Binding sequence-dependent regulation of the human proliferating cell nuclear antigen promoter by p53

Exposure of a lung epithelial cell line to ionizing radiation (IR) arrests cell cycle progression through 48 h post-exposure. Coincidentally, IR differentially activates expression of the cell cycle inhibitor, p21/WAF1, and the DNA replication protein, proliferating cell nuclear antigen (PCNA). p21/WAF1 mRNA levels remain elevated through 48 h post-exposure to IR, while PCNA mRNA levels increase transiently at early times. Since p21/WAF1 inhibits DNA replication by directly binding PCNA, the relative levels of the two proteins can determine cell cycle progression. The PCNA p53-binding site displayed reduced p53 binding affinity in vitro relative to the distal p21/WAF1 p53-binding site. Substitution of the p21/WAF1 site for the resident p53-binding site in the PCNA promoter altered the responses to increasing amounts of p53 or IR in transient expression assays. The p21/WAF1 p53-binding site sustained activation of the chimeric PCNA promoter under conditions (high p53 levels or high dose IR) that the PCNA p53-binding site did not. Binding site-specific regulation by wild-type p53 was not observed with mutant p53 harboring a serine to alanine change at amino acid 46. Limited activation of the PCNA promoter by p53 and its modified forms would restrict the amount of PCNA made available for DNA repair.     

Human NTH1 physically interacts with p53 and proliferating cell nuclear antigen

Thymine glycol (Tg) is one of predominant oxidative DNA lesions caused by ionizing radiation and other oxidative stresses. Human NTH1 is a bifunctional enzyme with DNA glycosylase and AP lyase activities and removes Tg as the first step of base excision repair (BER). We have searched for the factors interacting with NTH1 by using a pull-down assay and found that GST-NTH1 fusion protein precipitates proliferating cell nuclear antigen (PCNA) and p53 as well as XPG from human cell-free extracts. GST-NTH1 also bound to recombinant FLAG-tagged XPG, PCNA, and (His)6-tagged p53 proteins, indicating direct protein-protein interaction between those proteins. Furthermore, His-p53 and FLAG-XPG, but not PCNA, stimulated the Tg DNA glycosylase/AP lyase activity of GST-NTH1 or NTH1. These results provide an insight into the positive regulation of BER reaction and also suggest a possible linkage between BER of Tg and other cellular mechanisms.     

Induction of p53-dependent activation of the human proliferating cell nuclear antigen gene in chromatin by ionizing radiation

A human fibroblast cell line with conditional p53 expression displayed a p53-dependent increase in both the protein and mRNA levels of proliferating cell nuclear antigen (PCNA) after exposure to ionizing radiation (IR). The combination of p53 induction and IR cooperated to activate a transiently expressed human PCNA promoter-reporter gene via a p53-responsive element. Chromatin immunoprecipitation assays with antibodies specific for p53 or p300/CREB-binding protein revealed specific p53-dependent enrichment of PCNA promoter sequences in immunoprecipitates of sheared chromatin prepared from irradiated cells. Maximal and specific association of acetylated histone H4 with the PCNA promoter also depended on p53 induction and exposure to IR. These data demonstrate p53 binding to a target site in the PCNA promoter, recruitment of p300/CREB-binding protein, and localized acetylation of histone H4 in an IR-dependent manner. These molecular events are likely to play a role in mediating activation of PCNA gene expression by p53 during the cellular response to DNA damage. The analyses indicate that the combination of p53 induction and IR activate the PCNA gene via mechanisms similar to that of p21/wild-type p53-activated factor but to a lesser extent. This differential regulation of PCNA and p21/wild-type p53-activated factor may establish the proper ratio of the two proteins to coordinate DNA repair with cell cycle arrest.     

Backbone assignment of human proliferating cell nuclear antigen

PCNA is an essential factor for DNA replication, repair, chromatin metabolism, and effector of cell-cycle regulatory signals. The assignment of backbone ¹H_N, ¹³C_α, ¹³CO, and ¹⁵N, and sidechain ¹³C_β resonances of the human PCNA homotrimeric ring (∼90 kDa, 261 residues) is reported here.     

人增殖细胞核抗原的表达调控

增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)是一种生长调控蛋白,在DNA复制、修复、细胞周期调控、基因外遗传(epigenetic inheritance)等事件的协同机制中发挥重要功能。PCNA的表达调控发生在多个层次,涉及ATFl、CREB、RFXl、p53、E2F等转录因子以及内含子指导的反义RNA等等。     

Structure of the human gene for the proliferating cell nuclear antigen

The proliferating cell nuclear antigen (PCNA, cyclin) was originally defined as a nuclear protein whose appearance correlated with the proliferative state of the cell. It is now known to be a co-factor of DNA polymerase delta and to be necessary for DNA synthesis and cell cycle progression. cDNA clones of human PCNA have been isolated and, using one of these cDNA, we have now obtained from a lambda phage library a clone containing the entire human PCNA gene and flanking sequences. The human PCNA gene is a unique copy gene and has 6 exons. It spans, from the cap site to the poly(A) signal 4961 base pairs. We have identified, in the 5'-flanking sequence, a region with promoter activity, a well as other structural elements common to other promoters. An interesting feature of the PCNA gene is the presence of extensive sequence similarities among introns and between introns and exons.     

Programmed cell death, proliferating cell nuclear antigen and p53 expression in mouse colon mucosa during diet-induced tumorigenesis.

Western-style diets (WDs) trigger and sustain the early phases of tumorigenesis in mouse colon, and when continued throughout the life span lead to the development of dysplastic crypts. In order to evaluate the roles both of cell proliferation and programmed cell death (PCD) in WD-induced tumorigenesis, immunohistochemical detection of proliferating nuclear antigen (PCNA), in situ end labeling (TUNEL) of DNA breaks, and p53 protein were carried out in mouse colonic mucosa during prolonged feeding of two WDs. PCNA Labeling Index of colonic crypts was significantly higher in WD-treated animals than in controls only at the beginning of the nutritional study, the gap rapidly bridged by increased cell proliferation spontaneously occurring in the colonic mucosa during aging. A transient early homeostatic activation of PCD at the base of the crypt also was observed in WD groups. No changes in PCD were seen in the upper third of the crypt or in surface epithelium throughout the study, indicating that PCD in that colonic crypt segment produces a constant flux of cell loss, uninfluenced by homeostatic fluctuations. A major finding was an irreversible, progressive, age-related decline of PCD at the crypt base in both control and treated animals that occurred during the second half of the rodents' life span. p53 protein was not immunohistochemically detected, suggesting that neither overexpression of wild-type nor mutated forms of the protein are involved in the above mentioned changes.     

The immunohistochemical expression of stress-response protein (srp) 60 in human brain tumours: relationship of srp 60 to the other five srps, proliferating cell nuclear antigen and p53 protein

This study analyzed the expression of stress-response (heat-shock) protein 60 (srp 60) in a series of 158 human brain tumours. Immunohistochemical procedures were employed; cells of the human cervical cancer line HeLa S3 exposed to hyperosmolar stress served as positive controls. Deposits of reaction products were found in the cytoplasm. Approximately half of the glioblastomas multiforme (17/31), breast carcinoma metastases (6/10), and lung carcinoma metastases (5/11) as well as about one-third of the astrocytomas (5/13) and meningiomas (8/23) had tumour cells that expressed srp 60. A positive reaction for srp 60 was also seen in some medulloblastomas (2/16), primitive neuroectodermal tumours (PNETs) (2/11), schwannomas (2/21), and pituitary adenomas (2/7), but no positive reactions were observed with oligodendrogliomas and ependymomas. Compared with srp 60-negative tumours, srp 60-positive tumours coexpressed one or more stress-related proteins, among which srp 90, srp 72, srp 27, alphaB-crystallin and ubiquitin occurred with higher frequencies; a high correlation between srp 60 and the other five srps (0.88 - 0.97, p<0.01, Pearson correlation coefficient) was observed in srp 60-positive tumours. In contrast, the correlation coefficient in srp 60-negative tumours was not significant (-0.26 - 0.71). There was a tendency for the proliferating cell nuclear antigen (PCNA)-labeling index to be higher in glioblastomas, astrocytomas, medulloblastomas, PNETs, and breast and lung carcinoma metastases that expressed srp 60 than in those that did not. No significant immunohistochemical reactions of srp 60, PCNA and p53 protein were seen with sections of normal brain tissues. We conclude that primary and metastatic tumours of the brain produce srp 60 and that srp 60 in certain brain tumour cells may coexpress the other five srps. In addition, srp 60 expression might depend, in part, on proliferating potential.     

Regulation of proliferating cell nuclear antigen during the cell cycle

The proliferating cell nuclear antigen (PCNA), also known as cyclin and DNA polymerase delta auxiliary factor, is present in reduced amounts in nongrowing cells and is synthesized at a greater rate in the S phase of growing cells. The recently discovered involvement of PCNA in DNA replication suggested that this pattern of expression functions to regulate DNA synthesis. We have investigated this possibility further by examining the synthesis, stability, and accumulation of PCNA in HeLa cells fractionated by centrifugal elutriation into nearly synchronous populations of cells at various positions in the cell cycle. In these fractionated cells we found that there is an increase in the rate of PCNA synthesis with a peak in early S phase of the cell cycle, but the magnitude of the increase is only 2-3-fold. This change reflects similar changes in the amount of PCNA mRNA. The fluctuating synthesis of PCNA maintains this protein at a roughly constant proportion of the total cell protein, although the amount doubles/cell in the cell cycle. Consistent with this observation, the stability of PCNA does not differ significantly from that of total cellular protein in synchronized HeLa cells. We also observed that a maximum of one-third of the total PCNA is tightly associated with the nucleus, presumably in replication complexes, at the peak of S phase. We conclude that the cyclic synthesis of PCNA in cycling HeLa cells maintains PCNA in excess of the amount involved directly in DNA replication and the amount of the protein neither fluctuates significantly with the cell cycle nor is limiting for DNA synthesis.     

Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen

Uracil residues arise in DNA by the misincorporation of dUMP in place of dTMP during DNA replication or by the deamination of cytosine in DNA. Uracil-DNA glycosylase initiates DNA base excision repair of uracil residues by catalyzing the hydrolysis of the N-glycosylic bond linking the uracil base to deoxyribose. In human cells, the nuclear form of uracil-DNA glycosylase (UNG2) contains a conserved PCNA-binding motif located at the N-terminus that has been implicated experimentally in binding PCNA. Here we use purified preparations of UNG2 and PCNA to demonstrate that UNG2 physically associates with PCNA. UNG2 co-eluted with PCNA during size exclusion chromatography and bound to a PCNA affinity column. Association of UNG2 with PCNA was abolished by the addition of 100 mM NaCl, and significantly decreased in the presence of 10 mM MgCl(2). The functional significance of the UNG2.PCNA association was demonstrated by UNG2 activity assays. Addition of PCNA (30-810 pmol) to standard uracil-DNA glycosylase reactions containing linear [uracil-(3)H]DNA stimulated UNG2 catalytic activity up to 2.6-fold. UNG2 activity was also stimulated by 7.5 mM MgCl(2). The stimulatory effect of PCNA was increased by the addition of MgCl(2); however, the dependence on PCNA concentration was the same, indicating that the effects of MgCl(2) and PCNA on UNG2 activity occurred by independent mechanisms. Loading of PCNA onto the DNA substrate was required for stimulation, as the activity of UNG2 on circular DNA substrates was not affected by the addition of PCNA. Addition of replication factor C and ATP to reactions containing 90 pmol of PCNA resulted in two-fold stimulation of UNG2 activity on circular DNA.     

Immortalization of mutant p53-transfected human fibroblasts by treatment with either 4-nitroquinoline 1-oxide or x-rays

Summary The study of in vitro cell transformation is valuable for understanding the multistep carcinogenesis of human cells. The difficulty in inducing neoplastic transformation of human cells by treatment with chemical or physical agents alone is due to the difficulty in immortalizing normal human cells. Thus, the immortalization step is critical for in vitro neoplastic transformation of human cells. We transfected a mutant p53 gene (mp53: codon 273^Arg-His) into normal human fibroblasts and obtained two G418-resistant mp53-containing clones. These clones showed an extended life span but ultimately senesced. However, when they were treated with either 4-nitroquinoline 1-oxide or X rays, they were immortalized. The immortalized cells showed both numerical and structural chromosome abnormalities, but they were not tumorigenic. The expression of mutant but not wild type p53 was detected in the immortalized cells by RT-PCR. Expression of p21, which is located downstream of p53, was remarkably reduced in the immortalized cells, resulting in increased cdk2 and cdc2 kinase activity. However, there was no significant difference between the normal and immortalized human cells in expression of another tumor suppressor gene, p16. These findings indicate that the p53-p21 cascade may play an important role in the immortalization of human cells.     

ATM protein physically and functionally interacts with proliferating cell nuclear antigen to regulate DNA synthesis

Ataxia telangiectasia (A-T) is a pleiotropic disease, with a characteristic hypersensitivity to ionizing radiation that is caused by biallelic mutations in A-T mutated (ATM), a gene encoding a protein kinase critical for the induction of cellular responses to DNA damage, particularly to DNA double strand breaks. A long known characteristic of A-T cells is their ability to synthesize DNA even in the presence of ionizing radiation-induced DNA damage, a phenomenon termed radioresistant DNA synthesis. We previously reported that ATM kinase inhibition, but not ATM protein disruption, blocks sister chromatid exchange following DNA damage. We now show that ATM kinase inhibition, but not ATM protein disruption, also inhibits DNA synthesis. Investigating a potential physical interaction of ATM with the DNA replication machinery, we found that ATM co-precipitates with proliferating cell nuclear antigen (PCNA) from cellular extracts. Using bacterially purified ATM truncation mutants and in vitro translated PCNA, we showed that the interaction is direct and mediated by the C terminus of ATM. Indeed, a 20-amino acid region close to the kinase domain is sufficient for strong binding to PCNA. This binding is specific to ATM, because the homologous regions of other PIKK members, including the closely related kinase A-T and Rad3-related (ATR), did not bind PCNA. ATM was found to bind two regions in PCNA. To examine the functional significance of the interaction between ATM and PCNA, we tested the ability of ATM to stimulate DNA synthesis by DNA polymerase δ, which is implicated in both DNA replication and DNA repair processes. ATM was observed to stimulate DNA polymerase activity in a PCNA-dependent manner.