The Screening Of New Genes Associated With Cisplatin-induced Senescence And Function Identification

Background and ObjectiveSo far many kinds of human cell lines and zooblasts can be cultured in vitro. Moreover,the reproductive activity of most cultured cells was limited. When cells proliferated into acertain stage they would not divide any more, which meant senescence. Gradually peoplediscovered that senescence existed in many human cell lines and cell lines of other specieswhen Hayflick discovered the phenomenon in human fibroblast in 1960's. The local cellswould proliferated abnormally and carcinogenesis when the body received all kinds ofharmful stimulation. Through continuous study on senescence people gradually had a newrealization about the relation between senescence and tumor, that the anti-tumor effect ofsenescence was an important anti-tumor mechanism in the body. Many studies in vitro hasverified that the carcinogenesis was inhibited by the senescence pathway when cells werestimulated by oncogene, more studies in vivo also verified it. Nonetheless, many cellswhich were transforming into malignant cells could bypass the inhibition of senescence andchange into tumor cells ultimatedly owing to the senescence barriers. In vitro and in vivostudies has established that tumour cells can be induced into accelerated senescence bymany drugs which could cause DNA damage, such as cisplatin, camptothecin and VP16.And some studies have established that some senescence regulating genes in the ATMrelated DNA damage pathway take part in the accelerated senescence induced bychemotherapy. But the concrete mechanism is still unclear. Therefore in our study wechoose cisplatin which is the most common chemotherapy drug in clinic to induceNG108-15, Hela, A2780 cell lines respectively, which are different organization types. Theobjection is to make it clear that what roles these senescence regulating genes play in thesenescence process. Methods1. Apply cisplatin with different concentration gradient to affect tumour cells, choose theconcentration when the senescence rate was highest and without obvious apoptosis.2. Apply cell immunofluorescence to test the expression of HP1-γprotein during theprocess of cell senescence. Apply SA-βGal staining method to test the senescence oftumour cells induced by cisplatin.3. Apply MTT and CFSE staining method to test the proliferation of tumour cells duringsenescence.4. Apply caffeine to inhibit ATM, Apply PI staining method to test the cell cycle byFACS. Apply western blot to test the expression of senescence regulating genes such asP53, P21 and Cdc2.ResultsAll the NG108-15,Hela,A2780 cell lines showed senescence phenotype when theywere induced by certain low dose cisplatin. The senescent cells become large and flatten,increased vacuolus in cytoplasm, positive staining of SA-β-Gal, HP1-γexpression increasedand gathered in the nuclear as foci. The senescent cells stopped proliferating and weremainly blocked during G2/M period. Caffeine, as one of the ATM inhibitor, could make thetumour cells resistant to the senescence induced by cisplatin. The expression and activity ofP53, P21 and CDC2 genes changed obviously during the senescence process.ConclusionNG108-15, Hela and A2780 cells could be induced into accelerated senescence by acertain low dose cisplatin. The DNA damage pathway genes such as ATM, P53, P21 andCDC2 have taken part in the senescence process. The senescence induced by cisplatincould be reversed by inhibition of ATM. Background and ObjectiveTwo-dimensional gel electrophoresis proteomics technology is the most widely usedprotein separation technique in the study of proteomics up to now. Through isoelectriccondensation in the first dimension, different proteins concentrate in accordance with theirisoelectric point, and then the proteins of the same isoelectric point separate in accordancewith their molecular weight in the second dimension of the SDS-PAGE gel. On the basis ofabove theorem, two-dimensional gel electrophoresis proteomics technology can separatelarge amounts of proteins according their isoelectric point and molecular weight one time.Respecting its advantage of high-flux, large amounts of researches have studied relevantprotein in the tumorigenesis and progression of carcinoma using this technique. After theeffect of different kinds of factors, cells change from the state of active proliferative into thestate of irreversible growth arrest, which is named cell senescence. Tumor cells are generallyconsidered that they may have obstacles in the senescence pathway. However, recentresearch has shown that some DNA damage factors(such as cisplatin) can induce tumorcell senescence both in vivo and in vitro experiments. Nevertheless, it is still unknown thatwhich genes participate in the process of cell senescence. So we systematically screenrelevant differential expression protein in the cisplatin induced tumor senescent cell throughtwo-dimensional gel electrophoresis proteomics technology, in order to find some newsenescence-associate genes.Methods1. Using certain dose of cisplatin induced NG108-15 cell senescence, and then extractedtotal protein.2. Screened differential expression protein between NG108-15 normal cell and NG108-15 senescent cell through two-dimensional gel electrophoresis proteomics technology.Repeated the experiment three times and selected the spots whose difference was more than1.5 times for the follow-up analysis.3. Identified the selected protein by MALDI-TOF-MS.4. Verified expression of the selected protein screened by two-dimensional gelelectrophoresis proteomics technology through Western blot and Cell immunofluorescence;Constructed subcutaneous tumor model in nude mouse and then injected cisplatin in tumorsite. Detected the expression of GRP78 through immunohistochemitry and observed thesituation of tumor cell senescence through SA-β-Gal stainning.ResultsA total of 517±25 sports were detected in control group, while there were 474±21sports detected in NG108-15 senescent cell through two-dimensional gelelectrophoresis. Selected 5 significantly differential expression protein sports for thefollow-up analysis. After MALDI-TOF MS analysis, a total of 5 differential expressionprotein were detected. The results of western blot detecting the expression of Vimentin andGRP78 were coincidence with that of two-dimensional gel electrophoresis. GRP78 weredetected that it had a lower expression in cisplatin induced NG108-15 senescent cell bywestern blot, cell immunofluorescence and subcutaneous tumor model in nude mouse.ConclusionAs one of effective technique for selecting the differential expression protein,wo-dimensional gel electrophoresis can be successfully used for screening newcisplatin-induced senescence-associated gene. The expression of GRP78 decreasedsignificantly in the process of cisplatin-induced NG108-15 cell senescence and it may havesome link with cisplatin-induced cell senescence. Backgroud and objectionGlucose regulated protein 78(GRP78), which is also named the immu-noglobulinheavy chain binding protein(Bip), has high homology with GRP78 family. It is generallybelieved that GRP78 is one member of the GRP78 family. GRP78 takes part in manyphysiology and pathology process of cells, which has complicated biological function. It isnot only a molecular chaperon participating protein folding and transferring, but alsoup-regulated to maintain the stabilization of ER and protect cells as an ER stress proteinwhen low carbohydrates, hypoxia and low calcium. In recent years, many studiesestablished that GRP78 synthesis was high in many tumour cells which can endue them theanti-apoptosis characterization, and affect antigenicity of tumour cells. On the other hand,recent study show that GRP78 highly expressed in the ER of astrocyte exposed to heavymetal environment, which can protect sensitive neuron. Tumor is a age associated diseasewhich often occurred in old people. In 1961 Hayflick discovered that there was limitedproliferation in the culture cells in vitro, which was called cell senescence. So far thestudies about cell senescence often referred to cell cycle, free radical, relation betweenDNA damage and senescence, telomere theory, mitochondria and senescence, senescenceassociated genes and long life genes, cell signal transduction and so on. Nonetheless, therelation between GRP78 and senescence has not been seen in literature. Our previous workhas established by 2DE that GRP78 decreased obviously during senescence of NG108-15cells, which may be a new senescence regulating gene associated with cisplatin induction.Therefore, we are going to approach the role of GRP78 played during senescence inducedby cisplatin and to elucidate the regulating mechanism, which may afford clinic therapynew target and effective plan. Methods1. Western blot test the GRP78 expression during senescence induced by cisplatin inNG108-15, Hela, A2780 cells.2. GRP78 was transfected into tumour cells by lipofection. Apply Western blot to test thetransfection effect and expression of associated senescence genes. The senescencesensitivity for tumour cells induced by cisplatin after GRP78 transfection was detectedby SA-β-Gal staining method.3. Apply GRP78 inductor 2DG or A23187 to up-regulate GRP78 in different tumour cells.The sensitivity of senescence of tumour cells induced by cisplatin after GRP78up-regulation was detected by SA-β-Gal staining method.4. Apply laser confocal microscopy to test the relative concentration of calcium inkytoplasm, apply topoismerase inhibitor camptothecin and adriamycin to induce tumourcells senescence, senescence of tumour cells induced by different drugs were detectedby SA-β-Gal staining method. Apply Western blot to test GRP78 expression during thesenescence induced by different drugs.5. Apply immune histochemistry to test GRP78 and HP1-γexpression in cervical cancerand ovary cancer.Results1. GRP78 expression decreased during senescence induced by cisplatin in NG108-15,Hela, A2780 cells. CHOP and Caspase 7 expression also had a little decrease, whichmeant that there was no ER stress associated apoptosis occurred during senescence.2. P53 protein expression increased obviously when GRP78 was knock down by SiRNAin NG108-15, Hela, A2780 cells.3. The senescence sensitivity of NG108-15 and Hela cells induced by cisplatin had a littleincrease. But there was no positive staining of SA-β-Gal in A2780 cell after GRP78knockdown. 4. The senescence sensitivity of NG108-15 decreased obviously when GRP78 wasup-regulated by 2DG, Which can be reversed by GRP78 knockdown after 2DGinduction. Nonetheless, the phenomenon did not exist in Hela and A2780 cells. Thesenescence sensitivity of Hela and A2780 cells decreased obviously when GRP78 wasup-regulated by A23187, Which can be reversed by GRP78 knockdown after A23187induction.5. It was obviously that increase of P53, P21 and P-CDC2 expression tendency duringcisplatin induction was slow down by GRP78 up-regulation.6. The calcium concentration of kytoplasm increased after24h' cisplatin induction. But ifGRP78 was up-regulated by inductor, there was no change of hytoplasm calciumconcentration after24h' cisplatin induction. If GRP78 was knocked down afterup-regulation, the calcium concentration of kytoplasm can increase again with cisplatin.7. For immunohistochemistry results, GRP78 expressed highly in normal cervical tissuesand precancerous change and cancer in situ, but express lowly in cancer tissues. GRP78expression was higher in ovarian cancer than in normal and benign tissues. GRP78expression had high dependability with HP1-γexpression in these two cancers.Conclusion1. As a new discovered chemotherapy induced-senescence associated regulating gene,GRP78 has rivalry and promotion with DNA damage-repair genes such as P53, P21 andCdc2. Down-regulation of GRP78 could promote the senescence induced by cisplatin.Nonetheless, up-regulation of GRP78 can decrease sensitivity of cisplatin-inducedsenescence. The phenomenon demonstrated that GRP78 may play a resistant role insenescence induced by cisplatin.2. GRP78 may affect senescence sensitivity of tumour cells induced by cisplatin throughinfluencing DNA damage-repair pathway associated genes and change of kyroplasmcalcium concentration. GRP78 expression was higher in cervical precancerosis thanthat in normal tissues and cancer tissues. Moreover, GRP78 expression was highly relevant to HP1-γexpression, which meant that GRP78 may participate in thetransforming process from normal cells to malignant cells. GRP78 highly expressed inovarian cancer, which meant that it may influence chemotherapy effect of ovariancancer through anti-senescence. Background and objectiveIn 1961, Hayflick discovered that there was limited proliferating capability in culturedcell in vitro. He named it as cell senescence. Tumor cells showed to be immortalized andcan proliferate unlimitedly. So tumor cell were considered to have a certain senescenceblock.. Nonetheless, recent studies established that tumor cells can be induced to besenescent under some conditions. For example, lower than apoptosis-induced dose DNAdamage agent can induce cell to have accelerated senescence, which was also calledpremature senescence. Other studies showed that senescence was an important mechanismprotecting normal cell from malignant transforming.Esophageal carcinoma is one of the most common malignant tumor severily harmingpeople's life in our country. Esophageal carcinoma has a worse progonosis, nonetheless,which genes participate in its carcinogenesis and progression is still unknown. What rolethe senescence mechanism played in this process is also unclear. GRP78 gene was found tobe closely associated with senescence associated genes such as P53, which was discoveredas a new senescence regulating gene in our previous work. As one of the transcriptioninhibition factors, Heterochro-Matin-associated protein 1(HP1) is a component of thepolyprotein complex which could give rise to gene silencing. As a mark of senescenceassociated heterochromatin foci, HP1-γhas been widely used to show the cell and tissuesenescence. Owing to accessing paraffin section subjectively by staining intensity or area,the analysis results of traditional scoring method under light microscopy is lack of objectivity, which representativeness is not strong. Laser Scanning Cytometer(LSC)analyzes cells and morphous by sets of parameters, therefore it has been not only mainlyapplied to scan intracelluar materials and tissue quantitively, but also analyze the samepoint at different period of time. Because it can scan the selected region of entire paraffinsection at one time and can export objective and accurate result rapidly, LSC has beengradually used to analyze the immunohistochemistry results of tissue microarray. Based onstudy background and previous work above, we selected a tissue microarray containingdifferent grades of esophageal carcinoma and detected the expression of GRP78 and HP1-γprotein by immunohistochemistry. The results of immunohistochemistry were analyzedquantitatively by using laser scanning cytometer. We initially approach GRP78 andHP1-γexpression during malignant transformation of esophageal cell and their correlation.MethodsA tissue microarray containing different grades of esophageal carcinoma wasselected. GRP78 and HP1-γprotein was detected by immunohistochemistry. The results ofimmunohistochemistry were analyzed quantitatively by laser scanning cytometer. Thecorrelation of GRP78 expression and HP1-γexpression was analyzed by chi square test.ResultsThe phantoms and positive pixel percentage of selected areas were obtained afterscanning the entire tissue microarray. The positive rates of normal esophagus,moderate-severe atypical hyperplasia, carcinoma in site and squamous carcinoma were0%(0/8), 90.4%(19/21), 83.3%(5/6) and 47.4%(18/38), respectively. There was statisticaldiscrepancy of GRP78 expression among normal esophagus, moderate-severe atypicalhyperplasia plus carcinoma in site and squamous carcinoma(P＜0.01). HP1-γprotein wasprimarily expressed in the cell nucleus. The positive rate of normal esophagus,moderate-severe atypical hyperplasia, carcinoma in site and squamous cancer were 37.5%(3/8), 100% (21/21), 100%(6/6) and 23.7%(9/38), respectively. There was statistical discrepancy of HP1-γexpression among normal esophagus, moderate-severe atypicalhyperplasia plus carcinoma in site and squamous cancer(P＜0.01). There was highcorrelation between GRP78 expression and HP1-γexpression in esophageal carcinoma(P＜0.01).ConclusionThere are many advantages such as high resolution, high objectivity and accuracywhen using laser scanning cytometer to analyze the immunohistochemistry results.Senescence may be an important mechanism for prevent normal cells from malignanttransformation. GRP78 highly expressed in precancerosis of esophageal and its expressionwas extremely correlated with HP-γexpression. These results demonstrate that GRP78 mayplay an important role in protecting normal cells from malignant transformation withsenescence.