Two types of proliferating cell nuclear antigen (PCNA) complex formation in quiescent normal and xeroderma pigmentosum group A fibroblasts following ultraviolet light (uv) irradiation.

We examined the relationship between the formation of proliferating cell nuclear antigen (PCNA) complex with DNA and nucleotide excision repair in human fibroblasts following ultraviolet light (uv) irradiation. PCNA complex formation was detected by the immunofluorescence method after methanol fixation and nucleotide excision repair activity was detected as the unscheduled DNA synthesis (UDS) by autoradiography labeled with [3H]thymidine. Quiescent normal cells showed a strong punctuated pattern of PCNA staining 5 min to 3 h and UDS 3 h after 10 J/m2 of uv irradiation, but they no longer showed PCNA staining and UDS 24 h after irradiation. In contrast, xeroderma pigmentosum group A (XP-A) cells, which lack UDS activity, did not show PCNA staining up to 30 min after irradiation; however, unexpectedly, they were stained 3 h and even 24 h after irradiation with their staining pattern being different from that in normal cells. Namely, the fluorescence spots in XP-A cells were larger in size and much smaller in number than those in normal cells. When XP-A cells were fused with normal cells with polyethylene glycol treatment, nuclei of XP-A cells showed a PCNA staining pattern similar to that of normal cells at 30 min, which was no longer detected 24 h after irradiation. These results suggest that there exist two types of PCNA complex formation, nucleotide excision repair-related and -unrelated, in human fibroblasts following uv irradiation.     

Induction of proliferating cell nuclear antigen (PCNA) complex formation in quiescent fibroblasts from a xeroderma pigmentosum patient.

Accumulated evidence indicates that proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase delta and forms tight association with DNA replication sites during DNA replication or DNA repair synthesis. In this study, such PCNA complex formation was investigated by the indirect immunofluorescence method, using both normal human fibroblasts and those derived from a xeroderma pigmentosum group A (XP-A) patient. XP-A fibroblasts in both proliferating and quiescent states did not show any differences from normal fibroblasts in the properties of PCNA-staining in the untreated conditions. The PCNA complex formation was induced in quiescent normal fibroblasts by both ultraviolet light (UV)- and X-irradiation, whereas in XP-A fibroblasts it was induced by X-irradiation, but not by UV-irradiation. However, PCNA complex was induced in quiescent XP-A fibroblasts by UV-irradiation when the cells had previously incorporated 5-bromodeoxyuridine (BrdU). These observations indicate a close correlation of PCNA complex formation and unscheduled DNA synthesis (UDS). Thus, it was concluded that PCNA complex formation was commonly induced in at least three conditions to produce UDS in spite of different types of DNA damages and DNA repair mechanisms.     

Oxidative damage-induced PCNA complex formation is efficient in xeroderma pigmentosum group A but reduced in Cockayne syndrome group B cells.

Proliferating cell nuclear antigen (PCNA), a processivity factor for DNA polymerases delta and epsilon, is essential for both DNA replication and repair. PCNA is required in the resynthesis step of nucleotide excision repair (NER). After UV irradiation, PCNA translocates into an insoluble protein complex, most likely associated with the nuclear matrix. It has not previously been investigated in vivo whether PCNA complex formation also takes place after oxidative stress. In this study, we have examined the involvement of PCNA in the repair of oxidative DNA damage. PCNA complex formation was studied in normal human cells after treatment with hydrogen peroxide, which generates a variety of oxidative DNA lesions. PCNA was detected by two assays, immunofluorescence and western blot analyses. We observed that PCNA redistributes from a soluble to a DNA-bound form during the repair of oxidative DNA damage. PCNA complex formation was analyzed in two human natural mutant cell lines defective in DNA repair: xeroderma pigmentosum group A (XP-A) and Cockayne syndrome group B (CS-B). XP-A cells are defective in overall genome NER while CS-B cells are defective only in the preferential repair of active genes. Immunofluorescent detection of PCNA complex formation was similar in normal and XP-A cells, but was reduced in CS-B cells. Consistent with this observation, western blot analysis in CS-B cells showed a reduction in the ratio of PCNA relocated as compared to normal and XP-A cells. The efficient PCNA complex formation observed in XP-A cells following oxidative damage suggests that formation of PCNA-dependent repair foci may not require the XPA gene product. The reduced PCNA complex formation observed in CS-B cells suggests that these cells are defective in the processing of oxidative DNA damage.     

Induction of proliferating cell nuclear antigen (PCNA)-immunoreactive cells in goldfish retina following intravitreal injection with 6-hydroxydopamine

1. The dopaminergic neurotoxin, 6-hydroxydopamine (6-OHDA), was injected intravitreally into the eyes of juvenile (5- to 6-cm) goldfish. 2. Proliferation of rod neuroblasts caused by 6-OHDA (2 micrograms in 2 microliters saline) was detected in retinal wholemounts by immunofluorescence for proliferating cell nuclear antigen (PCNA) 3, 7, 14, 20, or 30 days after injection. 3. The injected dose of 6-OHDA was sufficient to cause permanent loss of dopaminergic interplexiform and serotonergic amacrine cells in the injected eye but not in the contralateral control eye. 4. 6-OHDA increased the density (mm-2) of PCNA-ir cells in the outer nuclear layer (ONL) of the injected eye to 2.65 times the initial density 20-30 days after injection, and it increased the density of PCNA-ir cells in the ONL of the contralateral, untreated eye, equally but after a delay of less than or equal to 7 days with respect to the injected eye. 5. 6-OHDA also increased the density of PCNA-ir cells in the inner nuclear layer (INL) to greater than 20 times the initial density 7 days after injection, followed by a rapid decline almost to control levels by 14 days after injection. 6. The sequence of responses to 6-OHDA, with PCNA-ir cells first scattered in the ONL and then clustered in the INL, suggests that neuroblasts from the ONL migrate to the INL to compensate for toxin-induced cell loss. 7. Double staining for 5-bromodeoxyuridine (BrUdR; a thymidine analogue) and PCNA, carried out on 7 days after intravitreal injection with 6-OHDA, showed that 77% of all PCNA-ir cells in the outer nuclear layer had been in S phase during the previous 24 hr. 8. Immunoreactivity for PCNA was found to be a valid marker for rod neuroblasts which have entered S phase within 1-2 days before sampling and was shown to be especially convenient for investigating the distribution of proliferating cells in whole mounts. 9. In controls injected unilaterally with saline or saline plus 1% dimethyl sulfoxide (DMSO), the differences in densities of PCNA-ir rod precursor nuclei 2-30 days after injection vs. day 0 (uninjected) were statistically insignificant in both injected and uninjected eyes (Negishi et al., 1991). Therefore the local effect of injecting 6-OHDA was due to 6-OHDA itself, not to mechanical damage or nonspecific actions of foreign substances.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of proliferating cell nuclear antigen (PCNA) isoforms in normal and cancer cells: there is no cancer-associated form of PCNA

In order to clarify the status of PCNA in normal and transformed cells, we performed analysis of this protein by 2D-PAGE, Western blot and mass spectrometry. All the cell lines examined contained the major PCNA form (pI 4.57/30kDa), that is not post-translationally modified. In addition to the major form, two minor isoforms (pI 4.52/30kDa and pI 4.62/30kDa) were also detected in all the cell lines examined. However, the level of PCNA in cancer cells is 5-6 folds higher than those in primary and most of the immortalized cells. Taken together, the significant difference in PCNA status between cancer and normal cells is not at the post-translational modifications but in the overall levels of PCNA.     

The promoter of the proliferating cell nuclear antigen (PCNA) gene is active in serum-derived cells.

mRNA levels for the Proliferating Cell Nuclear Antigen (PCNA) gene are growth regulated. PCNA promoters of different lengths were used to drive a linked reporter, the cDNA for human thymidine kinase (TK). After transfection into TK ts13 cells, stable cell lines were obtained. Regardless of promoter length, in all cell lines the levels of TK mRNA were roughly similar in serum-deprived and serum stimulated cells, confirming, by an independent method, that the growth regulation of PCNA mRNA levels doe not depend on the 5' flanking sequence of the PCNA gene.     

Immunochemical and biochemical analysis of the proliferating cell nuclear antigen (PCNA) in HeLa cells

PCNA, also known a cyclin, a protein of molecular weight (MW) 35 000, accumulates in the nuclei of dividing and transformed cells and reacts with autoantibodies from certain lupus patients. Using an indirect immunofluorescence technique, we show that lupus sera containing anti-PCNA antibodies reveal a heterogeneous nuclear fluorescence pattern upon reaction with asynchronous HeLa cells, whereas with synchronized cells a sequence of distinct patterns is disclosed on progression through the cell cycle. Cell-free translation of HeLa cell mRNA followed by immunoprecipitation with anti-PCNA sera shows a single protein with the same apparent MW as PCNA labelled in vivo, suggesting that PCNA is not derived from a larger precursor protein and not grossly modified by post-translational events. However, a group of at least nine nuclear polypeptides ranging in MW from about 12 000 to 110 000 are recognized by immunoblotting with anti-PCNA sera, indicating either that additional antigenic sites are produced on denaturation of native proteins or that additional autoantibodies are present in these sera. We also show that PCNA and several of these polypeptides are associated with nuclear structures containing chromatin.     

Immunohistochemical localization of proliferating cell nuclear antigen (PCNA) in the pig ovary

The aim of the study was to determine the expression of proliferating cell nuclear antigen protein (PCNA) in the pig ovary. The localization of PCNA was demonstrated in paraffin sections of pig ovarian tissue using primary mouse monoclonal anti-PCNA antibody. In primordial follicles, no remarkable staining for PCNA either in granulosa cells or in the oocytes was observed. In primary to secondary follicles, positive staining in oocytes and in some granulosa cells was detected. The advanced preantral and particularly actively growing small to large antral follicles showed extensive PCNA labeling in the layers of granulosa and theca cells and in the cumulus cells encircling the oocyte. PCNA labeling was expressed in nuclei of oocytes in preantral and small antral follicles. In atretic follicles, the level of PCNA protein expression was dependent on the stage of atresia. Follicles demonstrating advanced atresia showed only limited or no PCNA labeled granulosa and theca cells. The results of the study demonstrate that follicular growth and development in pig ovary may be effectively monitored by determining the granulosa cell expression of PCNA.     

Characterization of a second proliferating cell nuclear antigen (PCNA2) from Drosophila melanogaster

The eukaryotic DNA polymerase processivity factor, proliferating cell nuclear antigen, is an essential component in the DNA replication and repair machinery. In Drosophila melanogaster, we cloned a second PCNA cDNA that differs from that encoded by the gene mus209 (for convenience called DmPCNA1 in this article). The second PCNA cDNA (DmPCNA2) encoded a 255 amino acid protein with 51.7% identity to DmPCNA1, and was ubiquitously expressed during Drosophila development. DmPCNA2 was localized in nuclei as a homotrimeric complex and associated with Drosophila DNA polymerase delta and epsilonin vivo. Treatment of cells with methyl methanesulfonate or hydrogen peroxide increased the amount of both DmPCNA2 and DmPCNA1 associating with chromatin, whereas exposure to UV light increased the level of association of only DmPCNA1. Our observations suggest that DmPCNA2 may function as an independent sliding clamp of DmPCNA1 when DNA repair occurs.     

Analysis of proliferative activity in colorectal mucosa by immunohistochemical detection of proliferating cell nuclear antigen (PCNA)

Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) has been suggested as a new approach for determinating proliferative activity in paraffin-embedded tissue. In a prospective study PCNA immunostaining was performed in 284 colorectal biopsies using monoclonal antibodies 19F4 (Ogata et al. 1987) and PC10 (Waseem and Lane 1990) and compared with the Ki67 method. From each site three biopsies were taken and a variety of fixation regimens for frozen and paraffin-embedded samples tested. For frozen biopsies methanol fixation at −20° C proved best. In paraffin sections PCNA could be detected after methacarn fixation as well as after controled fixation at 4° C in 4% paraformaldehyde for 1 h and in most biopsies routinely fixed with 10% formalin. However, the latter fixation regimens revealed additional PCNA-positive cells in the normal superficial colonic mucosal epithelium. Although the percentage of cells positive for PCNA was generally lower than for Ki67, the rates correlated in a highly significant fashion, both in frozen methanolfixed biopsies, and in paraformaldehyde-fixed paraffinembedded samples. PCNA immunohistochemistry revealed a similar proliferative activity in different parts of the large bowel. A higher proliferative activity was found in inflamed mucosa, adenomas, carcinomas and even in normal mucosa from patients with colorectal neoplasms. In routinely fixed biopies, the monoclonal antibody PC10 was superior to 19F4 because of considerably less background staining. However, in the routine material only a rough estimate of the proliferative activity was possible by PCNA immunohistochemistry using these antibodies, because unpredictable numbers of non-S-phase cells were also stained. Thus, it was concluded that reliable results are only obtainable after careful control of the fixation conditions. Taking this reservation into account, PCNA immunohistochemistry still represents a convenient method for measurements of proliferative activity in paraffin-embedded colorectal mucosa and can be applied using methanol-containing fixatives as well as after 4% paraformaldehyde fixation. Supported by a grant of the Werner and Klara Kreitz-Stiftung, Kiel to J.D.     

Immunocytochemical localization of cyclin/proliferating cell nuclear antigen (PCNA) in fertilized eggs of mice.

Immunolocalization of cyclin/PCNA (proliferating cell nuclear antigen) was performed with monoclonal antibody using immunogold methods on ultrathin cryosections of fertilized mouse eggs. Immunolabeling in pronuclei was checked 20, 22, 24 and 26 h after HCG injection. A relation between onset of pronuclei migration (early S-phase) and appearance of colloidal particle clusters was found. Afterwards, (mid S-phase) the increase of labelling and the localization of cyclin/PCNA were found throughout the pronuclei, except in the nucleolar bodies. Lower labelling appeared at the time of close reciprocal pronuclei contact (late S-phase). It is concluded that bulk and distribution of cyclin/PCNA in pronuclei is closely related to the progression of first interphase after fertilization.     

Growth factor stimulation of proliferating cell nuclear antigen (PCNA) in human breast epithelium in organ culture.

Normal human breast explants were maintained in serum-free culture for 7 days in the presence of either insulin hydrocortisone and cholera toxin (I/H/CT), epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha). Explants were labelled with [3H] thymidine, fixed in methacarn and processed for autoradiography. Parallel sections were immunolabelled with anti-PCNA antibody and analysed with a CAS 200 image analyser. Thymidine labelling index (TLI) and PCNA expression produced similar results with both indices increased in response to I/H/CT, EGF and TGF-alpha. In sections double labelled for PCNA and autoradiography the majority of labelled cells were positive for both markers.     

A site-selective,irreversible inhibitor of the DNA replication auxiliary factor proliferating cell nuclear antigen (PCNA)

Proliferating cell nuclear antigen (PCNA) assumes an indispensable role in supporting cellular DNA replication and repair by organizing numerous protein components of these pathways via a common PCNA-interacting sequence motif called a PIP-box. Given the multifunctional nature of PCNA, the selective inhibition of PIP-box-mediated interactions may represent a new strategy for the chemosensitization of cancer cells to existing DNA-directed therapies; however, promiscuous blockage of these interactions may also be universally deleterious. To address these possibilities, we utilized a chemical strategy to irreversibly block PIP-box-mediated interactions. Initially, we identified and validated PCNA methionine 40 (M40) and histidine 44 (H44) as essential residues for PCNA/PIP-box interactions in general and, more specifically, for efficient PCNA loading onto chromatin within cells. Next, we created a novel small molecule incorporating an electrophilic di-chloro platinum moiety that preferentially alkylated M40 and H44 residues. The compound, designated T2Pt, covalently cross-linked wild-type but not M40A/H44A PCNA, irreversibly inhibited PCNA/PIP-box interactions, and mildly alkylated plasmid DNA in vitro. In cells, T2Pt persistently induced cell cycle arrest, activated ATR-Chk1 signaling and modestly induced DNA strand breaks, features typical of cellular replication stress. Despite sustained activation of the replication stress response by the compound and its modestly genotoxic nature, T2Pt demonstrated little activity in clonogenic survival assays as a single agent, yet sensitized cells to cisplatin. The discovery of T2Pt represents an original effort directed at the development of irreversible PCNA inhibitors and sets the stage for the discovery of analogues more selective for PCNA over other cellular nucleophiles.     

Molecular cloning of cDNA coding for rat proliferating cell nuclear antigen (PCNA)/cyclin.

The 'proliferating cell nuclear antigen' (PCNA), also known as cyclin, appears at the G1/S boundary in the cell cycle. Because of its possible relationship with cell proliferation, PCNA/cyclin has been receiving attention. PCNA/cyclin is a non-histone acidic nuclear protein with an apparent mol. wt of 33000-36000. The amino acid composition and the sequence of the first 25 amino acids of rabbit PCNA/cyclin are known. Using an oligonucleotide probe corresponding to the sequence of the first five amino acids, a cDNA clone for PCNA/cyclin was isolated from rat thymocyte cDNA library. The cDNA (1195 bases) contains an open reading frame of 813 nucleotides coding for 261 amino acids. The 3'-non-coding region is 312 nucleotides long and contains three putative polyadenylation signals. The mol. wt of rat PCNA/cyclin was calculated to be 28 748. The deduced amino acid sequence and composition of rat PCNA/cyclin are in excellent agreement with the published data. Using the cDNA probe, two species of mRNA (1.1 and 0.98 kb) were detected in rat thymocyte RNA. Southern blot analysis of total human genomic DNA suggests that there is a single gene coding for PCNA/cyclin. The deduced amino acid sequence of rat PCNA/cyclin has a similarity with that of herpes simplex virus type-1 DNA binding protein.     

Proliferating cell nuclear antigen (PCNA/cyclin) in plant proliferating cells: immunohistochemical and quantitative analysis using autoantibody and murine monoclonal antibodies to PCNA.

Proliferating-cell nuclear antigen (PCNA), also known as cyclin, is synthesized in proliferative cells and recently was identified as DNA polymerase-delta auxiliary protein. In this paper, the association of PCNA to the proliferative cells of plants was analysed using both autoantibodies to PCNA obtained from a patient with systemic lupus erythematosus (SLE) and murine monoclonal antibodies. By immunohistochemical analysis, nuclei of cells around the growing point in soybean root tips reacted strongly with autoantibodies to PCNA in the serum from a patient with SLE. The plant PCNA in root tip cells was purified by ammonium sulfate fractionation, DEAE chromatography, and affinity chromatography. The partially purified plant PCNA was tested by immunoblotting and a 34 kD polypeptide reacted with both the human anti-PCNA autoantibody and a mouse monoclonal antibody against human PCNA (TOB 7). In addition, the purified plant PCNA reacted with both antibodies in enzyme-linked immunosorbent assay (ELISA). The binding of anti-PCNA serum to the animal PCNA was blocked by the plant PCNA in this ELISA. The association of PCNA with growing cells in plants was further confirmed by quantitative sandwich type ELISA using two murine monoclonal antibodies to PCNA, TOB7 and TO17. Those results suggested that PCNA in both plant and animal cells had the same immunological and biochemical characteristics and the plant PCNA might play an important role in cell growth, existing as it does in proliferating plant cells. The concentration of PCNA in soybean germ extract before germination was less than 5 ng ml-1 (protein concentration, 6.8 mg ml-1), but that of the root tip stem including the growing point increased to 887 ng ml-1 (protein concentration 3.8 mg ml-1) in the second day after germination.     

Repair-deficient xeroderma pigmentosum cells made UV light resistant by fusion with X-ray-inactivated Chinese hamster cells.

Xeroderma pigmentosum (XP) is an autosomal recessive human disease, characterized by an extreme sensitivity to sunlight, caused by the inability of cells to repair UV light-induced damage to DNA. Cell fusion was used to transfer fragments of Chinese hamster ovary (CHO) chromosomes into XP cells. The hybrid cells exhibited UV resistance and DNA repair characteristics comparable to those expressed by CHO cells, and their DNA had greater homology with CHO DNA than did the DNA from XP cells. Control experiments consisted of fusion of irradiated and unirradiated XP cells and repeated exposure of unfused XP cells to UV doses used for hybrid selection. These treatments did not result in an increase in UV resistance, repair capability, or homology with CHO DNA. The hybrid cell lines do not, therefore, appear to be XP revertants. The establishment of these stable hybrid cell lines is an initial step toward identifying and cloning CHO DNA repair genes that complement the XP defect in human cells. The method should also be applicable to cloning genes for other diseases, such as ataxia-telangiectasia and Fanconi's anemia.