Establishment Of A Tumorsuppressor Gene Promoter-based Environmental Genotoxicity Screening System And Its Application In Screening Of Antitumor Drugs

Cancer,a complex disease that affects humans,is the most difficult to cure.At present,the tumorincidence in China continues to increase,and malignant tumors have become the deadliest disease.The occurrence and development of tumor is the result of the interaction of genetic and environmental factors.Environmental factors include a variety of carcinogens,radiation,and viruses,and genetic factors include activation of oncogenes and inactivation of tumorsuppressor.p53,as one of the most important tumorsuppressor genes,plays an important role in tumorsuppression.When the cells are subjected to DNA damage,the damage signal can activate p53 through transcriptional activation and protein phosphorylation,and then induce apoptosis,cell senescence,and other biological processes,which inhibit the occurrence and development of tumor.In a variety of malignant tumors,the level of p53 is very low,and its expression level is regulated by many factors.The increase of gene transcription level is closely related to the transcriptional activation of upstream transcription factor.Using the specific DNA damage-sensing ability of the p53 gene promoter,we cloned the p53 promoter and constructed a genetic toxicity screening system with green fluorescent protein(GFP)as the reporter gene.This system can be used to screen a variety of genetic toxicities in the environment and can also be used to screen anti-tumordrugs.In addition to p53 as an important tumorsuppressor gene,it has been proved that there was mutation of tumorsuppressor gene Ink4a/ARF in a variety of malignant tumors,including gene homozygous deletion,deletion,mutation and promoter methylation.Ink4a/ARF gene contains two tumorsuppressor genes,p16Ink4a and p19ARF,which share the last two exons,and each has its own first exon.P16 and p19 belong to the Rb and p53 pathways.P16 can directly bind to cell-cycle-dependent kinase(cyclin-dependent kinases,CDK),CDK4,and Cdk6;inhibit the phosphorylation of Rb1;and finally suppress E2F1,resulting in G1 phase growth arrest,which was found to play an important role in the inhibition of tumor.P19 can inhibit MDM2 activity,thereby inhibiting the degradation and causing indirect stabilization of p53.Similarly,we chose GFP as the reporter gene,and p16 and p19 as the targets of the anti-tumordrug-screening system.In the present study,we selected the fourth-generation promoter reporter vector,pGL4.82,and the stability of its expression in the cell was improved based on previous generations.To improve the detection efficiency,we changed the luciferase reporter gene to GFP,the construction of the pGL4-GFP vector.Then,we introduced three important tumor-suppressor gene promoters into the vector and transfected them into cells with clear background and through the resistance screening based on three tumor-suppressor gene promoters of screening cell lines p53-GFP,p16-GFP,and p19-GFP.After the screening system was established,we verified the function of the system.First,we chose the DNA damage-induced agent doxorubicin to verify the function of the p53-GFP screening system.The results show that doxorubicin can effectively activate the expression of the reporter gene,and then we introduced a variety of DNA damage such as adriamycin,cyclophosphamide,cisplatin,benzo pyrene into the cells.The results show that the system can accurately identify the damage signal.After the function of the system was proven,we used the system to select a large number of compounds.At the same time,a large number of cancer chemotherapy drugs are DNA damage agents,these drugs have great damage to normal cell,such as bone marrow hematopoietic cells,epithelial cells of the small intestine,thus there are a lot of side effects.The high throughput screening system we constructed is based on the transcriptional activation of the p53 promoter,so we are expected to be able to screen for the natural compounds that can effectively activate the expression of tumorsuppressor genes and have low cytotoxicity.Next,we used this system to screen a variety of natural compounds,including extracts from the Kunming Institute of Botany.The results showed that all kinds of Rhizoma Cimicifugae extracts could not effectively activate p53 promoter,but had less toxicity to wild-type MEF cells.While some of Buxus alkaloids compounds such as KBA01,KBA02,KBA03,KBA07 and KBR11 can effectively activate the expression of GFP.In order to find low cytotoxicity natural compounds that can effectively activate activates p53 in cells,we further treated the wild-type MEF cells with these compounds.We found that KBA03 can induce block of G1 phase,while KBR18 can induce cell apoptosis.Most of the screened alkaloids have different degrees of genotoxicity,while we found that KBA07 can effectively activate the p53 gene promoter,and has no obvious genotoxicity.There was no significant difference in the level of cycle and apoptosis in the treated wild-type MEF cells.We believe that this compound may activate the p53 gene promoter,but it does not have a strong genetic toxicity.Further studies on the mechanism of KBA07 are needed.Study on the anti tumormechanism of Pu'er Tea proved that Pu'er Tea can significantly improve the p 16 level of mRNA.The expression of GFP gene was observed in p16-GFP cells after treatment with Puer tea,but there is no dose effect.Inactivation of p16 was found in a variety of malignant tumors,so the compounds that activate the p16 promoter have certain antitumorpotential.We have used this system to screen compounds such as KY01,KY40,etc.,which can effectively activate p16,and the mechanism of their action needs further study.In the verification of p19-GFP screening system,we transfected the pBabe-RasG12V expression vector into the screening system.Overexpression of RasG12V can effectively activate the P19 gene promoter,and GFP expression was observed in 24h after transfection.Previous studies have shown that the P19 promoter is sensitive to the activation of proto oncogene and has specificity.Many compounds have been screened and few compounds have been found to activate P19 promoter,but we are pleased to find that KBA07 can activate the P19 promoter effectively,so we think that KBA07 has high research value.KBA07 has been proven to have anti-tumorpotential;thus,we dealt with a variety of tumorcells.The result shows that KBA07 has no genetic toxicity.It can activate P19 at the transcriptional level,and high expression of P19 can further stabilize p53 levels.Therefore,in some p53 gene normal tumorcells,KBA07 can effectively activate the p53 protein,and then,in the case of some DNA damage inducers,can effectively induce apoptosis or senescence.We found that after KBA07 treatment,the expression level of HIF-1 increased.We also proved that KBA07 can effectively activate HIF-1,and further research is ongoing.In summary,we developed three types of important tumor-suppressor gene-promoter-based biosensor systems that use GFP as reporter gene which named p53-GFP,p16-GFP,p19-GFP.These three screening systems were tested and proved to be rapid and specific for the production of small molecules of natural compounds that act on the promoters of these three tumorsuppressor genes.These systems can be used for rapid screening of various compounds or small molecules that has potential antitumoractivity.We also found that Buxus microphylla KBA07 has anti-tumorpotential,and its mechanism is being studied.At the same time,the molecular mechanism of KBA07 was studied by the competition of transcription factor binding sequence of p53 promoter.