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Results for “Proliferating Cell Nuclear Antigen”

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At least 19 recordsLinked to original sources

PubMed1993

[Importance of the colon in intestinal adaptation. Study with the proliferating cell nuclear antigen].

Proliferating cell nuclear antigen (PCNA) is an auxiliary protein to DNA polymerase delta necessary for tissue cellular proliferation. The colon releases several peptides or hormones which are probably related to intestinal proliferation. Colonic resection determines adaptive changes in the remnant bowel. In the present study, proliferative changes after colectomy were studied by means of the murine monoclonal PC10 antibody. A control group (n = 10 rats) and a 75% proximal colon resection group (n = 10 rats) were studied. 14 days after resection, jejunal, ileal and colon samples were taken and assayed for PCNA. Relationship between immunostained nuclei and the total number of nuclei was determined. The three intestinal segments showed statistically significant increases (p < 0.001) in the number of immunostained nuclei. PCNA proliferative index was greater in the remnant large bowel.

Adaptation, Physiological↗

PubMed1995

Influence of formalin fixation time and tissue processing method on immunoreactivity of monoclonal antibody PC10 for proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA), an auxiliary protein of DNA polymerase-delta, has recently been proposed as a marker of proliferation that is detectable in formalin-fixed paraffin-embedded tissue. Fixation time has been known to influence protein immunoreactivity and therefore can significantly influence the results of a quantitative immunohistochemical assay. In this study, we investigate the relationship between formalin fixation time and immunoreactivity for PCNA in paraffin-embedded sections and examine the effect of postfixation tissue treatment with modified Bouin's solution. Samples of small and large intestine from two freshly sacrificed rats were fixed in 10% buffered formalin for 6, 30, 54, 174, 340, and 508 h. Standard histological processing was performed on paired specimens whose treatment differed only by predehydration immersion in a picric acid and mercuric chloride-containing solution. Paraffin sections were reacted with monoclonal antibody PC10 in a standard immunoperoxidase assay. Staining intensity for PCNA was scored on a scale of 0 to 10, and the mean number of PCNA-positive cells per crypt (10 crypts counted) was determined. No difference between animals was found. PCNA immunoreactivity was maximal in specimens fixed for 6 to 30 h, exponentially declining with longer fixation time. The rate of decline was mitigated in the treated sections. Fixation-time dependence of PCNA immunoreactivity has immediate implications for intra- and interlaboratory comparisons, especially in experimental studies in which specimens can be stored in formalin for variable times followed by batch processing. With regard to surgical pathology specimens, this study suggests that sample comparisons are valid, since routine fixation time is within the optimal rage for PCNA immunodetection.

Animals↗

PubMed1995

Structure-function relationship of the eukaryotic DNA replication factor, proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA) is essential for eukaryotic DNA replication and functions as a processivity factor of DNA polymerase delta (pol delta). Due to the functional and structural similarity with the beta-subunit of Escherichia coli DNA polymerase III, it has been proposed that PCNA would act as a molecular clamp during DNA synthesis. By site-directed mutagenesis and biochemical analyses, we have studied the functional domains of human PCNA required for stimulation of replication factor C (RF-C) ATPase and DNA synthesis by pol delta. Short deletions from either the N or C termini caused drastic changes in extraction and chromatographic behaviors, suggesting that both of these terminal regions are crucial to fold the tertiary structure of PCNA. The short C-terminal stretch from Lys254 to Glu256 is necessary for stimulation of RF-C ATPase activity, but not for stimulation of DNA synthesis by pol delta. Nine basic amino acids that are essential for activating DNA synthesis by pol delta are positioned at the internal alpha-helices of PCNA. This result is in good agreement with the observation that PCNA has a ring structure similar to the beta-subunit and clamps a template DNA through this positively charged internal surface. Several other charged amino acids are also required to stimulate either RF-C ATPase or pol delta DNA synthesis. Some of them are positioned at loops which are exposed on one of the side surface of PCNA adjacent to the C-terminal loop. In addition, the beta-sheets composing the intermolecular interface of the trimeric PCNA are important for interaction with pol delta. Therefore, the outer surface of PCNA has multiple functional surfaces which are responsible for the interaction with multiple factors. Furthermore, the two side surfaces seem to be functionally distinguishable, and this may determine the orientation of tracking PCNA along the DNA.

Adenosine Triphosphatases↗

PubMed1999

Studies on the interactions between human replication factor C and human proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA) is a processivity factor required for DNA polymerase delta (or epsilon)-catalyzed DNA synthesis. When loaded onto primed DNA templates by replication factor C (RFC), PCNA acts to tether the polymerase to DNA, resulting in processive DNA chain elongation. In this report, we describe the identification of two separate peptide regions of human PCNA spanning amino acids 36-55 and 196-215 that bind RFC by using the surface plasmon resonance technique. Site-directed mutagenesis of residues within these regions in human PCNA identified two specific sites that affected the biological activity of PCNA. Replacement of the aspartate 41 residue by an alanine, serine, or asparagine significantly impaired the ability of PCNA to (i) support the RFC/PCNA-dependent polymerase delta-catalyzed elongation of a singly primed DNA template; (ii) stimulate RFC-catalyzed DNA-dependent hydrolysis of ATP; (iii) be loaded onto DNA by RFC; and (iv) activate RFC-independent polymerase delta-catalyzed synthesis of poly dT. Introduction of an alanine at position 210 in place of an arginine also reduced the efficiency of PCNA in supporting RFC-dependent polymerase delta-catalyzed elongation of a singly primed DNA template. However, this mutation did not significantly alter the ability of PCNA to stimulate DNA polymerase delta in the absence of RFC but substantially lowered the efficiency of RFC-catalyzed reactions. These results are in keeping with a model in which surface exposed regions of PCNA interact with RFC and the subsequent loading of PCNA onto DNA orients the elongation complex in a manner essential for processive DNA synthesis.

Amino Acid Substitution↗

PubMed1994

Inhibition of gastric cancer cell proliferation by antisense oligonucleotides targeting the messenger RNA encoding proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA) is a nuclear protein that regulates DNA synthesis by DNA polymerase delta, and is essential for DNA replication. PCNA expression level is related to the malignancy of gastric cancer cells. Seven different gastric cancer cell lines and two kinds of control cell lines were treated with antisense oligonucleotides complementary to the messenger RNA of PCNA. Treatment of each gastric cancer cell line with antisense oligonucleotides at concentration of 10-40 microM inhibited the cell growth, colony formation and PCNA protein production in a dose-dependent manner, but only affected normal cells slightly. A random sequence oligomer showed no effect. These results show that PCNA is essential for gastric cancer cell proliferation and that the use of synthetic oligonucleotides is an effective way of producing antisense-mediated changes in the behaviour of human gastric cancers.

Base Sequence↗

PubMed1996

Determination of the epitope of an inhibitory antibody to proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA) is an essential component for the normal processive DNA synthesis by DNA polymerase delta and is also required for DNA excision repair. Human PCNA autoantisera has been shown to inhibit the function of PCNA in vitro, in contrast to induced antibodies. A monoclonal IgG2 PCNA antibody, 74B1, that effectively inhibited DNA replication in vitro was identified. The inhibitory effect was dose dependent and using synthetic overlapping peptides of PCNA the 74B1 epitope was mapped to aa 121-135, a region of the PCNA protein containing the interdomain connector implicated in intermolecular interactions. Interestingly, a neighboring and partly overlapping peptide, aa 111-125, contains an immunodominant region recognized by a number of monoclonal PCNA antibodies with no inhibitory effect on DNA synthesis. The 74B1 antibody is a potentially useful antibody in future studies of PCNA and its interaction in complexes associated with cell cycle progress, DNA replication, and DNA repair.

Amino Acid Sequence↗

PubMed1992

Deletion studies to reveal the basis for size discrepancy in proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA), an essential component for DNA replication in eukaryotes, is a highly conserved nonhistone nuclear protein of 261 amino acids. The molecular weight of mammalian PCNA, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), differs notably from that predicted by the cDNA sequences, that is, 36,000 in comparison with 29,261 and 28,748 for human and rat PCNA, respectively. To investigate if this discrepancy is due to posttranslational modifications, we studied the PCNA protein synthesized by an in vitro transcription/translation system as well as the protein overproduced in bacteria. We found that both PCNA protein samples were indistinguishable from the authentic protein from the protein mobility in SDS-PAGE. The finding indicates that the size discrepancy is not due to the posttranslational modifications. Hence, the size discrepancy may be due to the protein sequence per se, namely a sequence-related anomaly in SDS-PAGE. Results from the analyses of a series of PCNA derivatives with various lengths of C- or N-terminal deletion indicate that the putative sequence is in the region of residues 128-150.

Amino Acid Sequence↗

PubMed2002

Direct interaction between mammalian DNA polymerase beta and proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA) plays an essential role in nucleic acid metabolism as a component of the DNA replication and DNA repair machinery. As such, PCNA interacts with many proteins that have a sequence motif termed the PCNA interacting motif (PIM) and also with proteins lacking a PIM. Three regions in human and rat DNA polymerases beta (beta-pol) that resemble the consensus PIM were identified, and we show here that beta-polymerase and PCNA can form a complex both in vitro and in vivo. Immunoprecipitation experiments, yeast two-hybrid analysis, and overlay binding assays were used to examine the interaction between the two proteins. Competition experiments with synthetic PIM-containing peptides suggested the importance of a PIM in the interaction, and studies of a beta-polymerase PIM mutant, H222A/F223A, demonstrated that this alteration blocked the interaction with PCNA. The results indicate that at least one of the PIM-like sequences in beta-polymerase appears to be a functional PIM and was required in the interaction between beta-polymerase and PCNA.

Amino Acid Motifs↗

PubMed1994

D-type cyclin-binding regions of proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA) is an auxiliary protein for DNA polymerase delta and is required for both DNA replication and DNA repair. PCNA forms complexes with D-type cyclins, candidate G1 cyclins in mammalian cells. To better understand the functions of the complexes, we examined interactions between PCNA and D-type cyclins, using in vitro-translated mouse PCNA and mouse cyclin D1 or D3 fused to glutathione S-transferase (GST). Analysis of a set of deletion mutants of PCNA revealed that either the N-terminal (residues 2-64) or the C-terminal (residues 197-228) region is necessary for association with D-type cyclins. The cyclin binding of the chimeric protein of the N-terminal (residues 1-68) or the C-terminal (residues 195-261) region of PCNA and rat DNA polymerase beta which does not bind to the cyclins by itself supports this notion. The purified recombinant mouse PCNA expressed in Escherichia coli bound to the D-type cyclin-GST fusion proteins, thereby suggesting that PCNA binds directly to D-type cyclins, without the requirement of other cellular factors. This is apparently the first report on the structure-function relationship of PCNA which may link DNA replication and DNA repair with cell cycle control.

Animals↗

PubMed2002

Saccharomyces cerevisiae RRM3, a 5' to 3' DNA helicase, physically interacts with proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA) plays an essential role in eukaryotic DNA replication, and numerous DNA replication proteins have been found to interact with PCNA through a conserved eight-amino acid motif called the PIP-box. We have searched the genome of the yeast Saccharomyces cerevisiae for open reading frames that encode proteins with putative PIP-boxes and initiated testing of 135 novel candidates for their ability to interact with PCNA-conjugated agarose beads. The first new PCNA-binding protein identified in this manner is the 5' to 3' DNA helicase RRM3. Yeast two-hybrid tests show that N-terminal deletions of RRM3, which remove the PIP-box but leave the helicase motifs intact, abolish the interaction with PCNA. In addition, mutating the two phenylalanine residues in the PIP-box to alanine or aspartic acid reduces binding to PCNA, confirming that the PIP-box in RRM3 is responsible for interaction with PCNA. The results presented here suggest that the RRM3 helicase functions at the replication fork.

Amino Acid Motifs↗

PubMed1992

Influence of free thiol group(s) on autoantibody-defined epitope of proliferating cell nuclear antigen.

Proliferating cell nuclear Ag (PCNA) is an intranuclear protein involved in DNA replication directed by DNA polymerase delta and is the target Ag of autoantibodies in some patients with SLE. There is evidence that the epitope on PCNA recognized by human autoantibodies is conformation-dependent and is not a continuous peptide sequence. Thimerosal, a mercury-containing sulfhydryl blocking compound, markedly reduced or abolished the reactivity of this autoantibody-defined PCNA epitope. The thimerosal effect was observed in various Ag-detecting systems including indirect immunofluorescence, immunodiffusion, immunoprecipitation, and flow cytometry. The mechanism of the thimerosal effect appeared to be mediated through free but not readily accessible sulfhydryl group or groups. The sulfhydryl-modification by thimerosal could be reversed by competition with thiol-containing compounds. Experimentally induced mAb to PCNA generated by immunization with purified PCNA have been shown to recognize epitopes that are continuous peptide sequences and these epitopes were not affected by thimerosal. It has been shown that the human autoantibody-defined epitope is related to the function of PCNA because autoantibodies are able to inhibit DNA polymerase delta directed DNA replication, whereas experimentally induced antibodies are not. These studies show that certain sulfhydryl groups in PCNA have a role in determining the antigenicity of the epitope recognized by autoantibody and raise the possibility that certain sulfhydryl groups might also be associated with its function.

Autoantibodies↗

PubMed2001

Evaluation of germ-cell kinetics in infertile patients with proliferating cell nuclear antigen proliferating index.

AIM: To explore the usefulness of proliferating cell nuclear antigen proliferating index (PCNA PI) in the pathological diagnosis and treatment of male infertility. METHODS: Testicular biopsy specimen obtained from 48 cases of male infertility and 2 normal controls were fixed and embedded. The sections were stained with anti-PCNA monoclonal antibodies or haematoxylin/eosin. Proliferating index (PI), expressed as the percentage of germ-cell nuclei positively stained with PCNA antibody, was assessed from more than 20 seminiferous tubules or 600 germ-cells. RESULTS: The infertile patients were divided into 4 groups: Group 1, normal spermatogenesis (14 cases); Group 2, hypospermatogenesis (16 cases); Group 3, germinal arrest (10 cases); Group 4, Sertoli cell only syndrome (8 cases). The PCNA PI of normal control testis was 86.5% (mean value). Group 3 had a significantly lower PCNA PI (29.8%) than normal testis; Group 1 and 2 had similar PIs (82.3% and 82.3%, respectively) as the control testis. PI of the negative control (Group 4) was 0 as no germ-cells were found. CONCLUSION: PCNA PI is useful for assessing germ-cell kinetics, especially for pathological diagnosis of germinal arrest which is difficult to differentiate by routine HE staining technique. In germinal arrest, there is a significantly lowered PCNA PI, which is an indication of DNA synthesis deterioration, suggesting the use of therapies be different from those for hypospermatogenesis.

Adult↗

PubMed1995

Clinicopathologic features and prognosis of resected hepatocellular carcinomas of varied sizes with special reference to proliferating cell nuclear antigen.

BACKGROUND: Proliferating cell nuclear antigen (PCNA) is an intranuclear protein that is linked closely to the cell cycle. An immunohistochemical study was performed on the expression of PCNA in various sized hepatocellular carcinomas (HCCs) to determine the relation between the proliferative activity of cancer cells and prognosis. METHODS: One hundred forty-one neoplasms resected from 141 patients who underwent hepatic surgery for HCC at Kyushu University hospital from April, 1991, to July, 1993, were studied immunohistochemically using monoclonal antibody for PCNA (PC10), and analyzed for a possible correlation between PCNA labeling index (PCNA-LI) and the prognosis of patients with HCC. RESULTS: Proliferating cell nuclear antigen reactive cancer cells were observed throughout the HCC. The PCNA-LI ranged from 1.2%-91.6%, with a mean of 37.7%. The high PCNA-LI (> 37.7) group showed a significantly higher incidence of tumor thrombus in the portal vein, higher Edmondson's Grade, and a higher recurrence rate than the low PCNA-LI (< 37.7) group. Hepatocellular Carcinomas were divided into three groups according to tumor size. Based on the clinicopathologic findings, in small (< 30 mm) HCCs, the high PCNA-LI (> 29.0) group showed a significant higher Edmondson's grade and a higher recurrence rate than the low PCNA-LI (< 29.0) group. In medium (30-60 mm) HCCs, the high PCNA-LI (> 36.1) group showed a significantly higher recurrence rate than the low PCNA-LI (< 36.1) group, although there was no difference in the pathologic findings between the high (> 59.4) and low (< 59.4) PCNA-LI groups. CONCLUSIONS: Proliferating cell nuclear antigen labeling index is closely related to cell differentiation and the prognosis of HCC. Furthermore, PCNA-LI was found to be useful in predicting the intrahepatic spread and prognosis of small (< 30 mm) and medium (30-60 mm) but not large (> 60 mm) HCCs.

Antigens, Neoplasm↗

PubMed1996

Biologic characteristics of esophageal epithelial dysplasia assessed by proliferating cell nuclear antigen.

BACKGROUND: Proliferating cell nuclear antigen (PCNA) correlates with the cell proliferative state. Using a PCNA specific monoclonal antibody, various malignant neoplasms have been assessed for cell proliferation and malignant potential. However, there are few studies regarding the expression of PCNA in esophageal dysplasia. METHODS: PCNA expression was immunohistochemically determined in resected specimens from 27 patients with superficial esophageal carcinoma that contained areas with various degrees of dysplasia, carcinoma in situ (CIS), and carcinoma invading the mucosal layer. The authors estimated the cell proliferative activity and biologic characteristics of the basal and parabasal cells of these esophageal epithelial disorders. RESULTS: The degrees of PCNA labeling index (PCNA LI) of the basal layers were lower than those of the parabasal layers in normal epithelium and dysplastic lesions, respectively, whereas no difference in CIS was recognized between the two layers. The proportion of PCNA LI in the basal layer of moderate dysplasia was lower than that in the basal layer of severe dysplasia. Conversely, there were no statistical differences between severe dysplasia and CIS in both basal and parabasal layers. In terms of lymphocytic infiltration beneath the dysplasia, there was a close relationship between the degree of lymphocytic infiltration and the PCNA index of the basal and parabasal layers. CONCLUSIONS: PCNA expression may be a useful marker to reflect the biologic characteristics of esophageal dysplasia, suggesting that a high grade of dysplasia is as serious a lesion as esophageal carcinoma.

Biomarkers↗

PubMed1992

Epithelial proliferation in Barrett's esophagus by proliferating cell nuclear antigen immunolocalization.

Proliferating cell nuclear antigen (PCNA) is an auxiliary protein to DNA polymerase delta and is an absolute requirement for cellular proliferation. Specialized-type Barrett's columnar-lined esophagus (CLE) is associated with adenocarcinomatous change. In the present study, the cellular proliferation of three histological types of CLE was assessed by semiquantitative evaluation of PCNA immunolocalization in 93 biopsy specimens from 45 patients using the murine monoclonal PC10. Statistical comparison was performed by the Mann-Whitney U test. Luminal surface cell labeling was uncommon in all histological types other than specialized CLE where 25 of the 43 biopsy specimens had at least occasional luminal surface cell labeling. Mean crypt labeling score of 4.06 for specialized type exceeded that for junctional (mean, 3.12; P < 0.001) and fundic types (mean, 1.6; P < 0.001). Gland cell PCNA staining scores for specialized-type CLE (mean, 3.18) exceeded that of junctional (mean, 1.97; P < 0.001) and fundic (mean, 1.04; P < 0.001). Summated PCNA scores for specialized-type, mean of 8.29, exceeded junctional mean score of 5.45 (P < 0.001) and fundic mean score of 2.76 (P < 0.001). PCNA immunolocalization reveals a high proportion of cells in cycle in the specialized-type CLE and expansion of the proliferative compartment, which may explain the association of specialized-type CLE with malignancy.

Antibodies, Monoclonal↗

PubMed1995

Expression and physicochemical characterization of human proliferating cell nuclear antigen.

Human proliferating cell nuclear antigen (PCNA) was overexpressed in Escherichia coli as a soluble protein. Recombinant PCNA was purified to homogeneity by phosphocellulose, Q-Sepharose, Sephacryl S-200, and hydroxylapatite chromatography. Approximately 20 mg of PCNA was isolated from E. coli cells derived from 2 L of culture. Characterization of the recombinant protein showed that it was functionally active and that its properties were similar to those of purified human placental PCNA. Recombinant PCNA stimulated human DNA polymerase delta activity at least 25-fold with poly(dA)/oligo-(dT) as the template. Recombinant PCNA eluted with a M(r) = 102,000 and a Stokes radius of 37 Angstrum by high-performance gel-permeation chromatography. The sedimentation coefficient determined by glycerol gradient ultracentrifugation was 6.3 S. The molecular weight calculated from the Stokes radius and S value was 96,800. The behavior of PCNA was entirely consistent with its being a trimeric protein. Analytical ultracentrifugation and gel filtration revealed the existence of a dimeric species at low dilution. Cross-linking experiments revealed the presence of PCNA dimers which predominated, as well as a trimeric species. These studies provide biophysical evidence that PCNA is an oligomeric protein which behaves as a trimeric species at high protein concentrations but dissociates to a dimer at low protein concentrations.

Base Sequence↗

PubMed1999

Drosophila replication and repair proteins: proliferating cell nuclear antigen (PCNA).

Proliferating cell nuclear antigen (PCNA), a protein intimately involved in both replication and repair, has been identified in eukaryotes at all levels of evolution. Is primary sequence, Drosophila melanogaster PCNA is 73% identical to mammalian PCNA. Moreover, it is able to substitute for mammalian PCNA in at least one intricate cell-free replication assay. Mutations in the gene for Drosophila PCNA, including some that are temperature sensitive, have been reported. Procedures are described for the biochemical purification of wild-type PCNA from a population of 6- to 18-h-old Drosophila embryos. Procedures were also developed for purification of unmodified wild-type Drosophila PCNA after induction of expression in Escherichia coli. An NH(2)-terminally His-tagged but otherwise wild-type Drosophila PCNA, as well as mutant His-tagged PCNA, were also engineered and purified to apparent homogeneity. Finally, an in situ polyacrylamide gel technique allows DNA polymerase assays to be performed on portions of single adults as well as single Drosophila embryos. This assay should tremendously facilitate systematic genetic studies of metazoan replication and repair.

Animals↗

PubMed1995

Overexpression and rapid purification of biologically active yeast proliferating cell nuclear antigen.

Yeast proliferating cell nuclear antigen (yPCNA) is a cell-cycle-regulated protein that has been shown to be required for the efficient elongation of primed DNA templates by DNA polymerase delta in vitro. We have expressed yPCNA to a high level (> or = 30% of the total cellular protein) with and without a six-residue histidine tag at its amino-terminus. Both forms of the recombinant protein were found to be biologically active and no significant differences were observed between the two forms. In this report we describe an efficient method of extraction of DNA binding proteins, such as yPCNA, overexpressed in Escherichia coli. The presence of a (His) delta tag on the polypeptide permitted rapid and high-yield single-step purification of the protein (approximately 60 mg of purified yPCNA per liter of induced cell culture) by immobilized metal affinity chromatography using an imidazole gradient elution procedure. The purified yPCNA was used to characterize the biological activity and tertiary structure of the protein. Chemical crosslinking and size-exclusion FPLC studies indicated that both forms of the protein have a trimeric-oligomeric structure in solution. Taken together these results indicate that both forms of the recombinant yPCNA were similar to the endogenous protein in their biochemical properties. The strategies presented here are designed to maximize the yield of recombinant protein and should prove useful to the purification of other recombinant DNA binding proteins.

Base Sequence↗