SEARCH · PubMed Health

Results for “PCNA”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 55 records · Page 3Linked to original sources

PubMed2001

Chromatin-bound PCNA complex formation triggered by DNA damage occurs independent of the ATM gene product in human cells.

Proliferating cell nuclear antigen (PCNA), a processivity factor for DNA polymerases delta and epsilon, is involved in DNA replication as well as in diverse DNA repair pathways. In quiescent cells, UV light-induced bulky DNA damage triggers the transition of PCNA from a soluble to an insoluble chromatin-bound form, which is intimately associated with the repair synthesis by polymerases delta and epsilon. In this study, we investigated the efficiency of PCNA complex formation in response to ionizing radiation-induced DNA strand breaks in normal and radiation-sensitive Ataxia telangiectasia (AT) cells by immunofluorescence and western blot techniques. Exposure of normal cells to gamma-rays rapidly triggered the formation of PCNA foci in a dose-dependent manner in the nuclei and the PCNA foci (40-45%) co-localized with sites of repair synthesis detected by bromodeoxyuridine labeling. The chromatin-bound PCNA gradually declined with increasing post-irradiation times and almost reached the level of unirradiated cells by 6 h. The PCNA foci formed after gamma-irradiation was resistant to high salt extraction and the chromatin association of PCNA was lost after DNase I digestion. Interestingly, two radiosensitive primary fibroblast cell lines, derived from AT patients harboring homozygous mutations in the ATM gene, displayed an efficient PCNA redistribution after gamma-irradiation. We also analyzed the PCNA complex induced by a radiomimetic agent, Bleomycin (BLM), which produces predominantly single- and double-strand DNA breaks. The efficiency and the time course of PCNA complex induced by BLM were identical in both normal and AT cells. Our study demonstrates for the first time that the ATM gene product is not required for PCNA complex assembly in response to DNA strand breaks. Additionally, we observed an increased interaction of PCNA with the Ku70 and Ku80 heterodimer after DNA damage, suggestive of a role for PCNA in the non-homologous end-joining repair pathway of DNA strand breaks.

Antigens, Nuclear↗

PubMed1999

The expression of proliferating cell nuclear antigen (PCNA) and p 53 protein correlate with prognosis of patients with oral squamous cell carcinoma.

Few Data are available regarding expression of proliferating cell nuclear antigen (PCNA) versus overexpression of p 53 protein and prognosis for oral squamous cell carcinoma (O-SCC). This study evaluated the relationship between expression of PCNA and p 53 protein and certain clinical and pathologic parameters to prognosis in O-SCC. Scattered PCNA immunoreactive cells were found in 50 of 51 (98.0%) tumors. The mean PCNA-labelling index (PCNA-LI) of O-SCC was 34.7 +/- 12.0% (n = 50). The PCNA-LI was significantly high in O-SCC when compared to normal squamous epithelium (3.9 +/- 2.1%, n = 10). Altogether, 28 of 51 (54.9%) tumors showed nuclear immunoreactive for the p 53 protein. p 53-positive nuclei were clearly stained as granular or reticular in O-SCC. Microscopically, it appeared that the staining patterns of PCNA- and p 53-positive cells were quite similar. The mean value of PCNA-LI for the p 53-positive cases was 37.9 +/- 11.9% (n = 28), which was significantly higher than the 30.8 +/- 11.1% of the 23 negative cases. The survival rates at 3-year and 5-year for patients with p 53-negative O-SCC were 78.3% and 73.4%, respectively, while those for patients with p 53-positive O-SCC were 51.9% and 43.2%, respectively. No significant correlation could be found between PCNA-LI and T classification, N classification and Stage classification. The expression of p 53 was detected in 48.5% (16/33) of cases without regional lymph node metastasis, and 66.7% (12/18) with regional lymph node metastasis. There was a significant difference between PCNA-LI and p 53-positive cases, and prognosis. In PCNA-LI, the Grade 4 by mode of invasion was significantly higher than Grade 1, 2 and 3. The PCNA-LI and p 53-positive cases were significantly correlated with histological grading of malignancy. The present study shows a relationship between PCNA and p 53 protein. It also suggests that both PCNA and p 53 positive rates are the malignant potential of the O-SCC.

Adult↗

PubMed1997

The value of proliferating cell nuclear antigen (PCNA)/cyclin in the assessment of cell proliferation in glomerulonephritis.

Proliferating cell nuclear antigen (PCNA)/cyclin is an acidic nuclear protein increasing from the late G1 to S phases of the cell cycle and whose detection parallels other standard methods for assessing cell proliferation. The aim of this study was to investigate PCNA expression in normal and diseased human kidneys, in order to clarify cell proliferation in renal tissue and to define a possible correlation of its expression with various types of glomerulonephritis (GN). The immunohistochemical avidin-biotin complex (ABC) method was used for the demonstration of PCNA applying the monoclonal antibody PC-10 to paraffin sections from: 10 normal kidneys, 55 renal biopsies with various types of proliferative GN (PGN), 44 renal biopsies with various types of non proliferative GN (NPGN). In PCNA-positive renal biopsies with GN the antigen showed a heterogenous nuclear expression in occasional or few mesangial and glomerular epithelial cells as well as in a greater number of tubular epithelial cells. PCNA was expressed in 20% of normal kidneys and in 38% of renal biopsies with GN. The frequency of PCNA expression was significantly increased in the cases of PGN (47%) compared to that observed in the cases of NPGN (27%) (p = 0.03). PCNA was detected in 10/24 cases of IgA nephropathy, in 3/4 cases of IgM nephropathy, in 5/14 of other types of primary PGN and in 8/13 of secondary PGN. PCNA expression was not correlated with the degree of mesangial cellularity in PGN. Moreover, there was no significant difference in PCNA expression between primary and secondary PGN. PCNA demonstrated an intense expression in the majority of epithelial cells forming cellular crescents in 8/11 cases of PGN. In conclusion, PCNA was observed more frequently in diseased than in normal kidneys. The significant increase in the frequency of PCNA intraglomerular expression in PGN suggests that PCNA has a certain value in the assessment of mesangial proliferation. Moreover, the increased PCNA expression in tubular epithelial cells especially in PGN, indicates their proliferative state and may be correlated with their proposed activation and role in the progression of renal injury.

Biopsy↗

PubMed2005

Prognostic value of proliferating cell nuclear antigen (PCNA) in infiltrating ductal carcinoma breast.

OBJECTIVE: To assess the independent and interdependent prognostic value of proliferating cell nuclear antigen (PCNA) in carcinoma of breast in our female population and its association with pathologic variables and disease outcome. DESIGN: A descriptive study. PLACE AND DURATION OF STUDY: Section of Histopathology, Department of Pathology and Microbiology, The Aga Khan University, Karachi from January 1992 to December 1997. PATIENTS AND METHODS: All cases diagnosed with invasive ductal carcinomas (IDC) of breast with lymph nodes sampling were included. The expression of PCNA was analyzed on tumor specimens of IDC breast. These patients also had axillary lymph node sampling. The expression of PCNA protein was analyzed immunohistochemically by PAP technique. Patients were followed for a median duration of 48 months. RESULTS: The percentage of PCNA positive tumor cells was estimated semi-quantitatively. Positivity was seen in every case, mean PCNA positivity was 27% (range 10-80) with a median of 28%. The <25% positivity was seen in 149 (47%) cases, and >25% positivity seen in 166 (53%) cases. According to the pathological grading lowest mean PCNA was seen in grade-I i.e., 26% tumor cells showed nuclear reactivity to PCNA followed by grade-II 30% and grade-III 33%. PCNA categorical expression was significantly correlated with histological differentiation, (p<0.05) and tumor size (p<0.01). Distant metastases were seen in>25% positive cases (p<0.05). PCNA expression when correlated with overall survival, showed significant correlation between categorical PCNA (p<0.05). At a median follow-up of 48 months, 66% of <25 PCNA positive patients died with an overall survival of 3.16 years and disease-free survival of 2.5 years, among >25% PCNA positive patients 77% died with an overall survival of 2.7 years and a disease-free survival of 2.2 years. CONCLUSION: In this study PCNA proved to be an independent prognostic indicator in predicting disease-free and overall survival in breast carcinoma patients.

Biomarkers, Tumor↗

PubMed1993

Comparison of three quantitation methods for PCNA immunostaining: applicability and relation to survival in 83 astrocytic neoplasms.

Recent studies on astrocytic tumours demonstrated a close association between patient prognosis and neoplastic proliferation estimated by such methods as Ki-67 and bromodeoxyuridine labelling. Novel monoclonal PCNA antibodies and special antigen-retrieval techniques have the advantage of working on routinely fixed and embedded specimens and thus make the estimation of proliferation simpler. In addition to PCNA-positive cell count expressed in percentages (PCNA-LI), we estimated the number of PCNA-immunopositive cells count expressed in percentages (PCNA-LI), we estimated the number of PCNA-immunopositive cells of 83 astrocytomas in two ways: (1) per mm2 of neoplastic tissue (uncorrected PCNA index); and (2) per mm2 of total neoplastic nuclear area (corrected PCNA index). Both of these methods were reproducible and showed a good correlation with PCNA-LI and malignancy grade (I-IV). With quantitation methods 1 and 2, the proliferative status of about 2000 cells could be estimated in about 7-10 min, whereas the PCNA count by PCNA-LI of 200 cells took approximately the same time. The proliferation indices obtained by all three quantitation methods were highly significantly related to patient prognosis. The corrected PCNA index, having a close association with the neoplastic cellularity, even divided the glioblastoma group (grade IV) into two significantly different prognostic groups in which 56 and 17 per cent of the patients were alive after 1-year follow-up. The combination of PCNA immunohistochemistry and morphometry seems to give important prognostic information about astrocytomas independent of the histopathological grade.

Astrocytoma↗

PubMed1995

Proliferating cell nuclear antigen (PCNA) detection of cellular proliferation in hypothyroid and hyperthyroid rat hearts.

Immunocytochemical techniques examining the expression of cell proliferation-related markers such as proliferating cell nuclear antigen (PCNA), may be employed to provide visual and quantitative evidence of cell proliferation. The efficacy of this method in frozen samples was tested on sections of 2-day-old rat heart. Mouse monoclonal anti-PCNA and goat anti-mouse IgG2a peroxidase-conjugated antibodies were applied to tissue cross-sections. PCNA positive (PCNA+) cells were evident in these sections as intensely stained, dark brown nuclei. This method was then applied to the hearts of four groups of adult rats (1) 1 month hyperthyroid, (2) 1 month hypothyroid, (3) 6 days hyperthyroid following 1 month hypothyroidism, or (4) euthyroid, in order to assess the ability of identifying and quantifying PCNA+cell types (myocyte, endothelial, or remaining, "other" cells). Serial cross-sections were prepared, half of the sections processed using the PCNA technique, while corresponding sections were stained with hematoxylin and eosin. Identification of PCNA+cell types was made by comparison of PCNA with H&E sections. PCNA labeling indices for a given cell type were expressed as the proportion of PCNA+nuclei per 1000 nuclei. No PCNA+myocyte nuclei were observed in any of the adult hearts. Only hypothyroid/hyperthyroid demonstrated significantly higher PCNA labeling of endothelial and "other" nuclei compared to euthyroid. This finding agrees with previous morphometric data which suggested capillary growth in these hearts. Results demonstrate that quantitation of PCNA expression on frozen heart tissue is possible, and can be used to provide direct, discernible evidence for cell proliferation.

Animals↗

PubMed1998

The prognostic value of proliferating cell nuclear antigen (PCNA) in the advanced cancer of larynx.

It was retrospectively examined whether the tumor growth fraction determined by PCNA score in the advanced cancer of larynx could be used as a prognostic factor. There was used immunohistochemical method to evaluate the PCNA score of positive cells in paraffin embedded tissues of laryngeal carcinoma obtained from 90 patients. They were treated by surgery in the Department of Otolaryngology at the Medical University in Cracow, Poland in 1987 and 1988. The follow-up period was not shorter than 5 years. In the examined material there were 59 patients with tumor T3 and 31 with T4. Neck dissection on one or on both sides was performed on patients. The metastases in regional lymph nodes was found in 26 patients. Histologic grading (G1 = 18, G2 = 52 and G3 = 20 patients) and number of mitosis was assessed. The PCNA score was defined by counting positive cells and presented as percentage of a cells. It ranged from 2.1 to 73.0% with the average value of 34.5%. The PCNA score below this level was defined as a low PCNA score (49 patients) and above this level as a high PCNA score (41 patients). There was a correlation between PCNA score and survival of the patients. 35 patients (71.4%) with low PCNA score survived and 15 (36.6%) with high PCNA score (P < 0.05). Similarly the high PCNA score influenced the number of metastases in lymph nodes. They occurred in low and high PCNA score respectively at nine (18.4%) and 17 patients (41.5%) (P < 0.05). There was not found any correlation between PCNA score and tumor differentiation, its size and mitosis number. The PCNA may be a prognostic factor for the patients with advanced cancer of larynx.

Adult↗

PubMed2000

A quantitative study of the in vitro binding of the C-terminal domain of p21 to PCNA: affinity, stoichiometry, and thermodynamics.

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication, repair, and control of cell proliferation, and its activity can be modulated by interaction with p21(Waf1/Cip1) [Cox, L. S., (1997) Trends Cell Biol. 7, 493-497]. This protein-protein interaction provides a particularly good model target for designing therapeutic agents to treat proliferative disorders such as cancer. In this study, the formation of complexes between PCNA and peptides derived from the C-terminus of p21 has been investigated at the molecular level and quantified using a competitive PCNA binding assay and isothermal titration calorimetry (ITC). The affinity constant for the interaction between p21 (141-160) peptide and PCNA has been determined to be 1.14 x 10(7) M(-)(1), corresponding to a K(d) of 87.7 nM. Measurement of the interaction of truncation and substitution analogues based on the p21 (141-160) sequence with PCNA revealed that the N-terminal part (residues 141-152) of the above peptide is the minimum recognition motif, required for PCNA binding. Truncation of the C-terminal region p21 (153-160), though, inhibited significantly the ability of the peptides to compete with the full-length p21 (141-160) for binding to PCNA. Alanine mutation of Met 147 or Asp 149 completely abolished or significantly decreased, respectively, the level of the PCNA binding and the inhibition of SV40 DNA replication. Comparison of the data obtained by the competitive PCNA binding assay and the ITC measurements demonstrated the usefulness of this assay for screening for compounds that could modulate the PCNA-p21 interaction. Using this assay, we have screened rationally designed peptides for binding to PCNA and interruption of the PCNA-p21 (141-160) complex. As a result of this screening, we have identified a 16-residue peptide (consensus motif 1 peptide) with the following sequence: SAVLQKKITDYFHPKK. Consensus motif 1 peptide and p21 (141-160) have similar affinities for binding PCNA and abilities to inhibit in vitro replication of DNA originated from SV40. Such peptides could prove useful in assessing p21-mimetic strategies for cancer treatment.

Amino Acid Sequence↗

PubMed1994

Nuclear distribution of proliferating cell nuclear antigen (PCNA) in fertilized eggs of the starfish Asterina pectinifera.

Previous studies (Nomura et al. (1991) Dev. Biol. 143, 289-296 (1993) Dev. Biol. 159, 288-297) determined the time of DNA replication period (S phase) in starfish eggs fertilized either during or after oocyte maturation. Here proliferating cell nuclear antigen (PCNA) localized within nuclei of starfish eggs was detected with an anti-PCNA human antiserum. Using a confocal laser scanning microscope, a three-dimensional structure of the PCNA region was analyzed. In eggs fertilized during maturation, PCNA started to localize within the nuclei at the same time as the initiation of the first S phase. During the S phase, the distribution of localized PCNA in a three-dimensional view coincided with the chromatin distribution. After the S phase, PCNA remained localized within the nuclei, but its distribution no longer coincided with the chromatin distribution. In eggs fertilized after maturation, however, PCNA started to localize within the female pronuclei about 10 minutes ahead of the first S phase. Localized PCNA occupied only a limited region of the nuclei without diffusing over the whole nuclear area. Chromatin distributed around the peripheral region of the nuclei mostly outside the PCNA region. When the first S phase was initiated, the chromatin distribution became coincident with the PCNA region. Later behavior of PCNA was the same as that of the eggs fertilized during maturation. The precocious localization of PCNA in those eggs fertilized after maturation simply demonstrates that the 'postactivation process' for preparing DNA replication is triggered by fertilization and PCNA localization and S phase are sequentially initiated with a time-lapse. On the other hand, the simultaneous occurrence of them seen in those eggs fertilized during maturation indicates that the postactivation process must be going on in parallel with the maturation process.

Adenine↗

PubMed1992

[Measurement of PCNA labeling index in astrocytic tumors].

PCNA (proliferating cell nuclear antigen) is said to be present specifically in the nucleus of proliferating cells. The PCNA labeling index (PCNA LI) of astrocytic tumors was measured and compared with histological types or prognosis. The specimens from 44 patients were fixed in a 10% formalin solution, and embedded in paraffin. The 3 microns-sections were stained immunohistochemically with anti-PCNA monoclonal antibody (PCIO, Novocastra) using an ABC method. The percentage of PCNA-positive-cells was determined by counting 2000 cells, and identified as PCNA LI. All of the PCNA-positive-cells showed diffuse nucleoplasmic staining. The averages of PCNA LIs in each pathological type were calculated and evaluated statistically. Although differences in averages of PCNA LIs among pilocytic, gemistocytic, fibrillary astrocytoma were not significant, there was a significant difference between anaplastic astrocytoma and glioblastoma. The relationship between PCNA LIs and the prognoses for 43 patients was studied. Forty-three patients were classified into 3 groups (over 22%, 7 to less than 22%, and less than 7%) according to PCNA LIs. The survival data in the 3 groups were analyzed, and differed significantly in the survival rates. Furthermore, twenty-three patients of anaplastic astrocytoma and glioblastoma were classified into two groups (over 22% and less than 22%). Likewise, the two groups differed significantly. In summary, pathological type and prognosis were closely related to PCNA LI in astrocytic tumors. Therefore, we thought measurement of PCNA LI would make it more possible to analyze clinically the proliferating activity of astrocytic tumors, and to care for patients more effectively.

Adolescent↗

PubMed1995

Characterisation of the interaction between PCNA and Gadd45.

We have examined the interaction between the DNA replication and repair protein PCNA, and the growth arrest and DNA damage induced protein Gadd45. An anti-Gadd45 polyclonal antibody co-immunoprecipitates PCNA but in reciprocal experiments, an anti-C terminal anti-PCNA antibody failed to co-immunoprecipitate Gadd45. We used a yeast two hybrid assay to demonstrate that human Gadd45 interacts with both human and S. pombe PCNA. We have determined that the N-terminal 94 amino acids of Gadd45 bind to PCNA, and using a series of N-terminal and C-terminal deletions of human PCNA we have mapped two potential Gadd45 binding sites. Deletion of the last 6 amino acids of PCNA ablated interaction, suggesting a role in Gadd45 binding. This explains the inability of an anti-C terminal PCNA antibody to co-immunoprecipitate Gadd45. Using a peptide ELISA approach, we showed that Gadd45 protein binds strongly to three regions of PCNA (residues 1-20, 61-80, and 196-215) and weakly to residues 121-170. The crystal structure of PCNA provides insight into our genetic and immunochemical data. Our results confirm an interaction between PCNA and Gadd45, define regions of both molecules involved in this interaction, and are consistent with a potential stoichiometry of 2 Gadd45 molecules to each PCNA monomer. These data provide support for the notion that PCNA-Gadd45 interactions co-ordinate cell cycle and DNA repair.

Amino Acid Sequence↗

PubMed1993

Flow cytometric analysis of the expression of PCNA during the cell cycle in HeLa cells and effects of the inhibition of DNA synthesis on it.

Flow cytometric bivariate DNA/PCNA analysis was performed to investigate the expression of PCNA during the cell cycle and the implication in DNA replication in HeLa cells, using a monoclonal antibody (PC10) to PCNA. The expression of PCNA was evident in almost all cells growing exponentially, when cells were fixed in methanol. The total amount of PCNA altered a little during the cell cycle. However, the treatment with Triton X-100 extracted 80-89% of total PCNA from the cells, resulting in the dramatic change of bivariate DNA/PCNA distribution pattern. PCNA was completely removed from nuclei in both G1 and G2 phases by the detergent treatment, whereas a certain amount of PCNA remained in S phase nuclei. After the treatment of cells with Triton X-100, PCNA was detected exclusively in S phase cells. The bivariate DNA/PCNA distribution pattern in cells treated with Triton X-100 was strikingly so similar to the DNA/BrdUrd distribution pattern that it was unable to differentiate one from the other. It is concluded that the detergent treatment of cells allows the rapid analysis of the cell cycle. The inhibition of DNA synthesis with 10 mM hydroxyurea elevated cellular PCNA content mainly due to the increase in the fraction of the detergent extractable PCNA. It was apparent, however, that in cells incubated with Triton X-100, the pattern of the bivariate DNA/PCNA distribution was not basically different from that in cells without HU treatment. The level of PCNA bound to nuclear structures (PCNA not extracted with detergent) increased in cells arrested at the G1/S boundary with the time of hydroxyurea treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗

PubMed2003

In human hepatocellular carcinoma in cirrhosis proliferating cell nuclear antigen (PCNA) is involved in cell proliferation and cooperates with P21 in DNA repair.

BACKGROUND: Proliferating cell nuclear antigen (PCNA) is a nuclear protein involved in DNA-synthesis and repair. During DNA-synthesis and repair the only active PCNA fraction is tightly bound to DNA. Similarly, during DNA-repair, a fraction of p21 colocalizes with PCNA in a detergent-insoluble form. AIM: The aim of the study was to analyze to what extent the presence of DNA-bound PCNA and p21 correlates with cell proliferation and DNA-repair in hepatocellular carcinoma (HCC). METHODS: Twenty-six HCCs and surrounding cirrhosis were studied. The DNA-bound and detergent-soluble fractions of PCNA and p21 were analyzed by immunoblotting. P53 and Ki67-Labeling Index (Ki67-LI) were evaluated by immunocytochemistry. RESULTS: Soluble fractions of PCNA and p21 were found in all samples. One out of 26 cirrhotic samples displayed a DNA-bound fraction of PCNA while no case expressed DNA-bound p21. Fourteen HCCs showed a DNA-bound PCNA fraction. A highly significant correlation was found between Ki67-LI and DNA-bound PCNA but not with detergent-soluble PCNA. DNA-bound p21 and PCNA, indicating ongoing DNA repair activity, were present in 6 of these 14 HCCs and correlated with a high histological grade and high Ki67-LI. CONCLUSIONS: Our results suggest that in HCC PCNA participates both in DNA synthesis and repair and that highly proliferating HCCs may display a sustained DNA-repair.

Adult↗

PubMed1997

Homologous regions of Fen1 and p21Cip1 compete for binding to the same site on PCNA: a potential mechanism to co-ordinate DNA replication and repair.

Following genomic damage, the cessation of DNA replication is co-ordinated with onset of DNA repair; this co-ordination is essential to avoid mutation and genomic instability. To investigate these phenomena, we have analysed proteins that interact with PCNA, which is required for both DNA replication and repair. One such protein is p21Cip1, which inhibits DNA replication through its interaction with PCNA, while allowing repair to continue. We have identified an interaction between PCNA and the structure specific nuclease, Fen1, which is involved in DNA replication. Deletion analysis suggests that p21Cip1 and Fen1 bind to the same region of PCNA. Within Fen1 and its homologues a small region (10 amino acids) is sufficient for PCNA binding, which contains an 8 amino acid conserved PCNA-binding motif. This motif shares critical residues with the PCNA-binding region of p21Cip1. A PCNA binding peptide from p21Cip1 competes with Fen1 peptides for binding to PCNA, disrupts the Fen1-PCNA complex in replicating cell extracts, and concomitantly inhibits DNA synthesis. Competition between homologous regions of Fen1 and p21Cip1 for binding to the same site on PCNA may provide a mechanism to co-ordinate the functions of PCNA in DNA replication and repair.

Amino Acid Sequence↗

PubMed2005

Nuclear dynamics of PCNA in DNA replication and repair.

The DNA polymerase processivity factor proliferating cell nuclear antigen (PCNA) is central to both DNA replication and repair. The ring-shaped homotrimeric PCNA encircles and slides along double-stranded DNA, acting as a "sliding clamp" that localizes proteins to DNA. We determined the behavior of green fluorescent protein-tagged human PCNA (GFP-hPCNA) in living cells to analyze its different engagements in DNA replication and repair. Photobleaching and tracking of replication foci revealed a dynamic equilibrium between two kinetic pools of PCNA, i.e., bound to replication foci and as a free mobile fraction. To simultaneously monitor PCNA action in DNA replication and repair, we locally inflicted UV-induced DNA damage. A surprisingly longer residence time of PCNA at damaged areas than at replication foci was observed. Using DNA repair mutants, we showed that the initial recruitment of PCNA to damaged sites was dependent on nucleotide excision repair. Local accumulation of PCNA at damaged regions was observed during all cell cycle stages but temporarily disappeared during early S phase. The reappearance of PCNA accumulation in discrete foci at later stages of S phase likely reflects engagements of PCNA in distinct genome maintenance processes dealing with stalled replication forks, such as translesion synthesis (TLS). Using a ubiquitination mutant of GFP-hPCNA that is unable to participate in TLS, we noticed a significantly shorter residence time in damaged areas. Our results show that changes in the position of PCNA result from de novo assembly of freely mobile replication factors in the nucleoplasmic pool and indicate different binding affinities for PCNA in DNA replication and repair.

Animals↗

PubMed1993

[Anti-PCNA antibody].

Antibodies to the proliferating cell nuclear antigen (PCNA) detected in patients with systemic lupus erythematosus (SLE) were allowed to react with a nuclear antigen expressed predominantly in proliferating cells such as cultured cells and mitogen-transformed cells. The characterization of both structure and function of PCNA has been studied. PCNA has been identified as a protein with a molecular weight of about 33 kD and isoelectric point of 4.8. The expression of PCNA increased in the cell from the late G1 and S phases of the cell cycle immediately preceding DNA synthesis. Recent studies have revealed that the auxiliary protein of DNA polymerase-d is identical to PCNA. Anti-PCNA antibodies can be detected by the methods of immunofluorescence (IF), double immunodiffusion (DID), counterimmunoelectrophoresis (CIE), enzyme-linked immunosorbent assay (ELISA) and immunoblotting (IB). Anti-PCNA antibodies were specifically detected in 2-5% of the patients with SLE by DID. In spite of their low frequency, anti-PCNA antibodies are useful as a clinical marker for SLE. Several reports indicated that patients with PCNA positive SLE showed a high frequency of renal and central nerve system (CNS) involvements and thrombocytopenia. In these patients, the anti-PCNA antibody titer was elevated before the development by proteinuria and the titer of anti-PCNA antibodies was decreased by treatment with corticosteroids. In addition, anti-PCNA antibodies have been known as a useful tool to detect activated lymphocytes and malignant proliferating cell.

Autoantibodies↗

PubMed1993

Proliferating cell nuclear antigen (PCNA), immunostaining and flow cytometric DNA analysis of renal cell carcinoma.

Nuclear PCNA immunoreactivity is found in the proliferating compartment of normal and tumor cells. The purpose of the present study was to examine immunohistochemical staining of PCNA in renal cell carcinoma and its relationship to tumor grading, flow cytometric DNA analysis and prognosis. The resected kidneys from 81 patients with renal cell carcinoma were used. Anti-PCNA monoclonal antibody was applied to paraffin sections to identify proliferating cells by the SAB technique. The proportion of PCNA-positive cells to negative cells was determined by counting the tumor cells stained by PCNA at 400x magnification and expressed as percentage of positive cells. In parallel with the PCNA immunostaining DNA flow cytometry was performed, using the cell suspension prepared from paraffin sections and stained with propidium iodide. 79 tumors were evaluable for PCNA immunohistochemistry and 54 tumors for DNA flow cytometry. Immunohistochemically, there was no significant difference in percentage of PCNA-positive cells between tumor grades G 1 and G 2 (1.4 +/- 1.3% in G 1 and 1.6 +/- 1.4% in G 2), while G 3 tumors contained high percentage of PCNA positive cells, the mean value being 7.6 +/- 5.3%. The percentage of PCNA-positive tumor cells was found to be correlated with histological grading of tumor and also with the S+G2+M phase fraction and the S phase fraction measured by flow cytometry (p < 0.01). The mean percentage of PCNA-positive tumor cells was higher in aneuploid tumors than in diploid tumors. There was no significant relationship between the percentage of PCNA positive cells and tumor staging. Sixty-one cases were available for survival analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Aneuploidy↗

PubMed2001

Targeting of PCNA to sites of DNA replication in the mammalian cell nucleus.

We have examined the targeting of proliferating cell nuclear antigen (PCNA), an integral component of the mammalian replicative enzyme DNA polymerase delta, with sites of DNA replication by using confocal microscopy and computer image analysis. Labeling (5 min pulse) of DNA replication sites in normal human diploid fibroblast cells (NHF1) with BrdU was followed by immunostaining with PCNA antibodies. A striking degree of colocalization was seen between PCNA and the characteristic patterns of DNA replication sites of early, middle and late S-phase (Nakayasu and Berezney [1989] J. Cell. Biol. 108:1-11). These observations were confirmed by quantitative computer image analysis which revealed that approximately 90% of the PCNA-stained area overlapped with DNA replication sites in early S-phase. Pulse-chase experiments, involving in vivo labeling for replication followed by PCNA staining at later time points, suggested that PCNA disassembles from previously replicated sites and targets to newly active sites of DNA replication. To further study this phenomenon in living cells, stable GFP-PCNA transfectants under the control of a tetracycline-inducible promoter were created in mouse 3T6 cells. Like the endogenous PCNA, GFP-PCNA targeted to sites of replication (approximately 80% colocalization) and demonstrated similar dynamic changes following pulse-chase experiments in fixed cells. Studies of living cells revealed progressive changes in the GFP-PCNA distribution that mimic the replication patterns observed in fixed cells. We conclude that GFP-PCNA targets to DNA replication sites in living cells and is an effective marker for tracking the spatio-temporal dynamics of DNA replication as cells transverse the S-phase.

Animals↗