Effect Of NPM On Chromosome Instability Induced By Irradiation

Genomic instability(GIN) induced by irradiation is considered to be a central aspect of carcinogenic process.A growing amount of evidence indicates that chromosome instability is a frequent phenomenon of genomic instability which might provide a route to aneuploidy and thereby contribute to the development of cancer.In many cancer cells the number and the structure of each chromosome can be highly variable. However,the regulating factors and mechanism of chromosome instability induced by irradiation are still unknown.Thus,investigation of key regulatory gene and mechanism of action is significant for the carcinogenesis induced by irradiation, meanwhile new detection methods and therapeutic strategy may be available for the early diagnosis and therapy of cancer.Nucleophosmin(NPM) is a multifunctional protein that has been proposed to function in ribosome assembly,pre-ribosomal RNA processing,DNA duplication, centrosome duplication and mitosis under the interaction with p53.It is frequently overexpressed in actively proliferating cells including tumor and stem cell.It is considered as one of the most important genes in cell proliferation and cell survial after DNA damaged,especially functioning in the start of centrosome duplication and keeping chromosome stability.The role of NPM in the chromosome instability induced by irradiation is not clear since there is few report correlated and also There is a debate on the relationship between NPM and p53.We took lymphoblast cell lines differed in p53 status as experimental material and observed the changes of NPM expression,apoptosis rate and chromosome number postirradiation.We studied the relationship between NPM expression and proliferation ability.Mutation of NPM gene was also detected to see whether proliferation ability had close relation to mutation.The study on the relationship among NPM,p53 and irradiation induced chromosome instability(CIN) was also done by using olomoucine to inhibite the phosphorylation of NPM.This research includes four parts as the following.Partâ… :Differential NPM expression and proliferation ability in lymphoblast cell lines Objective:To investigate the relationship between NPM expression and proliferation ability and chromosome number in different cell lines including lymphoblast cell lines differed in p53 status and human normal immortal lymphocyte line.Methods:Colony-formation in soft agar assays was used to detect the proliferation ability of lymphoblast cell lines TK6(wt p53),WTK1(mt p53) and human normal immortal lymphocyte line.Chromosome specimen of TK6 and WTK1 were made and also mRNA and protein expression of NPM was assayed respectively using RT-PCR and Westen Blot.The relationship between proliferation ability and NPM expression can be observed.Results:Colony-formation efficiency of WTK1 was as much as 2.75 times that of TK6;The two lymphoblast cell line,WTK1 and TK6,proliferate rapidly and have a high colony-formation efficiency,which was 41 and 15 folds higher than that of human normal immortal lymphocyte line respectively.The percentage of aneuploid cells were about 54%in both TK6 and WTK1 cell lines.Over 98%of TK6 cells were (near) diploid and no(near)tetraploid were found,however 83%of WTK1 cells were (near)diploid and the percentage of(near)tetraploid was as high as 11.4%.The relative quantity of NPM mRNA expression was HNILL＜TK6＜WTK1,but they were not significantly.Total and phosphorylated NPM protein of WTK1 was significantly higher than that of TK6 and both of them were much more than HNILL.Conclusions:WTK1 possessed much more proliferation ability and polyploid cells than the two other cell lines.There were positive correlation between NPM expression,CIN and proliferation ability.They are also related to the status of p53.Partâ…¡:Mutation analysis of NPM gene in human lymphocytesObjective:To investigate whether there were any chromosome translocations involved in NPM gene and mutations of the 12^th exon.We could study whether proliferation ability correlates to the mutation of NPM.Methods:Chromosome translocations were measured by chromosome G banding technology.Sanger approach was used to detect the 12^th exon mutation of different cell lines and fresh blood specimen.Results:There was heterozygotic T deletion in noncoding region of 12th exon of NPM in TK6 and WTK1,and it was the same as the result of one healthy volunteer. There was homozygous T deletion in noncoding region of 12th exon of NPM in human normal immortal lymphocyte line.However,there were no mutations in the coding region of 12th exon of NPM in the cells mentioned above.There was no mutations found in the other healthy volunteer,and it was absolutely the same as the sequences of GenBank.There were no chromosome translocations involved in 5^th chromosome including HNILL,TK6,WTK1 and blood lymphocyte specimen of healthy volunteers.Conclusions:Any functions involved in NPM in our study were related to the expression only.Partâ…¢:The effect ofγirradiation on NPM expression and chromosome instability of TK6 and WTK1.Objective:To investigate the relationship among apoptosis induced by irradiation,CIN,NPM expression and p53 status.Methods:TK6 and WTK1 were irradiated 4Gy by ¹³⁷Cs,the dose rate of which is 0.89Gy/min.Apoptosis rate was detected 24h postirradiation;chromosome number was observed at the time 0h,6h,12h,24h,48h postirradiation;mRNA expression of NPM was detected at the time 0h,1h,3h,6h,12h,24h postirradiation;Total and phosphorylated protein expression of NPM was also detected at the time 0h,3h,6h,12h,24h,36h,48h postirradiation.Results:Apoptosis rate was significant increased postirradiation both in TK6 and WTK1,and TK6 increased more than WTK1.TK6 and WTK1 emerged high rate of aneuploid 6～48h after 4Gy irradiation,from 54.3%in the control to 79～85%.The rate of polyploid increased sharply in both TK6 and WTK1.Polyploid rate of TK6 went from 1.4%to 13.5%,most of which were(near)triploid.But polyploid rate of WTK1 went higher,from 17.1%to 67%,most of which were(near)tetraploid.The level of mRNA,total and phosphorylated NPM protein of TK6 had little changes after 4Gy irradiation;but the mRNA of WTK1 went slightly to a lower level at 12～24h postirradiation.Total NPM protein level of WTK1 degraded at 12～48h after 4Gy irradiation,however the phosphorylated NPM protein went to a higher level at 12～24h postirradiation and then went to the baseline.Conclusions:Chromosome instability induced by irradiation in WTK1(mt p53) is significantly higher than that of TK6(wt p53).However,apoptosis rate of TK6 was much higher than that of WTK1.This phenomenon may be correlated with NPM expression induced byγirradiation and p53 status. Partâ…£:Influence of Olomoucine on the apoptosis rate induced byγirradiation and CIN.Objective:To evaluate the apoptosis rate and CIN after suppression of CDK2/cyclin E using olomoucine.Methods:Cells were irradiated after 6 hours of olomoucine treatment at the concentration of 10μmol/L.Changes of apoptosis rate and chromosome number were detected at 24h and 48h postirradiation respectively.Results:The apoptosis rate of TK6 and WTK1 was higher after Olomoucine treatment than control.The apoptosis of TK6 went from 33.5%to 43.9%,but the counterpart of WTK1 went from 16.8%to 25.1%.The apoptosis rate of TK6 is significantly higher than that of WTK1.The polyploid rate of TK6 went from 13.5% to 0 in the control of pure irradiation after olomoucine treatment.Therefore the counterpart of WTK1 degraded from 67%to 18%which is the level of unirradiated. Olomoucine had no effect on the aneuploid rate of TK6 and WTK1 induced by irradiation.Conclusions:Olomoucine raises the apoptosis rate of TK6 and WTK1 induced by irradiation to maintain chromosome stability by inhibiting phosphorylation of NPM protein.