P53R2 Impacts Mitochondrial Function In KB And PC-3 Cancer Cells And Its Expression Correlates With Prognosis Of Colorectal Cancer Patients

BackgroundRibonucleotide reductase (RR) plays an essential role in catalyzing the conversion of ribonucleoside diphosphates to the corresponding 2'-deoxyribonucleoside diphosphates, a rate-limiting step in the production of 2'-deoxyribonucleotide 5'-triphosphates (dNTPs) required for DNA synthesis and repair. In humans, a large subunit (RRM1) and two small subunits (RRM2 and p53R2) of RR have been identified. RRM1 contains substrate and allosteric effector binding sites that control the RR holoenzyme activity and substrate specificity. Recently cloned p53R2, a new RR family member, encodes a protein with striking similarity to RRM2. p53R2 participates in p53-directed repair of damaged DNA through forming a RR holoenzyme with RRM1 to synthesize dNTPs for DNA repair in response to UV light,γ-irradiation or adriamycin in a p53-dependent manner.A recent study showed that p53R2-null mice died from severe renal failure by 14 weeks of age. A greater number of apoptotic cells were observed in the kidneys of 8-week-old p53R2 mutant mice. Another study showed a direct correlation between p53R2 mutations and muscle mitochondrial DNA depletion in children with mitochondrial depletion syndrome. These results indicate the importance of p53R2-catalyzed ribonucleotide reduction for mtDNA synthesis in nonproliferating cells. In proliferating cells, deoxyribonucleotides can be imported into mitochondria from the cytoplasm; whereas nonproliferating cells also have to repair their DNA and synthesize mitochondrial DNA. Salvaging of deoxyribonucleosides by the intramitochondrial enzymes, deoxyguanosine kinase and thymidine kinase 2 is important, as mutations in either of these kinases result in the depletion of mitochondrial DNA in terminally differentiated, nonproliferating cells and cause severe diseases. Yet deoxyribonucleosides salvaging alone is insufficient, since proper mitochondrial DNA synthesis in nonproliferating cells also requires p53R2-catalyzed ribonucleotide reduction. Nonetheless, how p53R2 affects mtDNA synthesis, whether p53R2 localizes to mitochondria and p53R2 impacts mitochondrial function is still unclear.The disruption of the p53-p53R2 DNA repair system was associated with colon tumorigenesis in ulcerative colitis. Under irradiation, inhibition of p53R2 expression by siRNA causes three times higher mutation rate in TK6 cells. Our previous studies revealed that p53R2 is negatively related to invasion of cancer cells and metastasis of colorectal cancer. On other hand, inhibition of p53R2 could significantly enhance the invasion potential of KB, PC-3, HSC-3 and Ca9-22 cancer cells. Therefore, p53R2 might play an opposite role of RRM2 to suppress malignancy in cancer cells. So far, whether p53R2 impacts the outcome of human colorectal cancer is still unclear. PurposeTo address whether p53R2 impacts the mtDNA content and mitochondrial dNTP pools in KB and PC3 cells; to investigate if p53R2 localizes to mitochondria in KB and PC3 cells; to figure out whether the mitochondria have RR activity in KB and PC3 cells; we also determined the effect of p53R2 on mitochondrial function in KB and PC3 cells; to investigate whether p53R2 impacts the outcome on human colorectal cancers.ResultsPartⅠp53R2 Localizes to Mitochondria and Impacts the Mitochondrial Function in KB and PC3 Cells.1. p53R2 Expression Level is Associated with mtDNA Content and Mitochondrial Numbers in KB and PC-3 CellsPrevious studies revealed that mutations in p53R2 cause severe mitochondrial DNA depletion in mice and humans. To further confirm these findings in cancer cell lines, we suppressed p53R2 expression with p53R2 siRNA and measured the mtDNA content in KB and PC-3 cells. We measured the relative level of COX-1, a mitochondrial gene, to determine the influence of p53R2 suppression on mtDNA content by real-time PCR in KB and PC-3 cells. A decreased of 41.7% and 34.9% in mtDNA content (p<0.05) was measured in KB and PC-3 cells, respectively, after p53R2 expression was suppressed. mtDNA content decreased to 30.1% or 27.4% when ND1, another mitochondrial gene, was examined in a similar manner. In order to address whether the mitochondrial numbers decreased after p53R2 expression was suppressed. We used the electron microscopy to observe it. We found that the mitochondrial numbers decreased significantly (p<0.05) after p53R2 expression was suppressed. These results indicate that p53R2 expression is associated with mtDNA content and mitochondrial numbers in KB and PC-3 cells.2. p53R2 Localizes to Mitochondria and is Imported into Mitochondria depending on the TOM Machinery in KB and PC-3 CellsWestern blot analysis and cell fractionation were employed to detect the localization of p53R2 to the mitochondria. p53R2 was detected in the mitochondria, as well as in the cytoplasm. Further, coimmunostaining showed colocalization of p53R2 and cytochrome c in KB and PC-3 cells. Transiently expressed p53R2-EGFP fusion protein also colocalized with Mito-tracker red signals, confirming that p53R2 localizes to mitochondria. Zeiss LSM software determined that 66.8% and 61.2% of p53R2-EGFP colocalized to the mitochondria in KB or PC-3 cells, respectively. To confirm above findings, immunogold labeling of p53R2 was used to observe the localization of p53R2 to the mitochondria by electron microscopy. p53R2 labeling was apparent in the mitochondria and cytoplasm. It also showed an accumulation of p53R2 in mitochondria of KB and PC-3 cells. In order to determine whether the localization of p53R2 is associated with cell cycle, we used the serum-free medium to synchronize the cells. After 48h or 96h of serum starvation, medium with 10% serum was added and cellular localization of p53R2 was analyzed every 2 hours by immunofluorescence microscopy. The results showed no difference in each time point, suggesting that p53R2 localizes to the mitochondria regardless of cell cycle phase in KB and PC-3 cells.Nearly all mitochondrial preproteins are imported via the general entry gate, the translocase of the outer membrane (TOM complex). The central component of TOM is Tom40, an integral membrane protein that forms the channel for preprotein translocation across the outer membrane. Tom40 siRNA was used to suppress the function of TOM complex; the expression of p53R2 in KB and PC-3 cells was then detected. The expression of the p53R2 in mitochondria decreased profoundly after Tom40 was inhibited, whereas the expression level of p53R2 in the cytoplasm had no significant change. This result suggests that p53R2 is imported into mitochondria depending on the TOM machinery.3. RR Activity cannot be Detected in Mitochondria and Inhibition of p53R2 Expression Has Little Effect on Mitochondrial dNTP Pools in KB and PC-3 CellsTo explore the role of p53R2 in mitochondria, the RR activity of mitochondrial extracts (ME) and cytosol extracts (CE) were measured. Compared to the CE of KB and PC-3 cells, RR activity was almost undetectable in the ME. RR activity in the ME was 4.96% and 0.96% of RR activity in the CE in KB and PC-3 cells, respectively. RR small subunit p53R2 or RRM2 forms a complex with the large subunit RRM1 to generate enzyme activity. To further elucidate why RR activity cannot be detected in mitochondria, proteins extracted from the ME were analyzed by Western blot. RRM1 was barely detected in the ME suggesting that p53R2 has function in mitochondria independent of RRM1 and ribonucleotide reduction. To determine whether inhibiting p53R2 affects mitochondrial dNTP pools, the mitochondrial dNTP pools were determined in KB and PC-3 cells. After transfecting p53R2 siRNA in KB cells, the mitochondrial dATP, dTTP, dCTP, dGTP and total dNTP pools decreased from 7.3,12.6, 21.8,11.9 and 53.6 to 5.8,11.4,20.5,7.9 and 45.6 pmoles per million cells, respectively. Meanwhile, the p53R2 siRNA also reduced the mitochondrial dATP, dTTP, dCTP, dGTP and total dNTP pools from 4.0,2.6,1.9,1.7 and 10.2 to 2.7,2.2,1.4,1.5 and 7.8 pmoles per million cells in PC3 cells, respectively. More than 90% of dNTP pools remained after suppression of p53R2 expression in both cell lines suggesting that p53R2 has negligible impact on mitochondrial dNTP pools in normal condition in KB and PC-3 cells. 4. p53R2 Affects Mitochondrial Membrane Potential, Free ATP Synthesis and Cytochrome C Oxidase Activity in KB and PC-3 cellsWe investigated the influence of the p53R2 inhibition onΔΨmtt in KB and PC-3 cells via JC-1 staining. It showed that the transfection of p53R2 siRNA shifted these ratios toward lower values in KB and PC-3 cells. The percentage of R1 increased from 15.1% to 32.4% and 16.1% to 27.1% in KB and PC-3 cells, respectively. This indicates that suppression of p53R2 expression may cause mitochondrial membrane dysfunction in KB and PC-3 cells. At the same time, we measured mitochondrial free ATP synthesis and cytochrome c oxidase activity to probe the mitochondrial function in these cells. After suppressing p53R2 expression, free ATP was significantly reduced to 59.3% and 62.82% (p<0.05) in KB and PC-3, respectively. This result is compatible with mtDNA content data from our previous study. Mitochondrial cytochrome c oxidase activity was also detected after p53R2 suppression. In KB and PC-3 cells, its activity was decreased 88.8% and 87.3%, respectively. The above findings suggest that p53R2 may impact free ATP synthesis and cytochrome c oxidase activity on KB and PC-3 cells.PartⅡp53R2 Plays Essential Roles on Malignancy Suppression and Impacts Survival on Advanced Colorectal cancersA series of 220 consecutive colorectal cancer (CRC) patients who were received surgical treatment in 2nd affiliated hospital of Zhejiang University during 1999 and 2004 were entered in this outcome study. In addition,107 CRCs from City of Hope were collected as validation set. Patients were periodically followed up for survival. The immunohistochemistry (IHC) was conducted on tissue arrays to determine expression of p53R2. Both univariate and multivariate analysis indicated that p53R2 is negatively related to tumor invasion significantly among 220 CRCs (adjusted odds ratio, OR=0.5,95% CI 0.2-1.0). Survival analysis revealed that the p53R2 is significantly associated with overall survival (hazard proportional ratio, HR=0.56,95% CI 0.32-0.98). Further analysis indicated that RRM2B is negatively associated with survival of CRC atⅢ-ⅣTNM stage (adjusted HR=0.53,95% CI 0.28-1.01), rather thanⅠ-ⅡTNM stage (HR=1.02,95% CI 0.31-4.58). It was validated on stageⅣCRCs from City of Hope (HR=0.42,95% CI 0.20-0.91). Furthermore, p53R2 significantly reduces the HR of CRC at p53 mutant (HR=0.29,95% CI 0.14-0.63), but not in p53 wild type group (adjusted HR=0.99,95% CI 0.42-2.43).Conclusion1,p53R2 expression level is associated with mtDNA content and mitochondrial numbers in KB and PC-3 cells.2,p53R2 localizes to mitochondria and is imported into mitochondria depending on the TOM machinery in KB and PC-3 cells3,RR activity cannot be detected in mitochondria and inhibition of p53R2 expression has little effect on mitochondrial dNTP pools in KB and PC-3 cells.4,p53R2 affects mitochondrial membrane potential, free ATP synthesis and Cytochrome C Oxidase activity in KB and PC-3 cells.5,p53R2 might have malignancy-suppressing ability and impact outcome in advanced colorectal cancers.