Human APE2 protein is mostly localized in the nuclei and to some extent in the mitochondria, while nuclear APE2 is partly associated with proliferating cell nuclear antigen

In human cells APE1 is the major AP endonuclease and it has been reported to have no functional mitochondrial targeting sequence (MTS). We found that APE2 protein possesses a putative MTS. When its N-terminal 15 amino acid residues were fused to the N-terminus of green fluorescent protein and transiently expressed in HeLa cells the fusion protein was localized in the mitochondria. By electron microscopic immunocytochemistry we detected authentic APE2 protein in mitochondria from HeLa cells. Western blotting of the subcellular fraction of HeLa cells revealed most of the APE2 protein to be localized in the nuclei. We found a putative proliferating cell nuclear antigen (PCNA)-binding motif in the C-terminal region of APE2 and showed this motif to be functional by immunoprecipitation and in vitro pull-down binding assays. Laser scanning immunofluorescence microscopy of HeLa cells demonstrated both APE2 and PCNA to form foci in the nucleus and also to be co-localized in some of the foci. The incubation of HeLa cells in HAT medium containing deoxyuridine significantly increased the number of foci in which both molecules were co-localized. Our results suggest that APE2 participates in both nuclear and mitochondrial BER and also that nuclear APE2 functions in the PCNA-dependent BER pathway.     

Inhibition of colon cancer cell proliferation by the dietary compound conjugated linoleic acid is mediated by the CDK inhibitor p21CIP1/WAF1

Our previous studies indicated that dietary conjugated linoleic acid (CLA) inhibits colon tumor cell proliferation in vitro and in vivo. To identify mechanisms by which CLA regulates growth arrest, the HT-29 human colon carcinoma cell line was treated with various physiological concentrations of CLA and analyzed by flow cytometry. We detected a dose-dependent increase in the percentage of cells arrested in G1 after CLA treatment that was accompanied by induction of the cyclin dependent kinase (CDK) inhibitor p21CIP1/WAF. CLA addition also led to increased p21 expression in HCT116 and SW480 cells, indicating that p21 induction is a general consequence of CLA treatment in colon cancer cells. Since both HT-29 and SW480 cells have mutant p53, our data indicate that p53 is not essential for induction of p21. In addition to an increase in p21 levels, HT-29 cell growth arrest was also accompanied by moderate decreases in Cyclin A, D1, E, and proliferating cell nuclear antigen (PCNA) levels. Following CLA treatment, p21 associated with and inhibited CDK4 and CDK2, and this correlated with reduced phosphorylation of retinoblastoma proteins. Increased association of p21 with PCNA was also detected. Dietary CLA inhibits cell cycle progression by inducing p21, which negatively regulates the growth promoting activities of CDK/cyclins and PCNA. These studies indicate that physiological concentrations of CLA inhibit growth of colon cancer cells with either wild-type or mutant p53, and may have therapeutic benefits in vivo.     

Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells

The eukaryotic sliding DNA clamp, proliferating cell nuclear antigen (PCNA), is essential for DNA replication and repair synthesis. In order to load the ring-shaped, homotrimeric PCNA onto the DNA double helix, the ATPase activity of the replication factor C (RFC) clamp loader complex is required. Although the recruitment of PCNA by RFC to DNA replication sites has well been documented, our understanding of its recruitment during DNA repair synthesis is limited. In this study, we analyzed the accumulation of endogenous and fluorescent-tagged proteins for DNA repair synthesis at the sites of DNA damage produced locally by UVA-laser micro-irradiation in HeLa cells. Accumulation kinetics and in vitro pull-down assays of the large subunit of RFC (RFC140) revealed that there are two distinct modes of recruitment of RFC to DNA damage, a simultaneous accumulation of RFC140 and PCNA caused by interaction between PCNA and the extreme N-terminus of RFC140 and a much faster accumulation of RFC140 than PCNA at the damaged site. Furthermore, RFC140 knock-down experiments showed that PCNA can accumulate at DNA damage independently of RFC. These results suggest that immediate accumulation of RFC and PCNA at DNA damage is only partly interdependent.     

Regulation of proliferating cell nuclear antigen during the cell cycle

The proliferating cell nuclear antigen (PCNA), also known as cyclin and DNA polymerase delta auxiliary factor, is present in reduced amounts in nongrowing cells and is synthesized at a greater rate in the S phase of growing cells. The recently discovered involvement of PCNA in DNA replication suggested that this pattern of expression functions to regulate DNA synthesis. We have investigated this possibility further by examining the synthesis, stability, and accumulation of PCNA in HeLa cells fractionated by centrifugal elutriation into nearly synchronous populations of cells at various positions in the cell cycle. In these fractionated cells we found that there is an increase in the rate of PCNA synthesis with a peak in early S phase of the cell cycle, but the magnitude of the increase is only 2-3-fold. This change reflects similar changes in the amount of PCNA mRNA. The fluctuating synthesis of PCNA maintains this protein at a roughly constant proportion of the total cell protein, although the amount doubles/cell in the cell cycle. Consistent with this observation, the stability of PCNA does not differ significantly from that of total cellular protein in synchronized HeLa cells. We also observed that a maximum of one-third of the total PCNA is tightly associated with the nucleus, presumably in replication complexes, at the peak of S phase. We conclude that the cyclic synthesis of PCNA in cycling HeLa cells maintains PCNA in excess of the amount involved directly in DNA replication and the amount of the protein neither fluctuates significantly with the cell cycle nor is limiting for DNA synthesis.     

PCNA/cyclin expression and BrdU uptake define different subpopulations in different cell lines.

Monoclonal antibodies (MAb) to a 36 KD protein, proliferating cell nuclear antigen (PCNA/cyclin), have been previously shown to be capable of identifying proliferating cells in vitro as well as in alcohol-fixed, paraffin-embedded tissue specimens. The routine use of these anti-PCNA/cyclin MAb in investigative studies and in diagnostic pathology requires a clearer understanding of the distribution of PCNA/cyclin in the different cell populations found in tissue specimens. We therefore compared the ability of MAb to three nucleus-associated proliferation markers (MAb 19A2 to PCNA/cyclin; Ki-67 to an undefined proliferation-related marker; BU-1 to 5'-bromodeoxyuridine (BrdU) incorporated into DNA) to identify the proliferating cell fraction of various cells in vitro. The cell lines were chosen to represent a spectrum of proliferation rates (high to low) and cell lineage (mesenchymal vs epithelial, non-transformed vs malignant): (a) HeLa and A-431 (two malignant carcinoma cell lines with high proliferation rates); (b) SK-5 (a non-transformed fibroblast cell line with a low proliferation rate); (c) HUVE (a non-transformed human umbilical vein endothelial cell line with a low proliferation rate). Single and double labeling immunofluorescence studies were performed after uniform 1-hr incubations with BrdU. Comparison of the overlapping distributions of detectable PCNA/cyclin expression and BrdU incorporation demonstrated substantial qualitative and quantitative differences between the different cell lines. In two of the four cell lines (HeLa, A-431) the BrdU staining distributions formed inclusive subsets of the PCNA-positive cell populations. In the HUVE cell line the two populations overlapped incompletely. In one cell line, SK-5, the two populations were mutually exclusive. MAb Ki-67 demonstrated a pattern in the SK-5 cell line that was strongly predictive of PCNA positivity, while showing no associated patterns in the other three cell lines. We conclude that PCNA/cyclin expression detected by MAb may define different cell subpopulations in different cell types relative to those incorporating BrdU or expressing the target antigen for Ki-67. This has implications for the clinical study of mixed cell populations using these antibodies.     

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.     

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

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

Arsenic Inhibits DNA Mismatch Repair by Promoting EGFR Expression and PCNA Phosphorylation

Both genotoxic and non-genotoxic chemicals can act as carcinogens. However, while genotoxic compounds lead directly to mutations that promote unregulated cell growth, the mechanism by which non-genotoxic carcinogens lead to cellular transformation is poorly understood. Using a model non-genotoxic carcinogen, arsenic, we show here that exposure to arsenic inhibits mismatch repair (MMR) in human cells, possibly through its ability to stimulate epidermal growth factor receptor (EGFR)-dependent tyrosine phosphorylation of proliferating cellular nuclear antigen (PCNA). HeLa cells exposed to exogenous arsenic demonstrate a dose- and time-dependent increase in the levels of EGFR and tyrosine 211-phosphorylated PCNA. Cell extracts derived from arsenic-treated HeLa cells are defective in MMR, and unphosphorylated recombinant PCNA restores normal MMR activity to these extracts. These results suggest a model in which arsenic induces expression of EGFR, which in turn phosphorylates PCNA, and phosphorylated PCNA then inhibits MMR, leading to increased susceptibility to carcinogenesis. This study suggests a putative novel mechanism of action for arsenic and other non-genotoxic carcinogens.     

Proliferating cell nuclear antigen (PCNA) and human malignant tumor nucleolar antigens (HMTNA) in nucleoli of human hematological malignancies

Lymphoma (Lymphocytic non-Hodgkin's malignant lymphoma) and leukemic (chronic lymphocytic, acute and chronic myeloid, myelomonocytic leukemia) cells were studied by indirect immunofluorescence to evaluate the presence of proliferating cell nuclear antigen (PCNA) and human malignant tumor nuclear antigen (HMTNA) in their nucleoli. Most cells in lymph node smears of lymphocytic non-Hodgkin's malignant lymphoma (NHML) developed a bright nucleolar fluorescence with HMTNA antibodies. PCNA was detected in nucleoli of a limited number of cells which apparently represent the proliferating cell population in these lymphomas. Similarly, in the bone marrow smears of patients with chronic lymphocytic leukemia most cells possessed a nucleolar fluorescence for HMTNA and PCNA was present in nucleoli of a limited number of cells. In the bone marrow smears of patients with myeloid or myelomonocytic leukemias most blastic or monocytoid cells also developed a bright nucleolar fluorescence with HMTNA antibodies and PCNA was present only in a small percentage of these cells. Leukemic cells with PCNA in their nucleoli like thekhuntigen might represent a proliferating cell population in late G1-early S phase.     

Dietary fiber decreases colonic epithelial cell proliferation and protein synthetic rates in human subjects

Although it has been proposed that high fiber consumption can prevent proliferative diseases of the colon, the clinical data to support this hypothesis have been inconsistent. To provide a more robust measure of the effects of fiber on colonic mucosal growth than previous studies, we evaluated both cell proliferation and colonic mucosal protein synthesis in nine healthy volunteers after they consumed a typical Western diet (<20 g fiber/day) or a Western diet supplemented with wheat bran (24 g/day) in a randomized crossover design. Biopsies taken from the sigmoid colon were used to assess mucosal proliferation by determining proliferating cell nuclear antigen (PCNA) in crypt cells and to assess mucosal protein synthetic rate using stable isotopically labeled leucine infusion. Fiber supplementation produced a 12% decrease in labeling index (%crypt cells stained with PCNA) (P < 0.001) and an 11% decrease in mucosal protein fractional synthetic rate (FSR; P < 0.05). Moreover, mucosal protein FSR correlated directly with labeling index (r2= 0.22, P < 0.05). These data demonstrate that increased wheat bran consumption decreases colonic mucosal proliferation and support the potential importance of dietary fiber in preventing proliferative diseases of the colon.     

Nm23-H1 is responsible for SUMO-2-involved DNA synthesis induction after X-ray irradiation in human cells

Human cells derived from nevoid basal carcinoma syndrome (NBCCS) patients show increased levels of DNA synthesis activity after X-ray irradiation which is suggested to be casually related to reduction in cellular amounts of small ubiquitin-like protein modifier (SUMO-2/SMT-3A). In the present study, an increased level of DNA synthesis activity was found 8 h after X-ray irradiation in HeLa cells with reduction in SUMO-2 amounts by siRNA treatment for SUMO-2. When comparative proteomic analysis was performed between the siRNA and mimic control siRNA treated cells using two-dimensional (2D) electrophoresis and mass spectrometry, three proteins were identified as candidates. Our research focused on Nm23-H1, a nucleoside diphosphate kinase, whose amounts decreased after X-ray irradiation in HeLa cells treated with siRNA for SUMO-2. In the Nm23-H1 siRNA treated cells, induction of DNA synthesis was also detected. Furthermore, in synchronized HeLa cells, DNA synthesis was confirmed in the S phase. Moreover, increased expression of proliferating cell nuclear antigen (PCNA) was observed in Nm23-H1 siRNA treated HeLa cells after X-ray irradiation. In addition, Nm23-H1 was modified with SUMO-2 after X-ray irradiation. The present findings suggest that the reduction of Nm23-H1 is related to the decrease in sumoylation, which in turn, is involved in the induction of DNA synthesis via the regulation of PCNA expression after X-ray irradiation.     

Comparative analysis of the intracellular localization of c-Myc, c-Fos, and replicative proteins during cell cycle progression.

In eukaryotic cells, nucleus-cytoplasm exchanges play an important role in genomic regulation. We have analyzed the localization of four nuclear antigens in different growth conditions: two replicative proteins, DNA polymerase alpha and proliferating cell nuclear antigen (PCNA), and two oncogenic regulatory proteins, c-Myc and c-Fos. A kinetic study of subcellular localization of these proteins has been done. In cultures in which cells were sparse, these proteins were detected in the nucleus. When proliferation was stopped by the high density of culture cells or by serum starvation, these proteins left the nucleus for the cytoplasm with different kinetics. DNA polymerase alpha is the first protein to leave the nucleus, with the PCNA protein, c-Fos, and c-Myc leaving the nucleus later. In contrast, during serum stimulation c-Fos and c-Myc relocalize into the nucleus before the replicative proteins. We also noticed that in sparse cell cultures, 10% of the cells exhibit a perinuclear staining for the DNA polymerase alpha, PCNA, and c-Myc proteins but not for c-Fos. This peculiar staining was also observed as an initial step to nuclear localization after serum stimulation and in vivo in Xenopus embryos when the G1 phase is reintroduced in the embryonic cell cycle at the mid-blastula stage. We suggest that such staining could reflect specific structures involved in the initiation of the S phase.     

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.     

A positive role for the Ku complex in DNA replication following strand break damage in mammals

Ku70-Ku80 complex is the regulatory subunit of DNA-dependent protein kinase (DNA-PK) and plays an essential role in double-strand break repair following ionizing radiation (IR). It preferentially interacts with chromosomal breaks and protects DNA ends from nuclease attack. Here we show evidence that cells defective in Ku80 exhibit a significantly slow S phase progression following DNA damage. IR-induced retardation in S phase progression in Ku80-/- cells was not due to the lack of DNA-PK kinase activity because both wild-type cells and DNA-PKcs-deficient cells showed no such symptom. Instead, proliferating cell nuclear antigen (PCNA) dissociated from chromosomes following IR in Ku80-deficient cells but not in wild-type or DNA-PKcs-deficient cells. Treatment of HeLa cells with IR induced colocalization of the Ku complex with PCNA on chromosomes. Together, these results suggest that binding of the Ku complex at chromosomal breaks may be necessary to maintain the sliding clamps (PCNA) on chromatin, which would allow cells to resume DNA replication without a major delay following IR.     

帕比司他逆转前列腺癌细胞hepaCAM基因表达机制研究

目的:研究帕比司他(Panobinostat)逆转抑癌基因肝细胞粘附分子(hepatocyte cell adhesion molecule,hepaCAM)表达,协同hepaCAM抑制前列腺癌(prostate cancer,PCa)细胞生长。方法:不同浓度帕比司他作用于体外培养的PC3细胞,首先采用MTT法检测帕比司他对细胞增殖的影响,RT-PCR和Western blot法检测hepaCAM、组蛋白去乙酰化酶(histone deacetylases, HDACs)以及乙酰化组蛋白H3 赖氨酸9(Ac-H3K9)的表达变化。接着用不同因素处理细胞,MTT法检测细胞增殖活性,流式细胞术检测细胞周期改变,RT-PCR和Western blot法检测细胞周期调节因子cyclinD1 和增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的基因表达。结果:帕比司他抑制PC3细胞生长与作用浓度增加和作用时间呈正相关, hepaCAM mRNA、蛋白以及细胞核中Ac-H3K9表达随帕比司他浓度升高而增高,HDAC1、 HDAC3、 HDAC4 mRNA和蛋白的表达随浓度升高而降低;单独过表达hepaCAM腺病毒和单独使用帕比司他组PC3细胞生长明显受到抑制且随作用时间延长抑制率增加,两者联用更为明显,差异有统计学意义(P < 0.05);与单独hepaCAM 腺病毒组和帕比司他组相比,两者联用S期细胞比例明显增高,差异有统计学意义(P < 0.05),且可进一步下调cyclinD1、 PCNA mRNA和蛋白的表达,差异有统计学意义(P < 0.05)。结论:帕比司他可通过抑制HDACs活性,加强PCa PC3细胞组蛋白H3 N-端的赖氨酸残基乙酰化,逆转hepaCAM表达;hepaCAM腺病毒和帕比司他联用可通过阻滞细胞周期于S期协同抑制PC3 细胞生长,作用机制可能与下调cyclinD1 和PCNA 的表达有关。这对揭示抑癌基因hepaCAM在肿瘤缺失的原因及为将帕比司他应用于临床治疗肿瘤提供了科学支持。     

Interaction of proliferation cell nuclear antigen (PCNA) with c-Abl in cell proliferation and response to DNA damages in breast cancer

Cell proliferation in primary and metastatic tumors is a fundamental characteristic of advanced breast cancer. Further understanding of the mechanism underlying enhanced cell growth will be important in identifying novel prognostic markers and therapeutic targets. Here we demonstrated that tyrosine phosphorylation of the proliferating cell nuclear antigen (PCNA) is a critical event in growth regulation of breast cancer cells. We found that phosphorylation of PCNA at tyrosine 211 (Y211) enhanced its association with the non-receptor tyrosine kinase c-Abl. We further demonstrated that c-Abl facilitates chromatin association of PCNA and is required for nuclear foci formation of PCNA in cells stressed by DNA damage as well as in unperturbed cells. Targeting Y211 phosphorylation of PCNA with a cell-permeable peptide inhibited the phosphorylation and reduced the PCNA-Abl interaction. These results show that PCNA signal transduction has an important impact on the growth regulation of breast cancer cells.     

Characterisation and high-resolution distribution of a matrix attachment region-binding protein (MFP1) in proliferating cells of onion

The first matrix attachment region (MAR)-binding protein sequenced in plants, MFP1, has been characterised in two dicot species. Based on their antigenic relationship, we report here the conservation of MFP1-like proteins in proliferating root cells of onion (Allium cepa L). Two MFP1-like proteins with different molecular masses and solubilities were detected. The most abundant was a 90-kDa basic protein, presenting several separate spots in two-dimensional blots. The MFP1 was partially soluble and, similar to the proliferating cell nuclear antigen (PCNA)-labelled replication factories in the nucleus and nuclear matrix, was localised at discrete foci as detected by confocal microscopy. High-resolution immunolocalisation of MFP1 by electron microscopy identified the foci as nuclear structures, some of them containing PCNA, which are ultrastructurally similar to the replication factories described in animal cells. Our data provide the first report on MFP1-like proteins in the Alliaceae. In addition, we present evidence of the presence of AcMFP1 in the putative replication factories. Received: 12 May 2000 / Accepted: 13 September 2000