DNA repair and cell cycle regulation of human aging fibroblasts after UV damage
Duan Jianming Zhang Zongyu Tong Tanjun
(Department of Biochemistry and Molecular Biology, Beijing Medical University, Beijing 100083, China)
Abstract Human embryonic lung diploid fibroblasts (2BS) of different ages were cultured in vitro. After UV-induced DNA damage, cell morphology, proliferation characteristics, cell cycle, DNA repair and other cellular responses were observed, as well as gadd153 and p21. Expression of transcript levels in genes such as p53. Results: UV-induced DNA damage, senescence (>55 generations) 2BS cell shape
The changes of state and proliferative ability were not as significant as those of younger cells (<30 generations); G1 arrest occurred in cells of different ages, and the G1 arrest rate of young cells was significantly higher than that of senescent cells (P<0105); aging The total repair capacity of the cells was significantly lower than that of the younger cells (P<0101). At the same time, the inducibility of gadd153, p21, p53, etc. was lower than that of the young 2BS cells. Thus, the senescent cell cycle was reflected at the cell level and the gene level, respectively. The relationship between changes in cellular response and repair function after UV irradiation.
Key words senescent cells, DNA damage repair, cell cycle, checkpoint control
The accumulation of DNA damage and the decline of repair ability are one of the important causes of biological aging . p53, p21 and ATM are the check2point control genes of three DNA damage responses in human cells discovered in recent years . P53 and p21 not only play an important role as tumor suppressor genes, but also play a central role in the cellular response of DNA damage. They can regulate the transcription of many genes such as DNA damage repair, cell cycle arrest and apoptosis . Gadd45 and gadd153 in injury-inducible genes are also closely related to cell cycle arrest and repair after DNA damage, and may act as downstream genes of p53 . Whether these genes have expression changes and senescence in senescent cells Is the decline in cell repair ability related? It has not been reported at home and abroad. We studied human embryonic lung diploid fibroblasts (2BS) cultured in vitro and studied the DNA damage inducible gene gadd153 in senescent cells after UV irradiation. And the control point control gene p21, p53 expression changes, and its correlation with DNA repair capacity and cell cycle changes.
1 Materials and methods
1.1 Materials and main reagents Human embryonic lung diploid fibroblasts were obtained from the Institute of Biological Products of the Ministry of Health. The pB luesSK plasmid (gadd153 cDNA) was kindly provided by the People's Hospital of Beijing Medical University. p21WAF1.CIP1. SDI1 cDNA was donated by Baylor Medical College, USA. P53 and B2act in cDNA probes are retained in this room. DMEM dry powder medium: GIBCO.BRL, fetal calf serum: Beijing Beijiao Farm Blood Products Institute, 3H2TdR and A232P dCTP: Beijing Yahui Bioengineering Company, P rim e2a2Gene Lebeling System: Promege, hydroxyurea, salmon sperm DNA, polyvinylpyrrolidone, bovine serum albumin: Sigma, other reagents are Sig2ma products or domestic analytical pure.
1.2 Cell culture Human embryo lung diploid fibroblasts (2BS) were cultured in DMEM medium containing 10% fetal bovine serum, grown in a 37 Â°C incubator, and passaged to 62Â±4 generations and 24Â±6 generations as senescence and Young cells are used.
1.3 Cell morphology and proliferation characteristics After the cells were grown to logarithmic growth phase, the cell suspension was adjusted to 1Ã—104 cells.ml, and transferred to a 24-well plate for culture. After the cells were attached, the cells were exposed to ultraviolet light (0145 J.m 2.s). The cells were irradiated for 5 min , and the fresh medium was replaced at 37 Â° C. The morphological changes were observed every 12 h and the cells were counted. The senescence and young cells without UV irradiation were used as controls.
1.4 Cell cycle analysis The dose and time of UV irradiation were the same as above. After incubation for 24 hours at 37 Â°C, the cells were trypsinized, and the cells were collected, then digested with RNase at 37 Â° C for 1 h, then stained with propidium iodide (PI), and flowed with Bec2ton2 Dickinson. The instrument was subjected to FACScan (fluo res2cence act ivated cell sorting) analysis, and senescence and young cells without ultraviolet irradiation were used as controls.
1.5 UDS (un scheduled DNA syn thes is) After measuring the cell count, transfer the cells to a 6-well plate at 1Ã—105 cells. After the cells are completely attached, the cells are starved for 72 hours in DMEM containing 015% fetal bovine serum. The cells were synchronized, and the serum-free DMEM containing 5 mmo lL hydroxyurea was replaced at 37 Â° C for 1 h, then irradiated with ultraviolet light at the same dose and time as above. The medium was replaced (containing 5 mmo lL hydroxyurea, 1 LCi.m l3H2TdR, 10%). The fetal bovine serum DMEM) was collected at 37 Â° C for different time to collect 3H2TdR counts, and the control group was cells without ultraviolet irradiation .
1.6 Spot and Northern blot hybrid cells were irradiated with ultraviolet light in the logarithmic growth phase, time and dose were the same, then the cells were harvested at 37 Â° C for different time, and the total RNA was extracted by one step of guanidinium isothiocyanate, and then transferred to the membrane. Labeled probes were subjected to spot and Northern hybridization [6,7], and cells that were not irradiated with ultraviolet light were used as controls. Hybridization results were analyzed by integrated optical density scanning.
2.1 Comparison of cell morphology and proliferation characteristics Before UV irradiation, the morphology of senescent 2BS cells was significantly different from that of young cells. The senescent cells had hypertrophy, increased intracytoplasmic granules, cytoplasmic opacity, decreased refractive index, and extremely irregular cell alignment. The cell body is slender, the cytoplasm is clear, and the cells are arranged in a spiral shape. After ultraviolet irradiation, the morphological changes of senescent cells are not very obvious, only the cytoplasmic granules increase slightly; while the young cells show a significant increase in cytoplasmic granules and cytoplasmic transparency. Decreased, the cell body also increased slightly, but the cell morphology returned to normal after about 1 week of culture. The cell growth curve before and after UV irradiation is shown in Fig. 1. The proliferation rate of young cells is significantly inhibited by UV irradiation, but the proliferation ability after about 6 days. It returned to normal; the proliferation rate of senescent cells was significantly lower than that of young cells, and the proliferation changes before and after ultraviolet irradiation were not very obvious.
212 Cell cycle phase changes G1 arrest was observed in both senescent and young 2BS cells after UV irradiation, whereas the G1 phase cell arrest rate of young cells was higher than that of senescent cells (P < 0105) (Fig. 2).
213 DNA repair ability comparison 3H2TdR incorporation method for the determination of non-procedural DNA synthesis (UDS) to indicate the total DNA repair capacity. From F ig. 3, UDS peaks around 12 h after UV exposure. The ability of aging 2BS cells to repair Significantly lower than younger cells (P < 0101).
The results of 214 spot and Northern blot hybridization showed that the expression of gadd 153 peaked after 6 h of UV irradiation, and the gadd 153 of senescent cells was significantly less inducible than that of young cells. The results are shown in Fig. 4. Results The cells were collected 6 h after UV irradiation and Northern hybridization. The results showed that (Fig. 5), the inducibility of gadd153, p53 and p21 genes in senescent cells after UV irradiation was significantly lower than that of young cells, but p 53 Induction changes are minimal. In addition, the basic expression of these three genes before UV damage is also different in senescent cells and young cells. The basic expression of p 21 in senescent cells is significantly higher than that in young cells, and the expression of gadd153 is The senescent cells appear to be slightly higher than the young cells, but the expression of p53 is not significantly different.
Cellular senescence is a complex result of multiple factors and multiple pathways. The accumulation of DNA damage and the decline of repair ability may be one of the important causes of cell senescence . After DNA damage, senescent cell response and young cells have some Different. The morphological and proliferative curves of senescent cells are not obvious, and their recovery is slow. The results of flow cytometry show that the G1 arrest rate of senescent cells is lower than that of young cells; and the DNA repair ability of senescent cells Significantly lower than younger cells. These results indicate that senescent cells are significantly less responsive to DNA damage than younger cells. So, what is the underlying cause of the decline in aging cells' ability to respond to DNA damage? Gene expression status of p53, p21 and gaddl53 Changes before and after UV exposure may provide us with some valuable clues.
After DNA damage, it will induce cell reaction through certain signaling pathways, induce activation or inhibition of many gene expression, and activation of DNA repair system is an important mechanism for cells to maintain their functional integrity and cell survival . p 53 In the DNA damage monitoring, it is in the position controlled by the checkpoint and plays a central role. Both p21 and gadd153 are downstream genes. They share some common pathways and can act on the cell cycle and repair system through different pathways. The damage repair gene regulatory network interacts with each other and plays different roles . p21 is a cyclin kinase inhibitor, which acts as a downstream gene of p 53 and bridges between p 53 and cell cycle regulation. Role. High expression of p 21 leads to cell cycle arrest, and also affects DNA replication and repair by interaction with PCNA (proliferating cell nuclear antigen) . gadd153 is also dependent on p53 in UV-induced expression. As a downstream gene of p53, it has a certain influence on the cell cycle after injury, and may be coupled with DNA damage repair on the other hand. Gadd153 has a negative regulatory effect on the transcription factor C/EBP (CAAT enhancer binding protein) family . The experimental results show that the G1 phase arrest of both young and senescent cells after UV damage is accompanied by gadd153, The high expression of the p 21 and p 53 genes indicates that the inducible of these three genes is directly related to cell cycle arrest; however, it is interesting to note that senescent cells have significant G1 arrest before UV irradiation. Only high expression of p 21 was observed, suggesting that p 21 may play a major role in the G1 arrest of senescent cells, whereas the high expression of p21 is due to the accumulation of damage in senescent cells that cannot be effectively repaired. Because the senescent cells themselves That is, there is a significant G1 phase block, and p 53 , gadd 153, especially the inducible decrease of p 21 gene, may be the reason that the G1 phase of senescent cells is lower than that of young cells. Meanwhile, the decline of aging cell repair ability may also be The inducibility of DNA damage after gadd gene (including gadd153, gadd45, etc.), p 21 and p 53 is related to the decline of senescent cells. Experimental results show that although pi is in senescent cells 1 The high expression of this cell cycle inhibitor leads to G1 arrest of cells, and it takes time to repair the damage, but other genes directly related to repair can not effectively express and act. In the experiment, p 53 was also found. The expression changes are not very significant, suggesting that the high expression of p 21 in senescent cells may be regulated by the non-p 53 pathway ; on the other hand, this may be related to the effect of p 53 by the change of phosphorylation status [ 11 ] After the injury induction, the binding activity of p53 protein to various genes may be altered by the change of its phosphorylation status, which may also be one of the reasons why p 53 has no significant high expression in senescent cells. The damage inducibility of these genes in senescent cells is consistent, which may be one of the root causes of the decline of cell cycle regulation and repair ability of senescent cells after DNA damage. In summary, the investigation of senescent cells Cell cycle regulation, DNA damage repair, and DNA damage can induce changes in gene expression after DNA damage, facilitating the elucidation of DNA damage repair gene regulatory networks.
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