Molecular cloning and characterization of proliferating cell nuclear antigen (PCNA) from Chinese shrimp Fenneropenaeus chinensis

The proliferating cell nuclear antigen gene was cloned from Fenneropenaeus chinensis (FcPCNA). The full-length cDNA sequence of FcPCNA encodes 260 amino acids showing high identity with PCNAs reported in other species. FcPCNA expressed especially high in proliferating tissues of shrimp such as haematopoietic tissue (HPT) and ovary. In order to understand the response of HPT to bacteria and virus challenge, mRNA level of FcPCNA in HPT was analyzed after shrimp were challenged by Vibrio anguillarum and white spot syndrome virus (WSSV). FcPCNA expression in HPT of shrimp was responsive to WSSV and Vibrio challenge, but different expression profiles were obtained after challenge by these two pathogens. The data provide additional information to understand the defense mechanisms of shrimp against virus and bacteria. (c) 2008 Elsevier Inc. All rights reserved.

Immunohistochemical Analysis of Proliferating Cell Nuclear Antigen (PCNA) in Dental Follicles of Impacted Third Molars

This study investigated the immunodetection of PCNA in epithelial components of dental follicles associated with impacted third molars without radiographical and morphological signs of pathosis. A total of 105 specimens of dental follicles associated with impacted third molars with incomplete rhizogenesis (between Nolla`s stage 6 and 9) were surgically removed from 56 patients. Epithelial cell proliferating was determined by using immunohistochemical labeling. Statistical analysis was performed using the Fisher exact test. Of the 105 dental follicles collected, 6 were PCNA-positive (approximate to 6%). The specimens with squamous metaplasia and epithelial hyperplasia had higher rates of positivity for PCNA, as well as those with proliferative remnants of odontogenic epithelium. In conclusion, this study shows that dental follicles at this stage of development have low proliferative potential, but suggests that squamous metaplasia, hyperplasia of the epithelial lining and presence of proliferative odontogenic epithelial rests in the connective tissue may be early signs of developing lesions of odontogenic origin.

MyoD, myogenin and proliferating cell nuclear antigen expression in growing Nile tilapia (Oreochromis niloticus L.)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparative analysis of the expression of proliferating cell nuclear antigen, p53, bax, and bcl-2 in oral lichen planus and oral squamous cell carcinoma

Several epidermiologic studies have shown the malignant transformation potential of oral lichen planus; however, this potential is subject of much controversy. To evaluate the expression of proteins related to the cell proliferation and apoptosis processes in oral lichen planus, we compared oral lichen planus with oral squamous cell carcinoma. Twenty-four cases of each lesion were submitted according to streptavidin-biotin technique to evaluate the immunohistochemical expression of proliferating cell nuclear antigen, p53, bax, and bcl-2 proteins. X 2 test showed no statistically significant differences between the expression of p53, bax, and bcl-2 in oral lichen planus and oral squamous cell carcinoma (P > .05). However, the expression of proliferating cell nuclear antigen was significantly lower in oral lichen planus than in oral squamous cell carcinoma (P < .05). No statistically significant differences between the expression of p53, bax, and bcl-2 in oral lichen planus and oral squamous cell carcinoma were observed, which may be an evidence of the potential of malignant transformation of oral lichen planus. (C) 2009 Elsevier B.V. All rights reserved.

Estimation of growth fraction in fine needle aspirates from non-Hodgkin's lymphoma using anti-proliferating cell nuclear antigen - Correlation with the Kiel classification

OBJECTIVE: This study was undertaken to assess whether fine needle aspirates from non-Hodgkins lymphoma (NHL) could be used for growth fraction analysis with proliferating cell nuclear antigen (PCNA) staining and if there was a relationship between the growth fraction and cytomorphologic classification according to the Kiel classification.STUDY DESIGN: the study group consisted of 40 patients with NHL diagnosed by fine needle aspiration (FNA) cytology. The cytologic classification of the lymphomas was made by two cytopathologists on May-Grunwald-Giemsa-stained slides using the Kiel classification. There were 27 cases of low and 13 of high grade lymphoma. The estimation of the growth fraction was made by PCNA immunoreactivity. The PCNA index was quantitated in smears by counting an average of 1,000 cells, and the count teas correlated with the cytomorphologic classification.RESULTS: There was It strong correlation between the PCNA index and lymphoma grading. High grade lymphomas exhibited a mean PCNA positivity of 74.0%, which was significantly higher (P <.001) than that of low grade lymphomas (17.6%).CONCLUSION: Our study showed that PCNA evalua tion is suitable for smears obtained by FNA on NHL, correlates with increasing grades of lymphoma according to the Kiel classification and may offer a method of monitoring treatment of lymphoma.

The proliferating cell nuclear antigen index in breast carcinomas does not correlate with mitotic index and estrogen receptor immunoreactivity

To investigate the expression of a marker of cell proliferation (PCNA/Cyclin) and its putative relationship with histological grading, mitotic index and estrogen receptor immunoreactivity, we studied twenty-seven cases of invasive breast carcinoma in formalin-fixed, paraffin-embedded tissue sections. The PCNA and estrogen receptor were detected by the PC10 and H222 monoclonal antibodies respectively, using an avidin-biotin-pernxidase method. The median value of PCNA index was 20.9% with a range from 1.4 to 84.2%. We did not find any significant relationship between PCNA index anti the histological grading, mitotic index and estrogen receptor immunoreactivity. We conclude that PCNA detected by the monoclonal antibody PC10 in formalin-fixed material looks at present unrealiable as a proliferation marker in breast carcinoma.

Proliferating cell nuclear antigen (PCNA) in non-Hodgkin's lymphomas: Correlation with working formulation and Kiel classification in formalin-fixed paraffin-embedded material

PCNA is a 36-KD proliferating cell nuclear antigen associated with the cell cycle. The immunocytochemical detection of PCNA represents a useful tool for the study of tumor proliferation activity. This study documents the detection of PCNA, using antibody PC 10 in formalin-fixed, paraffin-embedded tissue, and correlates the proliferative activity of the non-Hodgkin's lymphomas (NHL) with histological grading assessed by the International Working Formulation (WF) and Kiel classification. In 92 cases of NHLs we found a strong correlation between the PCNA index and lymphoma grading. Statistically significant differences were also found between the proliferative index (PI) in low and high grade lymphomas according to the Kiel classification (t = 9.519; p < 0.001) and between low, intermediate and high grade lymphomas according to the WF classification (F = 79.01; p < 0.001). In the Kiel classification the mean of low grade lymphomas was 39.5% and of high grade 75.7%. In the WF the average of low grade lymphomas was 29.7%, intermediate 53.1% and high 75.1%. Although the differences among the groups had been significant, we found variations inside each histological subgroup in both classifications. The intermediate lymphomas were the most heterogeneous group, with PI inside the same histologic subtypes coincident with low and high grade lymphomas. Since PCNA may be used as a marker of cell proliferation in clinical studies to estimate the biological aggressiveness of lymphomas, its determination in intermediate grade NHL could be very useful to evaluate individual cases in this group and determine prognosis and probably the appropriate therapy.

Proliferating cell nuclear antigen immunoreaction in adrenal-tumors

Aims mid background: We studied, retrospectively, 33 cases of adrenal tumors of children at the Pediatric Endocrinology Unit, Children's Institute, Sao Paulo State University Medical School, from 1975 to 1993. Ail patients had at least 2 years of follow-up with a few exceptions. Methods: Clinical follow-up data were correlated with histopathologic review, laboratory data and cell kinetic evaluation (based on detection of proliferating cell nuclear antigens). Results: With one exception, all the patients had presented signs of androgen production and had high levels of dehydro-epiandrosterone-sulfate. Tumor weight evaluation represented a good parameter of neoplasm evolution: of 19 cases weighing less than 250 g, 17 had no evidence of disease after surgery, and 2 had an unfavorable prognosis. Of 14 cases weighing more than 250 g, only 1 had no evidence of disease and 13 had an unfavorable evolution. Conclusions: Proliferating cell nuclear antigen (PCNA) was not helpful to evaluate adrenal neoplasm evolution: our study did not show any correlation between PCNA score and prognosis.

An interaction between DNA ligase I and proliferating cell nuclear antigen: Implications for Okazaki fragment synthesis and joining

Although three human genes encoding DNA ligases have been isolated, the molecular mechanisms by which these gene products specifically participate in different DNA transactions are not well understood. In this study, fractionation of a HeLa nuclear extract by DNA ligase I affinity chromatography resulted in the specific retention of a replication protein, proliferating cell nuclear antigen (PCNA), by the affinity resin. Subsequent experiments demonstrated that DNA ligase I and PCNA interact directly via the amino-terminal 118 aa of DNA ligase I, the same region of DNA ligase I that is required for localization of this enzyme at replication foci during S phase. PCNA, which forms a sliding clamp around duplex DNA, interacts with DNA pol δ and enables this enzyme to synthesize DNA processively. An interaction between DNA ligase I and PCNA that is topologically linked to DNA was detected. However, DNA ligase I inhibited PCNA-dependent DNA synthesis by DNA pol δ. These observations suggest that a ternary complex of DNA ligase I, PCNA and DNA pol δ does not form on a gapped DNA template. Consistent with this idea, the cell cycle inhibitor p21, which also interacts with PCNA and inhibits processive DNA synthesis by DNA pol δ, disrupts the DNA ligase I–PCNA complex. Thus, we propose that after Okazaki fragment DNA synthesis is completed by a PCNA–DNA pol δ complex, DNA pol δ is released, allowing DNA ligase I to bind to PCNA at the nick between adjacent Okazaki fragments and catalyze phosphodiester bond formation.

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 δ (or ɛ)-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 δ-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 δ-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 δ-catalyzed elongation of a singly primed DNA template. However, this mutation did not significantly alter the ability of PCNA to stimulate DNA polymerase δ 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.

p21Cip1/Waf1 disrupts the recruitment of human Fen1 by proliferating-cell nuclear antigen into the DNA replication complex.

Fen1 or maturation factor 1 is a 5'-3' exonuclease essential for the degradation of the RNA primer-DNA junctions at the 5' ends of immature Okazaki fragments prior to their ligation into a continuous DNA strand. The gene is also necessary for repair of damaged DNA in yeast. We report that human proliferating-cell nuclear antigen (PCNA) associates with human Fen1 with a Kd of 60 nM and an apparent stoichiometry of three Fen1 molecules per PCNA trimer. The Fen1-PCNA association is seen in cell extracts without overexpression of either partner and is mediated by a basic region at the C terminus of Fen1. Therefore, the polymerase delta-PCNA-Fen1 complex has all the activities associated with prokaryotic DNA polymerases involved in replication: 5'-3' polymerase, 3'-5' exonuclease, and 5'-3' exonuclease. Although p21, a regulatory protein induced by p53 in response to DNA damage, interacts with PCNA with a comparable Kd (10 nM) and a stoichiometry of three molecules of p21 per PCNA trimer, a p21-PCNA-Fen1 complex is not formed. This mutually exclusive interaction suggests that the conformation of a PCNA trimer switches such that it can either bind p21 or Fen1. Furthermore, overexpression of p21 can disrupt Fen1-PCNA interaction in vivo. Therefore, besides interfering with the processivity of polymerase delta-PCNA, p21 also uncouples Fen1 from the PCNA scaffold.

Requirement of proliferating cell nuclear antigen in RAD6-dependent postreplicational DNA repair.

The proliferating cell nuclear antigen (PCNA) acts as a processivity factor for replicative DNA polymerases and is essential for DNA replication. In vitro studies have suggested a role for PCNA-in the repair synthesis step of nucleotide excision repair, and PCNA interacts with the cyclin-dependent kinase inhibitor p21. However, because of the lack of genetic evidence, it is not clear which of the DNA repair processes are in fact affected by PCNA in vivo. Here, we describe a PCNA mutation, pol30-46, that confers ultraviolet (UV) sensitivity but has no effect on growth or cell cycle progression, and the mutant pcna interacts normally with DNA polymerase delta and epsilon. Genetic studies indicate that the pol30-46 mutation is specifically defective in RAD6-dependent postreplicational repair of UV damaged DNA, and this mutation impairs the error-free mode of bypass repair. These results implicate a role for PCNA as an intermediary between DNA replication and postreplicational DNA repair.

Transcriptional activation of the human proliferating-cell nuclear antigen promoter by p53.

Proliferating-cell nuclear antigen (PCNA) is a DNA damage-inducible protein that performs an essential function in DNA replication and repair as an auxiliary factor for DNA polymerases delta and epsilon. Examination of the human PCNA promoter DNA sequence revealed a site with homology to the consensus DNA sequence bound by p53. PCNA promoter fragments with this site intact bound p53 in vitro and were transcriptionally activated by wild-type p53 in transient expression assays in SAOS-2 cells. The resident p53-binding site could be functionally substituted by a previously described p53-binding site from the ribosomal gene cluster. A plasmid expressing a mutated version of p53 derived from a patient with Li-Fraumeni syndrome failed to activate the PCNA promoter in the cotransfection assay. In different cell types, activation of the PCNA promoter by the p53-binding sequence correlated with the status of p53. Activation of the PCNA promoter by wild-type p53 depends upon the level of p53 expression. This concentration dependence and cell type specificity reconciles the observations presented here with prior results indicating that wild-type p53 represses the PCNA promoter. These findings provide a mechanism whereby p53 modulates activation of PCNA expression as a cellular response to DNA damage.

Redefining the Epstein-Barr virus-encoded nuclear antigen EBNA-1 gene promoter and transcription initiation site in group I Burkitt lymphoma cell lines.

The Epstein-Barr virus-encoded nuclear antigen EBNA-1 gene promoter for the restricted Epstein-Barr virus (EBV) latency program operating in group I Burkitt lymphoma (BL) cell lines was previously identified incorrectly. Here we present evidence from RACE (rapid amplification of cDNA ends) cloning, reverse transcription-PCR, and S1 nuclease analyses, which demonstrates that the EBNA-1 gene promoter in group I BL cell lines is located in the viral BamHI Q fragment, immediately upstream of two low-affinity EBNA-1 binding sites. Transcripts initiated from this promoter, referred to as Qp, have the previously reported Q/U/K exon splicing pattern. Qp is active in group I BL cell lines but not in group III BL cell lines or in EBV immortalized B-lymphoblastoid cell lines. In addition, transient transfection of Qp-driven reporter constructs into both an EBV-negative BL cell line and a group I BL cell line gave rise to correctly initiated transcripts. Inspection of Qp revealed that it is a TATA-less promoter whose architecture is similar to the promoters of housekeeping genes, suggesting that Qp may be a default promoter which ensures EBNA-1 expression in cells that cannot run the full viral latency program. Elucidation of the genetic mechanism responsible for the EBNA-1-restricted program of EBV latency is an essential step in understanding control of viral latency in EBV-associated tumors.

Endogenous Presentation of CD8+ T Cell Epitopes from Epstein-Barr Virus–encoded Nuclear Antigen 1

Epstein-Barr virus (EBV)-encoded nuclear antigen (EBNA)1 is thought to escape cytotoxic T lymphocyte (CTL) recognition through either self-inhibition of synthesis or by blockade of proteasomal degradation by the glycine-alanine repeat (GAr) domain. Here we show that EBNA1 has a remarkably varied cell type-dependent stability. However, these different degradation rates do not correspond to the level of major histocompatibility complex class I-restricted presentation of EBNA1 epitopes. In spite of the highly stable expression of EBNA1 in B cells, CTL epitopes derived from this protein are efficiently processed and presented to CD8(+) T cells. Furthermore, we show that EBV-infected B cells can readily activate EBNA1-specific memory T cell responses from healthy virus carriers. Functional assays revealed that processing of these EBNA1 epitopes is proteasome and transporter associated with antigen processing dependent. We also show that the endogenous presentation of these epitopes is dependent on the newly synthesized protein rather than the long-lived stable EBNA1. Based on these observations, we propose that defective ribosomal products, not the full-length antigen, are the primary source of endogenously processed CD8(+) T cell epitopes front EBNA1.