| Werner syndrome (WS) is a rare autosomal recessive disorder that displays symptoms of premature aging and increase risk for cancers. WS is regarded as the best model for studying human aging and cancers. In order to get WS mouse model, wrn knockout mice were produced. However, this mouse model did not display symptoms of premature aging. Further research found cells in human WS patients display telomere dysfunction that is essential for WS. Different from human, mouse somatic cells possess much longer telomere, and the telomerase keeps activity, thus can not manifest the phenotype of WS. Based on this finding, telomerase deficient and wrn null (mTerc-/-wrn-/-) mice were generated, which faithfully display human WS symptoms. Only late generation (G3-G5) of WS mice display WS symptoms when successive generations of telomerase deficient mice were produced to shorten telomeres that induced telomere dysfunction. This evidence further proved that telomere dysfunction is essential for WS.In our preliminary work, we found WS mouse embryonic fibroblast (MEF)(genotype:G5mTerc-/-Wrn-/-) entered soon into senescence in passaging, however, some senescent cells could escape fom senescence in long time culturing and become immortalized cell colonies. Within these immortalized cell colonies, some of them could form tumor by subcutaneous injection of SCID mouse. Because of negtive telomerase activity in these cells, the tumor formed by these cells belongs to ALT (Alternative lengthening of telomere) tumor. Overexpression of p21in these cells could efficiently inhibit cell proliferation and even induce cellular senescence. This result suggested that p21played a key role in inducing senescence and suppressing ALT tumor.To further research the role of p21in cellular senescence and tumor suppression, we crossed p21+/-wrn+/-mice with p21+/-wrn+/-mice to generate MEF with genotype of WT, Wrn-/-, p21-/-and p21-/-Wrn-/-. We overexpressed TRF2ΔBΔM in above cells to induce telomere dysfunction which simulating telomere dysfunction in WS MEF. By comparing the cellular responses in different genotype of cells, we explored the effects in cellular senescence and tumor formation caused by lack of p21.For the first time, our study try to understand the cellular responses and tumorigenesis in the condition of combining lack of p21function and lack of Wrn function, together with telomere dysfunction. One previous in vivo study has found that the p21null/Wrn mutant mice did not show an acceleration of tumorigenesis during the first year of life; Another study has shown that the deletion of p21prolongs the lifespan of telomerase-deficient mice with dysfunctional telomeres, however, p21deletion did not accelerate cancer formation. These two studies were conducted in the condition of "lack of p21and telomere dysfunction" or "lack of p21and lack of Wrn", respectively. Both studies showed that p21deletion would not accelerate cancer formation. So far, no study has been performed in the combination of all three factors: lack of p21, lack of Wrn, and telomere dysfunction". Our design of study could reflect the roles of p21in cellular senescence and tumor formation induced by telomere and Wrn dysfunction more precisely.Our main results are as following:First we compared cell proliferation ability of cells with the genotypes of WT, Wrn-/-, p21-/-or p21-/-Wrn-/-:we compared the growth curve of WT, Wrn-/-, p21-/-and p21-/-Wrn-/-in early generation (passage number=5) by crystal violet staining and found that cells lacking of p21growed faster than WT, Wrn-/-cells. We detected the protein level of PCNA (Proliferative Cell Nuclear Antigen) by western blot and found that PCNA expression increased in late generation cells lacking of p21, however, decreased in WT, Wrn-/-cells. Then we detected senescence in Wrn-/-, p21-/-and p21-/-Wrn-/-cells by senescence-associated β-galactosidase staining and found that Wrn-/-cells showed senescence staining, however, cells lacking of p21(p21-/-and p21-/-Wrn-/-cells) did not show senescence staining, suggesting that lack of p21rescued cellular senescence caused by Wrn deficience. Further, we detected senescence in cells with overexpression of TRF2ΔBΔM and found that Wrn-/-cells with overexpression of TRF2ΔBΔM showed senescence staining, however, cells lacking of p21with overexpression of TRF2ΔBΔM did not show senescence staining. The above results suggested that lack of p21could improve cells proliferation ability, and rescue the cellular senescence caused by telomere dysfunction (induce by either overexpression of TRF2ABAM or lack of Wrn function).Our data also suggested the possible mechanism that how lack of p21could rescue the cellular senescence:1. DNA damage response (DDR) reduction. We found p16and p19was lower in cells lacking of p21than Wrn-/-cells. In cells with transient overexpression of TRF2ΔBΔM, DDR will increase in response to telomere DNA damages induced by TRF2ΔBΔM. But in subsequent passages, those cells lacking of p21could down-regulate part of DDR by suppressing the expression of TRF2ΔBΔM, which suggested that lack of p21could down-regulate telomere DNA damages induced by TRF2ΔBΔM;2. DNA replication and repair potential improved. DNA damage is the key factor in inducing cellular senescence. DNA repair mechanism could reduce DNA damage and prevent cells from senescence. The PCNA could promote DNA replication and also involed in DNA repair, including mismatch repair and base excision repair. The p21could compete for PCNA binding with several PCNA-reliant proteins that are directly involved in DNA repair processe. We found that late generation cells lacking of p21have high expression level of PCNA, suggest this cells have high DNA replication and repair potential. To confirm this, we overepressed TRF2ΔBΔM in cells and found that the expression of PCNA was not changed in WT cells, but increased in cells lacking of p21. These results suggest that lack of p21can improve cells DNA replication and repair potential.3. Increased chromosome doubling. The karyotype in tumor cells are multiploid and mostly aneuploid. Tetraploidization has been proposed as an intermediate step toward aneuploidy in human cancer. Chromosome doubling may be an essential event in cell proliferation. By analysis of karyotype, we found that proportion of cells with chromosome doubling is12.5%in late generation of WT cells,65.9%in late generation of p21-/-cells and52.2%in late generation of p2r/-Wrn-/-cells. These results suggest that lack of p21increased the chromosome doubling. We then analyzed karyotype of p21-/-and p21-/-Wrn-/-cells with overepression of TRF2ΔBΔM, and found that telomere dysfunction increased chromosome doubling in p21-/-cells, the proportion of cells is as high as82.2%. However, the proportion is not increased in p21-/-Wrn-/-cells. These results indicate that telomere dysfunction and Wrn play an important role in chromosome doubling induced by lack of p21.Surprisingly, our data showed that expression of p16is higher in p21-/-cells than in WT cells. In cells with transient overexpression of TRF2ΔBΔM, expression of p16is higher in p21-/-cells than in WT cells. These data suggested that increase of p16alone could not induce cellular senescence when p21function is missing. We found increase of p16is related to chromosome doubling:p16expression level is higher in late generation of p21-/-cells than in late generation of WT cells. Coincidently, the chromosome doubling is65.9%in late generation of p21-/-cells, is around12.5%in late generation WT cells; The p16expression level is higher in p21-/-cells with transient overexpression of TRF2ΔBΔM than in control p21-/-cells. Again, the chromosome doubling is87.2%in p21-/-cells with TRF2ΔBΔM, but65.9%in control p21-/-cells. In late generation cells, p16is lower in p21-/-Wrn-/-cells than inp21-/-cells. In consistant with this, the chromosome doubling is52.2%in p21-/-Wrn-/-cells, is65.9%in p21-/-cells; p16is not increased inp21-/-Wrn-/-cells with TRF2ΔBΔM compared to control p21-/-Wrn-/-cells. The chromosome doubling is not increased in p21-/-Wrn-/-cells with transient overexpression of TRF2ΔBΔM compared to control p21-/-Wrn-/-cells; In cells with transient overexpression of TRF2ΔBΔM, p16is lower in p21-/-Wrrn-/-cells than in p21-/-cells. The chromosome doubling is46.3%in p21-/-Wrn-/-cells, lower than87.2%in p21-/-cells. These results suggest that increase of p16in p21-/-cells may be a stress response to chromosome doubling.We want to know if cells lacking of p21with TRF2ΔBΔM become cancerous, so we kept passaging p21-/-+TRF2ΔBΔM cells. We found that cells lacking of p21could reduce DDR by suppressing expression of TRF2ΔBΔM. The results from WB and fluorescent immunostaining showed that expression of TRF2ΔBΔM was reduced in cells lacking of p21. Since expression of TRF2ΔBΔM was reduced, DDR factors, such as p53, p19and γ-H2AX, were also reduced. However, in late generation cells, p19increased and γ-H2AX was recovered as well. We sequenced p53in late generation cells and found p53point mutation C138W occurred in these cells. Then we detected karyotype in cells with p53mutation and found that p53mutation increased cells’ genomic instability. By transformation assay and subcutaneous injection in SCID mice and nude mice, we found that although cells with p53mutation had strong ability of monoclonal formation in transformation assay, monoclonal was small and cells could not form tumors. The results suggested that lack of p21could prolong cells life span when suffered from telomere dysfunction induced by TRF2ΔBΔM, but will not induce tumorigenesis. Our results are consistent with results reported by Rudolph in mice.We passaged p21-/-Wrn-/-+TRF2ΔBΔM cells to observe the fate of cells when challenged with multiple telomere damages and study the role of p21in the whole process. By WB assay we found that although expression of TRF2ΔBΔM in p21-/-Wrn-/-+TRF2ΔBΔM cells was reduced gradually, DDR still increased gradually and cells showed increased selective pressure:p53, p19, p16and PCNA were all increased gradually.In p22-/-Wrn-/-(mid-term) cells with overexression of TRF2ΔBΔM, expression of p-p53was suddenly increased that suggested function of p53is activated! This time, others DDR signals, such as y-H2AX, p19, p16, p53and PCNA were also increased. Surprisingly, cells displayed strong proliferation capacity and did not show senescence phenotype. By p53sequecing, we found a p53point mutation R270L! These data suggested that lack of Wrn function induced accumlation of telomere damage in p21-/-+TRF2ΔBΔM cells and accelerated p53mutation. We checked the tumor formation ability of cells in late generation. We found that cells had strong ability of monoclonal formation in transformation assay and the colonies were big and cells could form tumor in SCID mice and nude mice. We cultured the tumor tissue and sequenced p53in tumor tissue. We found the same p53mutation R270L in those culture cells. By HE staining of tumor tissue and pathological analysis, we confirmed that this tumor was sarcoma. These results suggested that p21-/-Wrn-/-+TRF2ΔBΔM cells with p53mutation were cancerous. Then we checked DDR by WB assay in tumor tissues and tumor cultured cells. We found that p19and PCNA were highly expressed. The p53, p-p53, p16were not highly expressed in tumor tissue, however, they were highly expressed in tumor cells. These results indicate that in vitro culture environment is different from environment in vivo. The p53mutation also promoted genomic instability in p21-/-Wrn-/-+TRF2ΔBΔM cells, such as enhanced polyploidy and aneuploidy, increased chromosome fusion (36.6%) and breakage (14.6%), apprearing double minutes. But genomic stability is improved in cultured tumor cells which have less chromosome fusion (12.1%) and few chromosome breakage and double minutes Though with polyploidy and aneuploidy, number of chromosomes in about69%tumor cells were betweeen40-80. Only about3.4%tumor cells have more than160chromosomes. These results suggested that genomic instability was partially repaired while the tumor formed and numbers and structure of chromosome tend to stabilized in the tumor.Telomere dysfunction induced by TRF2ΔBΔM are similar to that induced by telomerase deficiency, but with some difference. To further verify the role of p21in WS ALT tumor, we will use MEF cells with negtive telomerase activity. In our previous data, we found WS MEFs G5mTerc-/-Wrn/-/-prone senescence in passaging, however, some senescent cells could escape fom senescence in long time culturing and become immortalized cell colonies. Within these immortalized cell colonies, some of them could not form tumor by subcutaneous injection of SCID mouse. In these cells expression of p21is high. We selected one of them,395-6B-1cells and transfected short hairpin RNA (shRNA) of p21(p21-shRNA) to reduce the expression of p21. Then we injected these cells into subcutaneous of SCID mouse. The result showed that these cells can form tumors in SCID mice. Because of negtive telomerase activity in these cells, the tumor induced by these cells belongs to ALT tumor. This data suggested that p21play a key role in suppression of ALT tumor in WS MEF cells with negtive telomerase activity.In conclusion, for the first time, our study try to understand the cellular responses and tumorigenesis in the condition of combining lack of p21function and lack of Wrn function, together with telomere dysfunction. We showed that p21play a primary role in cellular senescence induced by telomere dysfuncion; Increase of p16in cells lacking of p21is related with increased chromosome doubling; Lack of p21could prolong life span of cells suffered from telomere dysfunction induced by TRF2ΔBΔM, but will not induce tumor; Lack of p21could promote tumor formation induced by multiple telomere dysfunction induced by TRF2ΔBΔM and lack of Wrn. |
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