Roles Of PCNA Ubiquitination In Cisplatin Induced DNA Damage Response Pathways

DNA is highly vulnerable to spontaneous and environmental damage in living cells. If not repaired, DNA may cause damage -induced genetic instability, the cancer risk will be increased. Eukaryotic cells possess several DNA damage reponse mechanisms to protect the integrity of their genome, these include cell–cycle checkpoint, DNA repair pathways, apoptosis and also a distinct DNA damage-tolerance system that allows recovery of replication forks blocked at site of DNA damage. Guarding the integrity of DNA depends on a number of DNA-repair and cell cycle–control systems. Insight into how these pathways become activated is crucially important to the understanding of carcinogenesis and cancer treatments.Proliferating cell nuclear antigen (PCNA) was originally characterised as a DNA sliding clamp for replicative DNA polymerases. Subsequent studies have revealed it's striking ability to interact with multiple partners, which are involved in several metabolic pathways, including cell cycle regulation, DNA repair, apoptosis and translesion DNA synthesis. And many PCNA-binding proteins contain a common PCNA binding motif( PIP-box , PCNA interaction protein box). The discovery of the overlapping nature of the binding sites for these PCNA binding proteins suggested that the different partners must bind and dissociate sequentially to perform their functions. PCNA in mammalian cells thus appears to play a key role in controlling several reactions through the coordination and organization of different partners. However, two major questions have emerged: during DNA damage response, how do these proteins access PCNA in a coordinated manner, and how these pathways become activated?Recent studies of have revealed that PCNA can be ubiquitinated on the response to ultraviolet (UV) irradiation. This modification occurs on the lys164 of PCNA. It is discovered that this modification can mediate the polymerases switch from replicative polymerases to TLS polymeraseηunder the UV damage . Though it is thought that damage-tolerance synthesis is actived by ubiquitinated PCNA ,this modification may causes the recruit of damage tolerance polymerases to stalled replication forks , the actual mechanisms of this modification leaves largely unknown.Cisplatin (cis-diamminedichloro-platinum (II)) is one of the most commonly used and successful chemotherapeutic agents employed to date. The major cell-killing effect is due to cisplatin-DNA adducts.The main mechanism for the removal of cisplatin adducts from double-stranded DNA is nucleotide excision repair and is thought to involve the same processes that remove UV-induced DNA adducts. However, nucleotide excision repair is not the only DNA repair process that can influence the response to cisplatin, there are even some other metabolic pathways for that , including mismatch repair (MMR) and homologous recombination. Recent studies have indicated that tolerance to UV-induced DNA damage involves damage-induced PCNA monoubiquitination, this modification can mediate the polymerases switch from replicative polymerases to TLS polymerasesη, So far, there has a question is: similar to UV damage response, if the ubiquitinated PCNA even has a important role in cisplatin induced damage ? And as a coordinating protein, what is the actual function of ubiquitinated PCNA in cisplatin induced DNA damage response and how are these pathways co-ordinated?To evaluate the important cellular activities of ubiquitinated PCNA, here, we chose the dominant negative stategy. With the construction of mutant PCNA changing the K164 to R164, in which the PCNA had been mutated to abolish ubiqitination completely, the Hela cells stably overepressed of PCNA or mPCNA are generated. The sensitivity, apoptosis, DNA damage repair ability, cell cycle arrest and mutagenesis to cisplatin damage are compared between the wild type and the stable transfected cells. Our work can give an initial insight into the role of ubiquitinated PCNA in mediating mammalian cellular response to DNA damage .Methods:1. Construction and identification of PCNA and mutant PCNA eukaryotic expression plasmid:The full length cDNA of PCNA was generated by PCR by using pTAP-PCNA as a template,then the cDNA fragment of the PCNA was cloned in pGEM-T Easy vector. After digestion with HindⅢand XbaⅠ,the product was inserted into the eukaryotic expression vector pCDNA3.1/V5-HisA digested with HindⅢand XbaⅠ. pCDNA3.1/V5-His A-mPCNA (His-mPCNA, K164R)mutant cDNA was generated by one-step opposite direction PCR by using pGEM-T- Easy -PCNA as a template and the product was sub-cloned in the eukaryotic expression vector pCDNA3.1/V5-His A . The expression of the protein in the cell lines was determined by Western blot.2. Overexpression of His-PCNA and His-mPCNA in Hela cells2.1 Stable transfection: The Hela cells were seeded onto 35mm cell culture dishes and cultured to 80% confluence. The Hela cells were then separately transfected with the His -PCNA and His -mPCNA plasmid DNA using a cationic lipid lipofectamineTM 2000( 4ug plasmid DNA/ 4ul lipofectamineTM 2000/35mm dish) for 6 h. The medium was replaced with fresh cell growth medium and cultured for 48 h. The cells were further cultured in G418-containing medium (600μg/mL G4l8) for 2 weeks for selection of the G418-resistant colonies. The colonies were then subcultured, and the expression level of protein in the stably transfected cell lines was determined by Western blot.2.2 Analysis of Proliferating Cell Nuclear Antigen-ubiquitination:Two stable transfection Cells were grown to 70% confluence then incubated with 15μmol/L cisplatin for 4 hour. After treatment, the cells were placed in fresh serum-containing medium for 24 h,then harvested and analyzed by probing Western blot with the anti–his antibody (1:300,Santa Cruz) subsequently detected using enhanced chemiluminescence.3. Comparison of the sensitivity and apoptosis of wild type and stable transfection Hela cells in response to cisplatin treatment3.1 sensitivity analysis :1,000 cells (Hela,PCNA- Hela,mPCNA- Hela)were seeded in each well of a six-well plate and allowed to attach overnight. Drugs were added to wells in triplicate to give final concentrations as indicated. Cells were exposed to drugs for 4 hours before replacement with normal media. Cells were allowed to grow for a further 6 to 10 days in order to form colonies, after which time, medium was removed and colonies were fixed with methanol and stained with 5% Giemsa solution. Colonies of >50 cells were counted ,data shown are calculated from the mean values of three independent experiments.3.2 Annexin V-FITC apoptosis detection: Cells were plated in 100ml dishes and incubated at 37℃overnight. The cells were treated with either 15 or 20μmol/L cisplatin for 4 h. After washing completely, the cells were cultured in normal growth medium. The cells were harvested at various time points (12h and 24h) and washed twice with cold PBS. The cells were resuspended with 200μl 1×binding buffer at a concentration of 1×106 cells/ml. Add 5μl of Annexin V–FITC and 10μl of propidium iodide to tube and incubate for 15 min at room temperature in the dark, then analyze by flow cytometry as soon as possible (within 1 hr).4. Cell cycle analysis of wild type and mPCNA cell lines in response to cisplatin treatment:Cells were plated in 10 cm dishes and allowed to attach overnight. The cells were treated with 15μmol/L cisplatin for 4 h. After washing completely, the cells were cultured in normal growth medium. The cells were harvested at various time points and then the cellular DNA content was analyzed using a flow cytometer .5. Comparison of the DNA repair ability between Hela, PCNA-Hela, mPCNA-Hela cells. The plasmid PUG15 DNA was treated with cisplatin to generate DNA damage. Then both the undamaged and cisplatin-damaged plasmid were transfected into wild type and stable transfection Hela cells for DNA repair and luciferase gene expression.After forty-eight hours, the luc activity was detected to evaluate the DNA repair ability of different cells.6. Determination of the function of PCNA ubiquitination in cisplatin induced DNA Mutagenesis:The pSupFG1 plasmid DNA was treated with cisplatin to generate DNA damage.Then transfected into the wild type and stable transfection Hela cells.After forty-eight hours,we extracted the supF from these cells and used DPNⅠto digest for three hours,then transformed the digested supF into SY204 bacteria,planked the bacterias on the plates which contained X-gal,IPTG and ampicillin, the seconed day ,there are a lot of blue spots and a few of white spots on the plates. We count the amount of spots to get the frequency of mutations.Results:1. The eukaryotic expression plasmids were successfully constructed :The eukaryotic expression plasmids were identified by restriction enzyme and sequencing ,they were consistent with the sequences reported in GenBank.2. Overexpression of His-PCNA and His-mPCNA in Hela Cell Lines2.1 Stable transfection:To investigate the role of PCNA in response to cisplatin induced DNA damage , we established two stable transfection cell lines : His-PCNA- Hela and His-mPCNA- Hela (K164R).The results obtained from our Western blot using anti-his antibody indicated that these two cell lines can stable overexpress the recombination proteins.2.2 Ubiquitination of Proliferating Cell Nuclear Antigen after cisplatin treatment: In His-PCNA cells, a band of PCNA corresponding to 44 kD appeared in cisplatin dependent manner, while in His-mPCNA cells, the band remained at the 36KD even after cisplatin treated.These results clearly indicate that in cisplatin treated Hela cells ,the wild type PCNA was ubiquitinated but the mPCNA Hela cells can not be ubiquitinated after DNA damage.3. Comparison of the sensitivity and apoptosis of wild type and stable transfection Hela cells in response to cisplatin treatment3.1 sensitivity analysis :The mPCNA cells were found to be significantly more sensitive to cisplatin(colony formation:from 100% to 18% )compared with the wild type cells(colony formation:from 100% to 70% ), but the stable expressing wild type PCNA Hela cells (colony formation:from 100% to 65% )showed almost as normal cisplatin sensitive as the wild type cells.3.2 Apoptosis detection :Cisplatin treatment of mPCNA Hela cells has a negligible amount of DNA apoptosis increased in a cisplatin-dose and time-dependent ways, apoptosis from 5.7% to 36.74% ,compared to wild type cells from 5.18% to 12.24%.4. Cell cycle analysis of wild type and mPCNA cell lines in response to cisplatin reatment:The S-phase arrested of wild type Hela cells reached the highest point (59%) at 24 h after cisplatin treated. The mPCNA Hela cells failure to be ubquitianted were nearly have no S phase delay at 24 h (33%)after cisplatin treated compared to untreated cells(31%).5. Host cell reactivation assay:The relative rate of DNA repair ability were: WT- Hela:25%,Hela-PCNA:22%,Hela-mPCNA:23%。There is no significant difference compared with contral groups.6. In vivo mutation detection : We successfully transfected the pSupFG1 into different cells,then extracted the plasmid,and efficiently transformed it into SY204 bacterias.We get the frequency of mutations in different cells by cell counting:WT- Hela:2.58‰; PCNA-Hela:2.54‰;mPCNA-Hela:0.57‰. The mPCNA Hela cells has a significantly lower mutations compared to wild type and the PCNA Hela cells. Conclusions:1. The cell model of disrupting the PCNA ubiquitination was esbablisted.2. PCNA ubiquitination plays an important role in cisplatin induced damage response. Overexpression of mPCNA protein caused an increased apoptotic cell death after cisplatin treatment.3. PCNA ubiquitination has no impact on the DNA repair ability.4. Ubiqitinated PCNA may improve the S-phase cell cycle arrest on the ciplatin-mediated DNA damage response, and the intrinsic cisplatin drug resistance.5. PCNA ubiquitination is involved in mutations generation on the ciplatin-mediated DNA damage response.