inhibits the fire of replication.Checkpoint that do not rely on replication fork primarily recognizes DNA double strand breaks(DSBs)and achieve S-phase arrest via ATM-Chk2-Cdc25-CDK2 pathway;another way to repair DNA double strand breaks involves the phosphorylation of SMC1,which can block cell cycles and activate repair systems.The G2-M checkpoint can prevent cells from mitosis under the condition that the damaged DNA is not repaired.In the G2-M checkpoint,ATM and ATR are initiated by different DNA damage.ATM activates Chk2,and ATR activates Chkl.Activated Chkl and Chk2 inhibit the activity of Cdc2/cyclin B complex by Weel kinase and phosphorylate Cdc25B or Cdc25C to lead to G2 phase arrest.The whole process of DNA damage response,including mechanisms regulating cell cycle,is a complex network of signaling pathways involving multiple factors,and is precisely regulated by a variety of factors inside and outside cell.At present,the knowledge about DNA damage response is still limited.Therefore,identifying and discovering new genes involved in DNA damage response,clarifying their role in the DNA damage response signal network,and further completing the DNA damage response mechanism,can promote humans to understand,prevent and treat cancer and other diseases.DNA damage response,as a key mechanism to maintain genomic stability,is highly conserved among different species.Drosophila melanogaster.as a classical model organism,is one of the important tools for studying DNA damage repair,cell cycle checkpoint and apoptosis.Caliban(Clbn)is a new tumor suppressor found in Drosophila in recent years.It is homologous to human serum-defined colon cancer antigen 1(SDCCAG1)which 1s a tumor suppressor.Early studies and previous studies in our laboratory have shown that Clbn is a nuclear export factor that regulates the localization of the transcription factor Prospero in cells.Clbn participates in DNA damage response and regulate apoptosis induced by DNA damage through p53-dependent and p53-independent activity.Previous unpublished data confirmed that clbn mutant Drosophila melanogaster showed defect in S-phase checkpoint after irradiation,but they had intact G2/M checkpoint.Previous unpublished data also confirmed that over-expression of clbn decreased the numbers of S-phase cells in Drosophila compared with wild-type w1118 Drosophila,but the numbers of G2 and M-phase cells had no difference between the two kinds of Drosophila.All of the above data suggest that Clbn could be a new factor regulating cell cycle,but the detailed mechanism 1s still unclear.Objective:With melanogaster which 1s one of model organisms,to study the regulating role and specific mechanism of tumor suppressor Clbn in cell cycle,to elucidate the interaction between Clbn and E2F1 and their roles in cell cycle progression and cell cycle checkpoints.Methods:The wild-type(w1118)Drosophila were irradiated by X-ray to make DNA damage,and expression of Clbn protein before and after irradiation was detected and compared by Western-blot.Brain tissue,wing disc and eye disc of the third-stage larvae vwere dissected,and BrdU/EdU incorporation assay and immunofluorescent assay were carried out.With Flp-out cloning assay and UAS/GAL4 binary expression system,the effects of clbn over-expression on DNA replication were studied,and the effects of clbn over-expression on expression of cyclin E,E2F1 and Dacapo which are key factors in G1-S transition were detected,and the effects of clbn over-expression on expression of cyclin B which is a key factor in G2-M transition were detected too.With MARCM cloning assay,the effects of deletion on DNA replication and expression of cyclin E and E2F1 were studied.With Flp-out cloning assay and UAS/GAL4 binary expression system,the effects of E2F1 and P53 on expression of clbn were studied.Results:1.Irradiation up-regulates the expression of Clbn protein.2.Over-expression of clbn reduces DNA replication and blocks G1-S transition.3.Over-expression of clbn reduces cyclin E expression,but has no effect on cyclin B expression.4.Over-expression of clbn reduces expression of e2fl.5.E2F1 negatively regulate expression of clbn.6.Clbn antagonizes E2F1 activity to promote cyclin E expression and DNA replication.7.Over-expression of clbn promotes expression of Dacapo/p21.8.Over-expressing e2fl reduced the enhanced expression of Dacapo/p21 driven by clbn over-expression.9.p53 up-regulates expression of Clbn protein.Conclusions:1.Irradiation up-regulates expression of Clbn protein.2.Over-expression of clbn reduces expression of cyclin E and E2F1,but promotes expression of Dacapo/p21,which blocks Gl-S transition in cell cycle.3.E2F1 negatively regulate expression of clbn.