Expression of proliferating cell nuclear antigen (PCNA)/cyclin during the cell cycle

The expression of proliferating cell nuclear antigen (PCNA), also called cyclin, was quantified in the cell lines SP2/0 and MOLT-4 and in mouse splenocytes induced to proliferate in vitro with mitogens. Autoantibody from a patient with systemic lupus erythematosus was used to label PCNA in cell suspensions after the cells had been fixed and permeabilized. In the same cells DNA was stained by propidium iodide. The cells were then analysed by flow cytometry for PCNA and DNA content. The PCNA profiles in proliferating spleen cells and the cell lines were similar. Most G0-G1 cells did not express significant amount of PCNA. A dramatic increase in PCNA immunofluorescence was observed in late G1 cells, and further increases were observed in S-phase cells. G2-M cells showed a reduced level of PCNA immunofluorescence relative to S-phase cells but were still elevated relative to G0-G1 cells. Proliferating cells arrested at the G1-S boundary by exposure to cytosine arabinoside showed an increased PCNA immunofluorescence as compared to unstimulated cells.     

Intranuclear localization of proliferating cell nuclear antigen during the cell cycle in renal cell carcinoma

OBJECTIVE: To investigate, with laser scanning cytometry (LSC), proliferating cell nuclear antigen (PCNA) expression during the cell cycle in renal cell carcinoma. STUDY DESIGN: DNA ploidy and intracellular localization of PCNA in renal cell carcinoma were determined using LSC and immunohistochemistry. The subjects were nine patients who had received surgery for renal cell carcinoma. After DNA ploidy analysis, the glass slides were restained by immunohistochemistry of PCNA. LSC allowed direct observation of PCNA localization during the cell cycle because we could obtain immunohistochemical staining of PCNA as a function of cell cycle phase for individual cells. RESULTS: PCNA was not demonstrated in the nuclei of G0/G1 cells. PCNA expression increased from the S phase of the cell cycle. PCNA rapidly degraded at the end of the G2 phase. In the late G2 and M phase, PCNA was not detected in almost any nucleus. CONCLUSION: LSC allows morphologic observation of the intracellular distribution of PCNA during the cell cycle in renal cell carcinoma.     

Requirement for proliferating cell nuclear antigen expression during stages of the Chinese hamster ovary cell cycle

Proliferating cell nuclear antigen (PCNA/cyclin) is a nuclear protein that can stimulate purified DNA polymerase delta in vitro, and its synthesis correlates with the proliferation rate of cells. We have attempted to determine whether synthesis of PCNA/cyclin in Chinese hamster ovary cells is necessary to regulate entry into S phase. We have measured cellular PCNA/cyclin concentration of the mRNA or protein throughout the cell cycle. Cells were separated by centrifugal elutriation into populations enriched for G-1, S, and G-2/M phases. Quantitative Northern hybridization analysis was performed on RNA isolated from each cell population by using a cDNA clone of PCNA/cyclin as a probe. Results demonstrated that although intact PCNA/cyclin mRNA is present during all phases of the cell cycle, an induction of about 3-fold occurs during S phase. Two-parameter staining for PCNA/cyclin and DNA, and analysis by flow cytometry, confirmed that the quantity of PCNA/cyclin protein in the cells increases severalfold in G-1 or early S phase but generally is invariant in S and G-2/M phases. This cell cycle dependence of PCNA/cyclin expression suggests that the observed synthesis is a prerequisite for initiation of DNA replication. Introduction of an antisense oligonucleotide complementary to the PCNA/cyclin mRNA to inhibit PCNA/cyclin synthesis effectively prevented entry of G-1 phase cells into S phase. A complementary sense oligonucleotide used as a control did not have an inhibitory effect. This result suggests that a threshold concentration of PCNA/cyclin is necessary for entry into S phase.     

Regulation of apoptosis and cell cycle progression by MCL1. Differential role of proliferating cell nuclear antigen

MCL1 (ML1 myeloid cell leukemia 1), a Bcl-2 (B- cell lymphoma-leukemia 2) homologue, is known to function as an anti-apoptotic protein. Here we show in vitro and in vivo that MCL1 interacts with the cell cycle regulator, proliferating cell nuclear antigen (PCNA). This finding prompted us to investigate whether MCL1, in addition to its anti-apoptotic function, has an effect on cell cycle progression. A bromodeoxyuridine uptake assay showed that the overexpression of MCL1 significantly inhibited the cell cycle progression through the S-phase. The S-phase of the cell cycle is also known to be regulated by PCNA. A mutant of MCL1 that lacks PCNA binding (MCL1(Delta)(4A)) could not inhibit cell cycle progression as effectively as wild type MCL1. In contrast, MCL1(Delta)(4A) retained its anti-apoptotic function in HeLa cells when challenged by Etoposide. In addition, the intracellular localization of MCL1(Delta)(4A) was identical to that of wild type MCL1. An in vitro pull-down assay suggested that MCL1 is the only Bcl-2 family protein to interact with PCNA. In fact, MCL1, not other Bcl-2 family proteins, contained the PCNA-binding motif described previously. Taken together, MCL1 is a regulator of both apoptosis and cell cycle progression, and the cell cycle regulatory function of MCL1 is mediated through its interaction with PCNA.     

Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites

Pulse-chase experiments have revealed that cyclin, the auxiliary protein of DNA polymerase-delta, is stable during the transition from growth to quiescence in 3T3 cells. Immunoblotting together with immunofluorescence analysis has shown that the amount of cyclin after 24 h of quiescence is 30-40% of that of growing cells and that it presents a nucleoplasmic staining. Immunofluorescence studies show the existence of two populations of cyclin during the S phase, one that is nucleoplasmic as in quiescent cells and is easily extracted by detergent, and another that is associated to specific nuclear structures. By using antibromodeoxyuridine immunofluorescence to detect the sites of DNA synthesis, it was shown that the staining patterns of the replicon clusters and their order of appearance throughout the S phase are identical to those observed for cyclin. Two-dimensional gel analysis of Triton-extracted cells show that 20-30% of cyclin remains associated with the replicon clusters. This population of cyclin could not be released from the nucleus using high-salt extractions. This demonstrates that cyclin is tightly associated to the sites of DNA replication and that it must have a fundamental role in DNA synthesis in eukaryotic cells.     

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。     

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.     

Regulation of the proliferating cell nuclear antigen cyclin and thymidine kinase mRNA levels by growth factors

The enzymes of the DNA synthesizing machinery constitute a group of gene products that are generally expressed co-ordinately at the G1/S boundary of the cell cycle. We have investigated how growth factors regulate the steady-state mRNA levels of two of these genes, the PCNA (proliferating cell nuclear antigen)/cyclin and the thymidine kinase genes. To detect the PCNA/cyclin mRNA, we isolated a cDNA clone from a human library. Two different cell lines were used for these studies: BALB/c3T3 cells, which are exquisitely sensitive to growth factors, and ts13 cells, a temperature-sensitive (ts) mutant of the cell cycle, which arrests in G1 at the restrictive temperature. The steady-state levels of the RNAs for these two genes under different growth conditions were also compared with the levels of histone H3 RNA which are good indicators of the fraction of cells in S phase. Both PCNA/cyclin and thymidine kinase genes share two fundamental characteristics, i.e. they are not inducible in a G1-specific ts mutant of the cell cycle at the restrictive temperature and their expression is inhibited by cycloheximide, indicating that unlike early growth-regulated genes, they require the previous expression of other growth-regulated genes. However, the two genes also show differences, the most notable being that PCNA/cyclin is inducible by epidermal growth factor alone, while thymidine kinase is not.     

Changes in cyclin/proliferating cell nuclear antigen distribution during DNA repair synthesis

UV irradiation of quiescent human fibroblasts immediately triggers the appearance of the nuclear protein cyclin/proliferating cell nuclear antigen (PCNA) as detected by indirect immunofluorescent staining after methanol fixation. This was found to be independent of new synthesis of cyclin/PCNA by two-dimensional gel analysis and cycloheximide treatment. The intensity of the immunofluorescent staining of cyclin/PCNA observed in UV-irradiated cells corresponded with the UV dose used and with the DNA repair synthesis detected by autoradiography. The nuclear staining remains as long as DNA repair activity is detected in the cells. By extracting the UV-irradiated quiescent cells with Triton X-100 and fixing with formaldehyde, it was possible to demonstrate by indirect immunofluorescence rapid changes in the cyclin/PCNA population after irradiation, a small proportion (5-10%) of which is tightly associated to the nucleus as determined by high salt extraction. By incubating at low temperature and depleting the ATP pools of the cells before UV irradiation, we have demonstrated that the changes in cyclin/PCNA distribution observed involve at least two different nuclear associations.     

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.     

Flow cytometric multiparameter analysis of proliferating cell nuclear antigen/cyclin and Ki-67 antigen: a new view of the cell cycle

Flow cytometric multiparameter analysis of two proliferation-associated nuclear antigens (proliferating cell nuclear antigen (PCNA)/cyclin and Ki-67) was performed on seven human hematopoietic cell lines. PCNA/cyclin, an S phase-related antigen, was detected using an autoantibody and a fluorescein isothiocyanate-labeled anti-human antibody. The Ki-67 antigen, which in cycling cells is expressed with increasing levels during the S phase with a maximum in the M phase, was detected using a monoclonal antibody and a phycoerythrin-conjugated anti-mouse antibody. In some experiments the PCNA/Ki-67 staining was combined with a DNA stain, 7-amino actinomycin D, and simultaneous detection of the three stains was performed by a single laser flow cytometer. Using this technique four distinct cell populations, representing G1, S, G2, and M, respectively, could be demonstrated in cycling cells on the basis of their PCNA/cyclin and Ki-67 levels. The cell cycle phase specificity could be verified using metaphase (vinblastine, colcemide) and G2 phase (mitoxantrone) blocking agents, as well as by stainings with a mitosis-specific antibody (MPM-2). Also, G0 cells could be discriminated from G1 cells in analysis of a mixture of resting peripheral mononuclear blood cells and a proliferating cell line. This technique can be valuable in detailed cell cycle analysis, since all cell cycle phases can be visualized and calculated using a simple double staining procedure.     

Expression of proliferationg cell nuclear antigen during the cell cycle of human diploid fibroblasts

Summary Proliferating cell nuclear antigen mRNA levels were determined in human diploid fibroblasts as they progressed through the cell cycle. PCNA message levels were low at G0, gradually increased following entrance into G1, peaked at G1/S, and declined during S phase. PCNA mRNA was determined to have a half life of 12 hours when cells were blocked at the G1/S interface. PCNA protein levels increased two- to three-fold as cells moved from G0 to S phase.     

Detection of proliferating cell nuclear antigen in diagnostic histopathology.

We describe the effects of tissue preservation, fixation time, and hydrolytic treatment on the detection of proliferating cell nuclear antigen (PCNA) by immunoperoxidase staining with three commercial anti-PCNA antibodies (19A2, 19F4, PC10). Our goal was to provide guidelines for PCNA immunohistochemistry in formalin-fixed, paraffin-embedded specimens. In proliferative cell compartments, nuclear staining was achieved with all three antibodies. In some cases PCNA was also expressed in non-proliferative, histologically normal tissues associated with tumors or other lesions elsewhere. In most autopsy specimens PNCA immunoreactivity was markedly diminished as compared with similar surgical specimens. Incubation overnight with primary antibody at 4 degrees C enhanced PCNA immunoreactivity over incubation at 42 degrees C for 45 min. Pre-treatment with 2 N HCl did not increase staining. Staining with the PC10 antibody was much better preserved than staining with the antibodies 19A2 and 19F4 after prolonged formalin fixation of surgical specimens and in tissues obtained at autopsy. With all three antibodies, however, PCNA immunoreactivity was well preserved during formalin fixation for 8-24 hr and during fixation delays for 8 hr at room temperature. This indicates that PCNA is stable under conditions routinely encountered in diagnostic surgical pathology and facilitates its potential use as a diagnostic proliferation marker.     

Mechanism whereby proliferating cell nuclear antigen stimulates flap endonuclease 1

Human flap endonuclease 1 (FEN1), an essential DNA replication protein, cleaves substrates with unannealed 5'-tails. FEN1 apparently tracks along the flap from the 5'-end to the cleavage site. Proliferating cell nuclear antigen (PCNA) stimulates FEN1 cleavage 5-50-fold. To determine whether tracking, binding, or cleavage is enhanced by PCNA, we tested a variety of flap substrates. Similar levels of PCNA stimulation occur on both a cleavage-sensitive nicked substrate and a less sensitive gapped substrate. PCNA stimulates FEN1 irrespective of the flap length. Stimulation occurs on a pseudo-Y substrate that exhibits upstream primer-independent cleavage. A pseudo-Y substrate with a sequence requiring an upstream primer for cleavage was not activated by PCNA, suggesting that PCNA does not compensate for substrate features that inhibit cleavage. A biotin.streptavidin conjugation at the 5'-end of a flap structure prevents FEN1 loading. The addition of PCNA does not restore FEN1 activity. These results indicate that PCNA does not direct FEN1 to the cleavage site from solution. Kinetic analyses reveal that PCNA can lower the K(m) for FEN1 by 11-12-fold. Overall, our results indicate that after FEN1 tracks to the cleavage site, PCNA enhances FEN1 binding stability, allowing for greater cleavage efficiency.     

Deviating the level of proliferating cell nuclear antigen in Trypanosoma brucei elicits distinct mechanisms for inhibiting proliferation and cell cycle progression

The DNA replication machinery is spatially and temporally coordinated in all cells to reproduce a single exact copy of the genome per division, but its regulation in the protozoan parasite Trypanosoma brucei is not well characterized. We characterized the effects of altering the levels of proliferating cell nuclear antigen, a key component of the DNA replication machinery, in bloodstream form T. brucei. This study demonstrated that tight regulation of TbPCNA levels was critical for normal proliferation and DNA replication in the parasite. Depleting TbPCNA mRNA reduced proliferation, severely diminished DNA replication, arrested the synthesis of new DNA and caused the parasites to accumulated in G2/M. Attenuating the parasite by downregulating TbPCNA caused it to become hypersensitive to hydroxyurea. Overexpressing TbPCNA in T. brucei arrested proliferation, inhibited DNA replication and prevented the parasite from exiting G2/M. These results indicate that distinct mechanisms of cell cycle arrest are associated with upregulating or downregulating TbPCNA. The findings of this study validate deregulating intra-parasite levels of TbPCNA as a potential strategy for therapeutically exploiting this target in bloodstream form T. brucei.     

Regulation of DNA replication and repair proteins through interaction with the front side of proliferating cell nuclear antigen.

The DNA polymerase accessory factor proliferating cell nuclear antigen (PCNA) has been caught in interaction with an ever increasing number of proteins. To characterize the sites and functions of some of these interactions, we constructed four mutants of human PCNA and analysed them in a variety of assays. By targeting loops on the surface of the PCNA trimer and changing three or four residues at a time to alanine, we found that a region including part of the domain-connecting loop of PCNA and loops on one face of the trimer, close to the C-termini, is involved in binding to all of the following proteins: DNA polymerase delta, replication factor C, the flap endonuclease Fen1, the cyclin dependent kinase inhibitor p21 and DNA ligase I. An inhibition of DNA ligation caused by the interaction of PCNA with DNA ligase I was found, and we show that DNA ligase I and Fen1 can inhibit DNA synthesis by DNA polymerase delta/PCNA. We demonstrate that PCNA must be located below a 5' flap on a forked template to stimulate Fen1 activity, and considering the interacting region on PCNA for Fen1, this suggests an orientation for PCNA during DNA replication with the C-termini facing forwards, in the direction of DNA synthesis.     

Molecular cloning of Phaseolus vulgaris cDNA encoding proliferating cell nuclear antigen

A cDNA fragment encoding a common bean (Phaseolus vulgaris) proliferating cell nuclear antigen (PCNA) was isolated using rapid amplification of cDNA 3' end (3' RACE) method, cloned and sequenced. The nucleotide sequence of this clone contains an open reading frame of 798 nucleotides encoding a protein of 265 amino acids. Alignment of the common bean PCNA predicted sequence shows its high degree of identity with PCNA from other plant species. Analysis of PCNA content in the germinating embryos of common bean showed a decrease in the protein level after 60h of germination. Moreover, PCNA was not detected in the tested plant organs (root, stem, leaf and flower). The presence of PCNA in the germinating seeds and its absence from mature plants suggests that this protein plays a crucial role during early stages of plant development.