Cell cycle dependent distribution of proliferating cell nuclear antigen/cyclin and cdc2-kinase in mouse T-lymphoma cells

The aim of the present study was to investigate bromodeoxyuridine (BrdU) uptake and coordinated distribution of proliferating cell nuclear antigen (PCNA) and p34-cdc2-kinase, two important proteins involved in cell cycle regulation and progression. Flow cytometric analysis of marker proteins in freshly plated mouse T-lymphoma cells (Yac-1 cells), using fluorescein isothiocyanate (FITC)-labeled specific antibodies, showed PCNA distributed throughout the cell cycle with increased intensity in S-phase. PCNA is essential for cells to cycle through S-phase and its synthesis is initiated during late G1-phase before incorporation of BrdU and remains high during active DNA replication. The intensity of PCNA fluorescence increases with the duration of incubation after plating. The cdc2-kinase was detectable in all phases of the cell cycle and the G2-M-phase appears to have the maximum concentrations. The cell cycle analysis of high dose colcemid (2 μg/ml) treated Yac-1 cells showed an aneuploid or hypodiploid population. Although the G2-M-phase seems to be the dominating population in aneuploid cells, the concentrations of cdc2-kinase were variable in this phase of cell cycle. The colcemid treatment at 25 ng/ml arrested 96% of cells in S-phase and G2-M-phase, but PCNA expression was evident in a portion of the cell population in G2-M-phase. Although cells blocked in M-phase seem to have high levels of cdc2-kinase, colcemid renders them inactive. From these data, it appears that the down regulation and/or inactivation of cdc2-kinase could be responsible for the colcemid arrest of cells in M-phase.     

Novel Interaction between Proliferating Cell Nuclear Antigen and HLA I on the Surface of Tumor Cells Inhibits NK Cell Function through NKp44

NK cell function is closely regulated by numerous inhibitory and activating receptors binding corresponding ligands on the surface of target cells, providing vital first line defenses against infections and cancer. NKp44, originally discovered as an activating NK cell receptor, was recently found to elicit inhibitory effects on NK cell effector function through recognition of cell surface PCNA. Other reports have pointed to potential associations between NKp44 and HLA I molecules, as well as HLA I and Damage Associated Molecular Pattern molecules (DAMPs) on the surface of tumor cells. In this report, we have identified novel interaction between HLA I and PCNA on the surface of human tumor cells by confocal microscopy and immunoprecipitation. In addition to previous reports, we show PCNA on the cell surface where novel association with HLA I does not require the presence of NKp44 expressing NK cells and occurs with endogenous PCNA. The association of HLA I and PCNA forms the inhibitory ligand for NKp44, resulting in inhibition of NK cell cytotoxicity. We further postulate NCR ligands are composed of DAMP molecules localized to the cell surface, colocalizing with HLA I, and potentially heparin sulfate proteoglycans.     

A PCNA-Derived Cell Permeable Peptide Selectively Inhibits Neuroblastoma Cell Growth

Proliferating cell nuclear antigen (PCNA), through its interaction with various proteins involved in DNA synthesis, cell cycle regulation, and DNA repair, plays a central role in maintaining genome stability. We previously reported a novel cancer associated PCNA isoform (dubbed caPCNA), which was significantly expressed in a broad range of cancer cells and tumor tissues, but not in non-malignant cells. We found that the caPCNA-specific antigenic site lies between L126 and Y133, a region within the interconnector domain of PCNA that is known to be a major binding site for many of PCNA''s interacting proteins. We hypothesized that therapeutic agents targeting protein-protein interactions mediated through this region may confer differential toxicity to normal and malignant cells. To test this hypothesis, we designed a cell permeable peptide containing the PCNA L126-Y133 sequence. Here, we report that this peptide selectively kills human neuroblastoma cells, especially those with MYCN gene amplification, with much less toxicity to non-malignant human cells. Mechanistically, the peptide is able to block PCNA interactions in cancer cells. It interferes with DNA synthesis and homologous recombination-mediated double-stranded DNA break repair, resulting in S-phase arrest, accumulation of DNA damage, and enhanced sensitivity to cisplatin. These results demonstrate conceptually the utility of this peptide for treating neuroblastomas, particularly, the unfavorable MYCN-amplified tumors.     

Cell cycle-related proteins: a flow cytofluorometric study in human tumors

We used 2-parameter flow cytometry (FCM) to investigate the relationship between the cell cycle phases and 3 proteins whose expression is known to increase in proliferating cells: the surface antigen transferrin receptor (Trf-r), the "cyclin" (a proliferating cell nuclear antigen, PCNA), and the nuclear antigen recognized by the monoclonal antibody (MoAb) Ki-67. FITC-labeled antibodies against Trf-r, PCNA, and the Ki-67-reactive antigen, as well as propidium iodide-DNA distribution, were simultaneously measured on human leukemia HL-60 and K562, and breast carcinoma MCF-7 cell lines and on fresh human leukemic and glioblastoma cells. The 70% ethanol fixation for Trf-r and PCNA and the 95% acetone fixation for Ki-67 plus permeabilization (with 0.1% and 1% Triton X100, respectively, for the surface and the nuclear antigens) produced cell suspensions with negligible cell clumping, high-quality DNA profiles, and bright specific immunofluorescent staining. The investigated proteins have different relationships with the proliferative state of the cell. Trf-r is expressed mainly at the transition from G0/G1 to S-phase. PCNA expression is prominent in late G1 and through S-phase and decreases in G2-M. The Ki-67-reactive antigen is widely distributed in G1, S, and G2-M phases. Knowledge regarding the relationships between proliferation-associated antigens and cell cycle phase in normal and neoplastic cells could improve our understanding of the mechanisms underlying growth regulation and neoplastic transformation. Bivariate FCM is an easy method for obtaining these data from large numbers of cells.     

增殖细胞核抗原蛋白在Spodoptera frugiperda昆虫细胞中的表达及纯化

目的:利用昆虫细胞表达系统生产重组的人增殖细胞核抗原(proliferating cell nuclear antigen,PCNA),并进行纯化和抗体结合特性鉴定。方法:以HeLa细胞逆转录的cDNA为模板,扩增人PCNA基因,并插入杆状病毒载体AcMNPV。利用昆虫细胞得到PCNA基因的重组杆状病毒。病毒感染细胞表达蛋白,联合镍柱亲和层析和离子交换层析获得高纯度的重组人PCNA蛋白。ELISA法测定抗体结合特异性。结果:以HeLa细胞cDNA为模板得到的基因序列同GenBank的人PCNA基因序列一致。草地贪夜蛾细胞(Spodoptera frugiperda,Sf9)表达重组人PCNA(recombinant human PCNA,rPCNA)的最佳感染值(MOI)和感染时间分别为0.05h和144h。rPCNA的产量高达110mg/L细胞,纯度95%。间接ELISA法检测抗体结合特性,rPCNA的敏感性和特异性分别为93.3%和85.0%。结论:建立了rPCNA的表达和纯化方法,获得了高效表达、高度抗体结合特异性的PCNA蛋白,该蛋白质能进一步开发为PCNA相关疾病的体外诊断试剂盒,具较大的应用价值。     

Detection of Proliferating Cell Nuclear Antigen in Tissues of Three Small Fish Species

An immunohistochemical assay for proliferating cell nuclear antigen (PCNA) identifies cells in all active phases of the cell cycle. In this study, PCNA methodology, which was developed primarily for mammalian tissues, was adapted to three small fish species, medaka (Ory-zias latipes), guppy (Poecilia reticulata), and western mosquitofish (Gambusia affiinis) that are used in carcinogenesis bioassays and environmental sentinel studies. Our study showed that PCNA can be identified in routinely processed, paraffin embedded specimens of these fishes. Optimum staining conditions were dependent on fixative, primary antibody, antigen retrieval processing. and protein blocking reagent. Best results were achieved using 10% neutral buffered formalin as the fixative, clone PC10 as the primary antibody, and a combination of powdered milk and bovine serum albumin as a protein block. Except for medaka specimens, antigen retrieval was not required for specimens preserved in 10% neutral buffered formalin. but was required for the other fixatives tested. In whole fish specimens, PCNA marked cells in normally proliferating tissues such as testis. ovary, primary filament epithelium of the gill, hematopoietic tissues, thymus, retina and alimentary tract. The study demonstrated the successful application of mammalian-based PCNA technology to these aquatic species. Further applications of the assay will aid in understanding the role of cell proliferation in normal, diseased, and toxicant-affected tissues of aquatic animals.     

幽门螺杆菌感染促进胃炎儿童胃粘膜细胞的增殖

本研究旨在探讨幽门螺杆菌感染对小儿慢性胃炎患者细胞增殖的影响,使用内镜检查消化不良患者的上消化道症状,使用改良的Giemsa染色检测胃粘膜活组织中幽门螺杆菌,用苏木精/曙红和改良的吉姆萨染色活组织,并通过光学显微镜研究染色后胃粘膜样品组织病理学变化,RT-PCR检测各组胃粘膜细胞中调控细胞凋亡的Bcl-2、Bcl-xl、Bax和PCNA的mRNA表达,提取胃粘膜细胞蛋白质,利用蛋白质免疫印迹分析蛋白质浓度。组织化学染色结果表明,与对照相比,患有胃炎和幽门杆菌感染后的胃炎患者胃粘膜细胞明显增加,且幽门螺杆菌感染后细胞增殖更显著(p<0.05);幽门螺杆菌感染后Bcl-2和Bcl-xl,PCNA在患者体内表达显著上调(p<0.05),而细胞促凋亡因子Bax基因在胃炎患者感染幽门螺杆菌后被显著下调(p<0.05),蛋白免疫印迹分析Bcl-2,Bcl-xl,Bax和PCNA蛋白表达趋势与基因表达一致,说明结果可靠。幽门螺杆菌感染会显著提高慢性胃炎儿童患者胃粘膜细胞的增殖。     

Epidermal Growth Factor Receptor Protects Proliferating Cell Nuclear Antigen from Cullin 4A Protein-mediated Proteolysis

Proliferating cell nuclear antigen (PCNA) is an essential component for DNA synthesis upon growth stimulation. It has been shown that phosphorylation of PCNA at Tyr-211 by the EGF receptor (EGFR) protects PCNA from polyubiquitylation and degradation, whereas blocking phosphorylation induces ubiquitylation-mediated degradation of the chromatin-bound, but not the -unbound, PCNA, and suppresses cell proliferation. However, the ubiquitin E3 ligase linking growth signaling to the proteolysis of PCNA and the underlying regulatory mechanism remain to be identified. Here we show that, in the absence of Tyr-211 phosphorylation, PCNA is subject to polyubiquitylation at Lys-164 by the CUL4A E3 ligase, resulting in the degradation of PCNA. Mutation of Lys-164 to arginine prevents PCNA ubiquitylation and rescues the degradation of the K164R/Y211F PCNA double mutant. Activation of EGFR inhibits the interaction of PCNA with CUL4A, whereas inhibition of EGFR leads to increased CUL4A-PCNA interaction and CUL4A-dependent ubiquitin-mediated degradation of PCNA. Substitution of endogenous PCNA with the Y211F mutant PCNA conveys enhanced sensitization to EGFR inhibition. Our findings identify CUL4A as the ubiquitin ligase linking the down-regulation of cell surface receptor tyrosine kinase to the nuclear DNA replication machinery in cancer cells.     

Nuclear-to-cytoplasmic Relocalization of the Proliferating Cell Nuclear Antigen (PCNA) during Differentiation Involves a Chromosome Region Maintenance 1 (CRM1)-dependent Export and Is a Prerequisite for PCNA Antiapoptotic Activity in Mature Neutrophils

Neutrophils are deprived of proliferative capacity and have a tightly controlled lifespan to avoid their persistence at the site of injury. We have recently described that the proliferating cell nuclear antigen (PCNA), a nuclear factor involved in DNA replication and repair of proliferating cells, is a key regulator of neutrophil survival. In neutrophils, PCNA was localized exclusively in the cytoplasm due to its nuclear-to-cytoplasmic relocalization during granulocytic differentiation. We showed here that leptomycin B, an inhibitor of the chromosome region maintenance 1 (CRM1) exportin, inhibited PCNA relocalization during granulocytic differentiation of HL-60 and NB4 promyelocytic cell lines and of human CD34⁺ primary cells. Using enhanced green fluorescent protein fusion constructs, we have demonstrated that PCNA relocalization involved a nuclear export signal (NES) located from Ile-11 to Ile-23 in the PCNA sequence. However, this NES, located at the inner face of the PCNA trimer, was not functional in wild-type PCNA, but instead, was fully active and leptomycin B-sensitive in the monomeric PCNAY114A mutant. To test whether a defect in PCNA cytoplasmic relocalization would affect its antiapoptotic activity in mature neutrophils, a chimeric PCNA fused with the SV40 nuclear localization sequence (NLS) was generated to preclude its cytoplasmic localization. As expected, neutrophil-differentiated PLB985 cells expressing ectopic SV40NLS-PCNA had an increased nuclear PCNA as compared with cells expressing wild-type PCNA. Accordingly, the nuclear PCNA mutant did not show any antiapoptotic activity as compared with wild-type PCNA. Nuclear-to-cytoplasmic relocalization that occurred during myeloid differentiation is essential for PCNA antiapoptotic activity in mature neutrophils and is dependent on the newly identified monomerization-dependent PCNA NES.     

Cell cycle specific plasmid DNA replication in the nuclear extract of Saccharomyces cerevisiae: modulation by replication protein A and proliferating cell nuclear antigen

Plasmid DNA replication in nuclear extracts of Saccharomyces cerevisiae in vitro has been shown to be S-phase specific, similar to that observed in vivo. We report here a reconstituted in vitro system with partially purified replication proteins, purified replication protein A (RPA), and recombinant proliferating cell nuclear antigen (PCNA). Nuclear extracts from S-phase, G(1)-phase, and unsynchronized yeast cells were fractionated by phosphocellulose chromatography. Protein fraction (polymerase fraction) enriched with replication proteins, including DNA polymerases (alpha, delta, etc.), was isolated, which was not capable of in vitro replication of supercoiled plasmid DNA. However, when purified yeast RPA and recombinant PCNA together were added to the polymerase fraction obtained from S-phase synchronized cells, in vitro plasmid DNA replication was restored. In vitro plasmid DNA replication with polymerase fractions from unsynchronized and G(1)-phase cells could not be reconstituted upon addition of purified RPA and PCNA. RPA and PCNA isolated from various phases of the cell cycle complemented the S-phase polymerase pool to the same extent. Reconstituted systems with the S-phase polymerase pool, complemented with either the RPA- and PCNA-containing fraction or purified RPA and recombinant PCNA together, were able to produce replication intermediates (ranging in size from 50 to 1500 bp) similar to that observed with the S-phase nuclear extract. Results presented here demonstrate that both RPA and PCNA are cell cycle-independent in their ability to stimulate in vitro plasmid DNA replication, whereas replication factors in the polymerase fractions are strictly S-phase dependent.     

Cell proliferation and death of Hertwig's epithelial root sheath in the rat

Hertwig's epithelial root sheath (HERS) degenerates immediately after root dentin is formed. However, odontogenic tumors or cysts may originate from residual cells, although little is known about how HERS proliferates and disappears. This study investigated whether cell death is provoked in the tissues surrounding the root during eruption of the rat upper molar. We employed the TdT-mediated-dUTP nick end labeling (TUNEL) method and transmission electron microscopy (TEM) to observe the morphological features of cell death. We examined the activity of cell proliferation immunohistochemically using proliferative cell nuclear antigen (PCNA) and the continuity of HERS using polyclonal keratin antibody (PK). Cell death resembling apoptosis and apoptotic bodies phagocytosed by neighboring mesenchymal cells were detected in only a few cells by both TUNEL and TEM. We also found cells with electron-lucent cytoplasm which contained dilated or ruptured mitochondria and remarkably dilated rough endoplasmic reticulum (rER) which lay sparsely along the root. These cells seemed to be dead HERS cells based on their ultrastructural features, location, and stage. PCNA-positive cells were found in the apical end of the HERS cells, fibroblasts of the periodontal ligament, and odontoblasts. PK reacted with HERS; however, PK-positive cells partially disappeared after the 15th postnatal day when the root dentin had formed slightly. These results may indicate that HERS cells migrate into the periodontal ligament or die immediately after root dentin is formed and that various types of cell death such as apoptosis and cytoplasmic type occur in the tissues surrounding the root during tooth development.     

Flow cytometric analysis of proliferation associated nuclear antigens using washless staining of unfixed cells.

The expression of different proliferation associated nuclear antigens was analyzed using a washless double-staining method and flow cytometry. It is a simple and rapid two-step procedure which can be performed on low cell numbers. A series of hematopoietic cell lines and fresh lymphoma cells were tested and the methodology was found to be applicable to a number of nuclear antigens (PCNA, Ki-67, p105, MPM-2, fibrillarin). For PCNA, the detectability was dependent on the type of antibody used. The immunofluorescence pattern observed by microscopy was altered for antigens stained by the washless technique in comparison with the pattern obtained with fixed cells. With the washless method, detailed cell cycle analysis could be obtained by dual parameter analysis of PCNA and Ki-67.     

Targeting Proliferating Cell Nuclear Antigen and Its Protein Interactions Induces Apoptosis in Multiple Myeloma Cells

Multiple myeloma is a hematological cancer that is considered incurable despite advances in treatment strategy during the last decade. Therapies targeting single pathways are unlikely to succeed due to the heterogeneous nature of the malignancy. Proliferating cell nuclear antigen (PCNA) is a multifunctional protein essential for DNA replication and repair that is often overexpressed in cancer cells. Many proteins involved in the cellular stress response interact with PCNA through the five amino acid sequence AlkB homologue 2 PCNA-interacting motif (APIM). Thus inhibiting PCNA’s protein interactions may be a good strategy to target multiple pathways simultaneously. We initially found that overexpression of peptides containing the APIM sequence increases the sensitivity of cancer cells to contemporary therapeutics. Here we have designed a cell-penetrating APIM-containing peptide, ATX-101, that targets PCNA and show that it has anti-myeloma activity. We found that ATX-101 induced apoptosis in multiple myeloma cell lines and primary cancer cells, while bone marrow stromal cells and primary healthy lymphocytes were much less sensitive. ATX-101-induced apoptosis was caspase-dependent and cell cycle phase-independent. ATX-101 also increased multiple myeloma cells’ sensitivity against melphalan, a DNA damaging agent commonly used for treatment of multiple myeloma. In a xenograft mouse model, ATX-101 was well tolerated and increased the anti-tumor activity of melphalan. Therefore, targeting PCNA by ATX-101 may be a novel strategy in multiple myeloma treatment.     

Intranuclear localization of proliferating cell nuclear antigen during the cell cycle in renal cell carcinoma

OBJECTIVE: To investigate, with laser scanning cytometry (LSC), proliferating cell nuclear antigen (PCNA) expression during the cell cycle in renal cell carcinoma. STUDY DESIGN: DNA ploidy and intracellular localization of PCNA in renal cell carcinoma were determined using LSC and immunohistochemistry. The subjects were nine patients who had received surgery for renal cell carcinoma. After DNA ploidy analysis, the glass slides were restained by immunohistochemistry of PCNA. LSC allowed direct observation of PCNA localization during the cell cycle because we could obtain immunohistochemical staining of PCNA as a function of cell cycle phase for individual cells. RESULTS: PCNA was not demonstrated in the nuclei of G0/G1 cells. PCNA expression increased from the S phase of the cell cycle. PCNA rapidly degraded at the end of the G2 phase. In the late G2 and M phase, PCNA was not detected in almost any nucleus. CONCLUSION: LSC allows morphologic observation of the intracellular distribution of PCNA during the cell cycle in renal cell carcinoma.     

高温湿热酸法破壁提取法夫酵母胞内虾青素

法夫酵母是一种能积累虾青素的红酵母, 对其进行破壁是当前虾青素工业化提取生产的瓶颈工艺。研究在高温湿热条件下,低浓度盐酸对法夫酵母破壁而提取其胞内虾青素的工艺。探讨了不同破壁温度、盐酸浓度、液料比与破壁处理时间等因素对法夫酵母破壁后虾青素及类胡萝卜素提取率的影响, 确定了高温湿热酸法破壁提取虾青素的最佳条件为: 饱和蒸汽压力 0.1 MPa (121°C), 盐酸浓度0.5 mol/L, 液料比 (V/W)30 mL/g, 破壁时间2 min。在最佳条件下进行中试放大实验, 可得到虾青素与类胡萝卜素的提取率分别为: (84.8±3.2)%, (93.3±2)%。经优化后的新破壁工艺安全高效, 不会有毒性残留, 具有良好的工业应用前景。     

A Small Molecule Inhibitor of Monoubiquitinated Proliferating Cell Nuclear Antigen (PCNA) Inhibits Repair of Interstrand DNA Cross-link,Enhances DNA Double Strand Break,and Sensitizes Cancer Cells to Cisplatin

Small molecule inhibitors of proliferating cell nuclear antigen (PCNA)/PCNA interacting protein box (PIP-Box) interactions, including T2 amino alcohol (T2AA), inhibit translesion DNA synthesis. The crystal structure of PCNA in complex with T2AA revealed that T2AA bound to the surface adjacent to the subunit interface of the homotrimer of PCNA in addition to the PIP-box binding cavity. Because this site is close to Lys-164, which is monoubiquitinated by RAD18, we postulated that T2AA would affect monoubiquitinated PCNA interactions. Binding of monoubiquitinated PCNA and a purified pol η fragment containing the UBZ and PIP-box was inhibited by T2AA in vitro. T2AA decreased PCNA/pol η and PCNA/REV1 chromatin colocalization but did not inhibit PCNA monoubiquitination, suggesting that T2AA hinders interactions of pol η and REV1 with monoubiquitinated PCNA. Interstrand DNA cross-links (ICLs) are repaired by mechanisms using translesion DNA synthesis that is regulated by monoubiquitinated PCNA. T2AA significantly delayed reactivation of a reporter plasmid containing an ICL. Neutral comet analysis of cells receiving T2AA in addition to cisplatin revealed that T2AA significantly enhanced formation of DNA double strand breaks (DSBs) by cisplatin. T2AA promoted colocalized foci formation of phospho-ATM and 53BP1 and up-regulated phospho-BRCA1 in cisplatin-treated cells, suggesting that T2AA increases DSBs. When cells were treated by cisplatin and T2AA, their clonogenic survival was significantly less than that of those treated by cisplatin only. These findings show that the inhibitors of monoubiquitinated PCNA chemosensitize cells by inhibiting repair of ICLs and DSBs.     

Flow cytometric multiparameter analysis of proliferating cell nuclear antigen/cyclin and Ki-67 antigen: a new view of the cell cycle

Flow cytometric multiparameter analysis of two proliferation-associated nuclear antigens (proliferating cell nuclear antigen (PCNA)/cyclin and Ki-67) was performed on seven human hematopoietic cell lines. PCNA/cyclin, an S phase-related antigen, was detected using an autoantibody and a fluorescein isothiocyanate-labeled anti-human antibody. The Ki-67 antigen, which in cycling cells is expressed with increasing levels during the S phase with a maximum in the M phase, was detected using a monoclonal antibody and a phycoerythrin-conjugated anti-mouse antibody. In some experiments the PCNA/Ki-67 staining was combined with a DNA stain, 7-amino actinomycin D, and simultaneous detection of the three stains was performed by a single laser flow cytometer. Using this technique four distinct cell populations, representing G1, S, G2, and M, respectively, could be demonstrated in cycling cells on the basis of their PCNA/cyclin and Ki-67 levels. The cell cycle phase specificity could be verified using metaphase (vinblastine, colcemide) and G2 phase (mitoxantrone) blocking agents, as well as by stainings with a mitosis-specific antibody (MPM-2). Also, G0 cells could be discriminated from G1 cells in analysis of a mixture of resting peripheral mononuclear blood cells and a proliferating cell line. This technique can be valuable in detailed cell cycle analysis, since all cell cycle phases can be visualized and calculated using a simple double staining procedure.     

Cyclin D1 Inhibits Cell Proliferation through Binding to PCNA and Cdk2

Cyclin D1 is known as a promoting factor for cell growth. We previously showed, however, that the expression of cyclin D1 increases markedly in senescent human fibroblastsin vitro.Here we investigate whether the overexpression of cyclin D1 inhibits cell proliferation. Colony formation after transfection with the cyclin D1 expression vector was repressed in NIH-3T3, TIG-1, CHO-K1, and HeLa cells, compared with those with mock and cyclin E expression vectors. A transient transfection assay demonstrated that the overexpression of cyclin D1 inhibited DNA synthesis of TIG-1 cells. The complexes of cyclin D1 with PCNA and cdk2 increased remarkably in senescent cells, compared with young counterparts. Excessive glutathioneS-transferase (GST)–cyclin D1 inhibited DNA replication and repressed cdk2-dependent kinase activityin vitro.DNA synthesis of NIH-3T3 transfectants with PCNA or cdk2 expression vectors was not inhibited by the overexpression of cyclin D1. These results indicate that an excessive level of cyclin D1 represses cell proliferation by inhibiting DNA replication and cdk2 activity through the binding of cyclin D1 to PCNA and cdk2, as it does in senescent cells.     

Proliferating cell nuclear antigen (PCNA) as a proliferative marker during embryonic and adult zebrafish hematopoiesis

We investigated the expression of proliferative cell nuclear antigen (PCNA) in zebrafish to delineate the proliferative hematopoietic component during adult and embryonic hematopoiesis. Immunostaining for PCNA and enhanced green fluorescence protein (eGFP) was performed in wild-type and fli1-eGFP (endothelial marker) and gata1-eGFP (erythroid cell marker) transgenic fish. Expression of PCNA mRNA was examined in wild-type and chordin morphant embryos. In adult zebrafish kidney, the renal tubules are surrounded by endothelial cells and it is separated into hematopoietic and excretory compartments. PCNA was expressed in hematopoietic progenitor cells but not in mature neutrophils, eosinophils or erythroid cells. Some PCNA+ cells are scattered in the hematopoietic compartment of the kidney while others are closely associated with renal tubular cells. PCNA was also expressed in spermatogonial stem cells and intestine crypts, consistent with its role in cell proliferation and DNA synthesis. In embryos, PCNA is expressed in the brain, spinal cord and intermediate cell mass (ICM) at 24 h-post fertilization. In chordin morphants, PCNA is significantly upregulated in the expanded ICM. Therefore, PCNA can be used to mark cell proliferation in zebrafish hematopoietic tissues and to identify a population of progenitor cells whose significance would have to be further investigated.     

Apoptotic cell death in neuronal differentiation of P19 EC cells: Cell death follows reentry into S phase

Apoptotic cell death was observed during aggregate culture of the mouse embryonal carcinoma cell line P19 exposed to all-trans retinoic acid (tRA). This finding was confirmed by genomic DNA agarose gel electrophoresis and transmission electron microscopy. Apoptosis was associated with P19 cell neuronal differentiation; alternative causes of cell death, i.e., cavitation-related, cytotoxicity of tRA, or spontaneous cell death were excluded. Analysis by flow cytometry revealed that the apoptosis was likely to occur in multiplying cells that underwent to reentering into S phase. We therefore examined 5-bromo-2′-deoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) expression and localization in the aggregates by immunofluorescent staining. Although the P19 cells in the aggregates exposed to tRA incorporated BrdU at an equivalent level to those not exposed to tRA, the cells showed diminished PCNA expression and nuclear accumulation. We propose that P19 apoptosis during neuronal differentiation is a model system in which programmed cell death occurs simultaneously with cell division leading to differentiation. J. Cell. Physiol. 172:25–35, 1997. © 1997 Wiley-Liss, Inc.