Downregulation Of Proliferating Cell Nuclear Antigen Research Articles

The E301R protein of African swine fever virus functions as a sliding clamp involved in viral genome replication.

Sliding clamp is a highly conserved protein in the evolution of prokaryotic and eukaryotic cells. The sliding clamp is required for genomic replication as a critical co-factor of DNA polymerases. However, the sliding clamp analogs in viruses remain largely unknown. We found that the ASFV E301R protein (pE301R) exhibited a sliding clamp-like structure and similar functions during ASFV replication. Interestingly, pE301R is assembled into a unique ring-shaped homotetramer distinct from sliding clamps or proliferating cell nuclear antigens (PCNAs) from other species. Notably, the E301R gene is required for viral life cycle, but the pE301R function can be partially restored by the porcine PCNA. This study not only highlights the functional role of the ASFV pE301R as a viral sliding clamp analog, but also facilitates the dissection of the complex replication mechanism of ASFV, which provides novel clues for developing antivirals against ASF.

Panlongqi Tablet (PLQT) Inhibits LPS-Induced Abnormal Proliferation of RA-HFLS by Regulating PI3K/ AKT and MAPK Signaling Pathways

Panlongqi Tablet (PLQT), a traditional Chinese formula, is effective in the clinical treatment of Rheumatoid Arthritis (RA) in China for several decades. However, the underlying mechanism of PLQT’s therapeutic effect remains unclear. The main chemical constituents of PLQT were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The human fibroblast-like synovial cells in rheumatoid arthritis (RA-HFLS) were stimulated with LPS (10 μg/mL) to establish a inflammatory model in vitro. The cells were then treated with PLQT-medicated serum to verify the protective effect of the herbal compound. MTT assay was used to detect the viability of RAHFLS and abnormal cell proliferation. The inflammatory factors levels including interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-17 (IL-17) in RAHFLS were measured by ELISA. In addition, the cell cycle progression was analyzed by flow cytometry and protein expression of Proliferating Cell Nuclear Antigen (PCNA) was detected using immunohistochemistry and western blot. The protein expression levels associated with PI3K/AKT and MAPK signaling pathways in RA-HFLS were detected by western blot analyses. Our results revealed that PLQT treatment effectively inhibited the abnormal proliferation of RA-HFLS and decreased the inflammatory factors levels. The proportion of cells in S phase was significantly increased with the treatment of PLQT. Meanwhile, the expression levels of PCNA, p-PI3K, p-AKT, p-mTOR, p-JNK/JNK, p-ERK/ ERK, and p-P38/P38 proteins from RA-HFLS were dramatically inhibited by PLQT treatment. PLQT inhibits LPS-induced abnormal proliferation of RAHFLS, which may be related to the down regulation of PCNA, and the inhibition of abnormally activated PI3K/AKT and MAPK signaling pathways.

Anti-tumor effects of isoliquiritigenin in Bcl-2/Bax and PCNA expression of T24 human bladder cancer cells

IntroductionThe aim of this study was to investigate the proliferation and apoptosis of T24 after treatment with isoliquiritigenin (ISL) and to explore the underlying mechanism.Material and methodsT24 cells were divided into 6 groups and cultured. The five experimental groups were seeded into medium with 10, 20, 40, 80, and 160 µM ISL and the control group was administered with 0.01% DMSO. The cell morphology was observed using an optical microscope. The proliferation of cells and half maximal inhibitory concentration (IC50) were determined and calculated by MTS. Cell cycle and apoptosis were evaluated by flow cytometry. The migration ability of cells was detected by wound healing assay. The expression of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and proliferating cell nuclear antigen (PCNA) was determined by RT-qPCR and western blot.ResultsAt 24 h after treatment with ISL, morphology revealed that cell number diminished and the cell volume shrank with increasing concentration. According to the results of MTS, ISL significantly inhibited the T24 cell proliferation in a dose- and time-dependent manner. Flow cytometry showed that ISL could block the replication of T24 cell DNA from S phase to G2, therefore promoting cell apoptosis. Wound healing assay showed that at 48 h after treatment with ISL, the migration of T24 cells was remarkably inhibited. The results of RT-qPCR and Western blot revealed that after 24 h of treatment, the expression levels of Bcl-2 and PCNA were down-regulated, and the expression level of Bax was increased in the different dose of the ISL treated group.ConclusionsISL possesses detrimental effects on the viability of BC cell T24 in a dose-dependent manner via blocking the cell cycle and inducing apoptosis by down-regulation of PCNA and Bcl-2 expression and up-regulation of Bax expression.

Expression of microRNA- 942 in liver cancer tissues and its effect on proliferation and invasion of hepatocellular carcinoma

Objective To investigate the expression of microRNA (miRNA, miR)- 942 in liver cancer tissues and its effect on proliferation and invasion of hepatocellular carcinoma (HCC). Methods miR- 942 expression was detected in HCC and peritumoral tissues by real-time quantitative polymerase chain reaction (Real-time PCR) from 70 patients. The Huh7 cells were transfected with scrambled miRNA sequence (negative control group), miR- 942 mimics (miR- 942 mimics group) and miR- 942 inhibitors (miR- 942 inhibitors group) respectively. The proliferative activity and invasion abilities of Huh7cells were measured by methyl thiazol tetrazolium (MTT) and Transwell assays. The expression levels of proliferating cell nuclear antigen (PCNA) and metalloproteinase- 9 (MMP- 9) were detected by Western blotting. Results miR- 942 was significantly up-regulated in HCC tissues as compared with matched peritumoral tissues (0.92±0.08 vs. 0.42±0.05, t=5.712, P=0.000). The results of cell proliferation showed that the absorbance (A) value of Huh7 cells (A) in miR- 942 inhibitors group was significantly lower than that of the negative control group and the miR- 942 mimics group after 5 days (0.79±0.02 vs. 1.02±0.04, t=7.513, P=0.001; 0.79±0.02 vs. 1.51±0.12, t=8.621, P=0.001). The results of Transwell assays showed that the number of metastatic cells in the miR- 942 inhibitors group was significantly decreased compared to the negative control group and the miR- 942 mimics group respectively [(59.13±5.41) cells vs. (97.26±11.13) cells, t=11.712, P=0.000; (59.13±5.41) cells vs. (99.42±12.13) cells, t=12.113, P=0.000]. The expression of PCNA and MMP- 9 protein in miR- 942 inhibitors group was reduced by 80.1%and56.3% respectively (t=5.231, P=0.000; t=6.415, P=0.000). Conclusion The expression of miR- 942 in HCC tissues was significantly higher than that in adjacent tissues, and dysregulation of miR- 942 can suppress proliferation and invasion of HCC cells, which may be related to down-regulation of PCNA and MMP- 9 expression. Key words: Carcinoma, hepatocellular; MicroRNA-942; Proliferation; Invasion

Effects of resveratrol on ARPE-19 cell proliferation and migration via regulating the expression of proliferating cell nuclear antigen, P21, P27 and p38MAPK/MMP-9.

To explore whether resveratrol (Res) can inhibit human retinal pigment epithelial cell (ARPE-19 cell) proliferation and migration, and to research the molecular mechanisms. ARPE-19 cells were pretreated with various concentrations at 0, 50, 100, 150, 200 and 300 µmol/L of Res, and with 0 µmol/L Res as the control for 24, 48 and 72h. The cell proliferation, apoptosis and migration were measured with cell counting kit-8 (CCK-8), flow cytometry, and wound-healing and Transwell assays, respectively. The expression of proliferating cell nuclear antigen (PCNA), P21 and P27, as well as matrix metalloproteinase-9 (MMP-9) and p38 mitogen-activated protein kinases (p38MAPK) was identified by Western blot. Cell proliferation was effectively inhibited by Res (P<0.05). When pretreated with Res, cells arrested in S-phase increased remarkably (P<0.05), but the apoptosis ratios showed no significant difference between the treatment and control groups (P>0.05). Cell migration was suppressed by Res both in wound-healing assay and Transwell migration assay (P<0.05). Decreases of PCNA, MMP-9 and p38MAPK, as well as increases of P21 and P27 were detected by Western blot (P<0.05). Res can inhibit APRE-19 cell proliferation and migration in a concentration-dependent manner with up-regulation of the expression of P21 and P27, and down-regulation of PCNA, MMP-9 and p38MAPK.

Damage to dopaminergic neurons is mediated by proliferating cell nuclear antigen through the p53 pathway under conditions of oxidative stress in a cell model of Parkinson's disease.

Oxidative stress is widely considered as a central event in the pathogenesis of Parkinson's disease (PD). The mechanisms underlying the oxidative damage-mediated loss of dopaminergic neurons in PD are not yet fully understood. Accumulating evidence has indicated that oxidative DNA damage plays a crucial role in programmed neuronal cell death, and is considered to be at least partly responsible for the degeneration of dopaminergic neurons in PD. This process involves a number of signaling cascades and molecular proteins. Proliferating cell nuclear antigen (PCNA) is a pleiotropic protein affecting a wide range of vital cellular processes, including chromatin remodelling, DNA repair and cell cycle control, by interacting with a number of enzymes and regulatory proteins. In the present study, the exposure of PC12 cells to 1-methyl-4-phenylpyridinium (MPP+) led to the loss of cell viability and decreased the expression levels of PCNA in a dose- and time-dependent manner, indicating that this protein may be involved in the neurotoxic actions of MPP+ in dopaminergic neuronal cells. In addition, a significant upregulation in p53 expression was also observed in this cellular model of PD. p53 is an upstream inducer of PCNA and it has been recognized as a key contributor responsible for dopaminergic neuronal cell death in mouse models of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD. This indicates that MPP+-induced oxidative damage is mediated by the downregulation of PCNA through the p53 pathway in a cellular model of PD. Thus, our results may provide some novel insight into the molecular mechanisms responsible for the development of PD and provide new possible therapeutic targets for the treatment of PD.

Defective CFTR-regulated granulosa cell proliferation in polycystic ovarian syndrome

Polycystic ovarian syndrome (PCOS) is one of the most frequent causes of female infertility, featured by abnormal hormone profile, chronic oligo/anovulation, and presence of multiple cystic follicles in the ovary. However, the mechanism underlying the abnormal folliculogenesis remains obscure. We have previously demonstrated that CFTR, a cAMP-dependent Cl(-) and HCO3 (-) conducting anion channel, is expressed in the granulosa cells and its expression is downregulated in PCOS rat models and human patients. In this study, we aimed to investigate the possible involvement of downregulation of CFTR in the impaired follicle development in PCOS using two rat PCOS models and primary culture of granulosa cells. Our results indicated that the downregulation of CFTR in the cystic follicles was accompanied by reduced expression of proliferating cell nuclear antigen (PCNA), in rat PCOS models. In addition, knockdown or inhibition of CFTR in granulosa cell culture resulted in reduced cell viability and downregulation of PCNA. We further demonstrated that CFTR regulated both basal and FSH-stimulated granulosa cell proliferation through the HCO3 (-)/sAC/PKA pathway leading to ERK phosphorylation and its downstream target cyclin D2 (Ccnd2) upregulation. Reduced ERK phosphorylation and CCND2 were found in ovaries of rat PCOS model compared with the control. This study suggests that CFTR is required for normal follicle development and that its downregulation in PCOS may inhibit granulosa cell proliferation, resulting in abnormal follicle development in PCOS.

Epigallocatechin-3-Gallate Prevents Autoimmune-Associated Down- Regulation of p21 in Salivary Gland Cells Through a p53-Independent Pathway

The submandibular salivary glands of non-obese diabetic (NOD) mice, a model for Sjogren's syndrome and type-1 diabetes, show an elevated level of proliferating cell nuclear antigen (PCNA), a protein involved in cell proliferation and repair of DNA damage. We reported previously that epigallocatechin-3-gallate (EGCG), the most abundant green tea catechin, normalizes the PCNA level. PCNA's activity can be regulated by the cyclin-dependent kinase inhibitor p21, which is also important for epithelial cell differentiation. In turn, expression of p21 and PCNA are partially regulated by Rb phosphorylation levels. EGCG was found to modulate p21 expression in epithelial cells, suggesting that EGCG-induced p21 could be associated with down-regulation of PCNA in vivo. The current study examined the protein levels of p21 and p53 (which can up-regulate p21) in NOD mice fed with either water or EGCG, and the effect of EGCG on p21 and p53 in cell line models with either normal or defective Rb. In NOD mice, the p21 level was low, and EGCG normalized it. In contrast to HSG cells with functional Rb, negligible expression of p21 in NS-SVAC cells that lack Rb was not altered by EGCG treatment. Inhibition of p53 by siRNA demonstrated that p21 and p53 were induced independently in HSG cells by a physiological concentration range of EGCG, suggesting p53 could be an important but not conditional factor associated with p21 expression. In conclusion, PCNA and p21 levels are altered inversely in the NOD model for SS and in HSG cells, and warrant further study as candidate new markers for salivary dysfunction associated with xerostomia. Induction of p21 by EGCG could provide clinically useful normalization of salivary glands by promoting differentiation and reducing PCNA levels.

Knockdown of survivin contributes to antitumor activity in cisplatin-resistant ovarian cancer cells

Survivin (SVV) is an important member of the inhibitor of apoptosis family. It is overexpressed in a number of cancer types, including human ovarian carcinomas. SVV promotes invasion, metastasis, growth and survival of malignant cells and confers resistance to specific chemotherapeutic drugs. The present study aimed to elucidate the role and possible mechanisms of SVV in cisplatin-resistant ovarian cancer cells (A2780/CP). Using a loss-of-function approach, we investigated the effects of adenovirus-mediated knockdown of SVV by small hairpin RNA (ad5-SVV) on the expression of pro-caspase-3, cleaved caspase-3, proliferating cell nuclear antigen (PCNA) and matrix metalloproteinase-2 (MMP-2) in A2780/CP cells by real-time PCR and western blot analysis. Proliferation was measured by MTT assay, invasive potential by Transwell, and cell apoptosis by FITC-Annexin V and propidium iodide for the functional analysis of A2780/CP cells following infection with ad5-SVV. As a result, knockdown of SVV downregulated the expression of PCNA and MMP-2 and upregulated the expression of pro-caspase-3 and cleaved caspase-3. In addition, knockdown of SVV enhanced cisplatin-induced proliferative activities, induced cell apoptosis and inhibited the invasive potential in A2780/CP cells. The present findings demonstrate that knockdown of SVV contributes to antitumor activity in cisplatin-resistant ovarian cancer cells via the downregulation of PCNA and MMP-2 expression and the upregulation of caspase-3 expression and indicate that SVV is a potential target for therapeutic anticancer drugs.

Silver nanoparticles induce toxicity in A549 cells via ROS-dependent and ROS-independent pathways

Silver nanoparticles (AgNPs) are incorporated into a large number of consumer and medical products. Several experiments have demonstrated that AgNPs can be toxic to the vital organs of humans and especially to the lung. The present study evaluated the in vitro mechanisms of AgNP (<100nm) toxicity in relationship to the generation of reactive oxygen species (ROS) in A549 cells. AgNPs caused ROS formation in the cells, a reduction in their cell viability and mitochondrial membrane potential (MMP), an increase in the proportion of cells in the sub-G1 (apoptosis) population, S phase arrest and down-regulation of the cell cycle associated proliferating cell nuclear antigen (PCNA) protein, in a concentration- and time-dependent manner. Pretreatment of the A549 cells with N-acetyl-cysteine (NAC), an antioxidant, decreased the effects of AgNPs on the reduced cell viability, change in the MMP and proportion of cells in the sub-G1population, but had no effect on the AgNP-mediated S phase arrest or down-regulation of PCNA. These observations allow us to propose that the in vitro toxic effects of AgNPs on A549 cells are mediated via both ROS-dependent (cytotoxicity) and ROS-independent (cell cycle arrest) pathways.

Expression and Yes-Associated Protein in Gastric Adenocarcinoma and Inhibitory Effects of its Knockdown on Gastric Cancer Cell Proliferation and Metastasis

Yes-associated protein (YAP) has been implicated as an oncogene in multiple human cancers. In the present study, human gastric adenocarcinoma tissues of different grades (N=78) were collected and the mRNA and protein expression of YAP and phosphorylated YAP (p-YAP) in gastric adenocarcinomas were evaluated using immunohistochemistry, Real-time PCR and Western blot assays. Then, human gastric cancer SGC-7901 cells were stably transfected with lentivirus-mediated YAP small hairpin RNA (shRNA). The expression levels of YAP, proliferating cell nuclear antigen (PCNA) and metalloproteinase-2 (MMP-2) were detected and the effects of shRNA-mediated knockdown of YAP on cell proliferation and metastasis were assessed in gastric cancer cells. As a result, the expression of YAP was observed in 69.23 percent gastric adenocarcinoma tissues, elevating with the ascending order of tumor malignancy. Knockdown of YAP could down-regulated the expression of PCNA and MMP-2, and inhibit the proliferation and metastasis of gastric cancer cells. In conclusion, YAP is strongly expressed in gastric adenocarcinomas, and knockdown of YAP may inhibit gastric cancer cell proliferation and metastasis through down-regulation of PCNA and MMP-2 expression, suggesting that YAP represents an important therapeutic target in human gastric cancer.

Inhibitory Effects and Underlying Mechanism of 7-Hydroxyflavone Phosphate Ester in HeLa Cells

Chrysin and its phosphate ester have previously been shown to inhibit cell proliferation and induce apoptosis in Hela cells; however, the underlying mechanism remains to be characterized. In the present study, we therefore synthesized diethyl flavon-7-yl phosphate (FP, C19H19O6P) by a simplified Atheron-Todd reaction, and explored its anti-tumor characteristics and mechanisms. Cell proliferation, cell cycle progression and apoptosis were measured by MTS, flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling techniques, respectively in human cervical cancer HeLa cells treated with 7-hydroxyflavone (HF) and FP. p21, proliferating cell nuclear antigen (PCNA) and cAMP levels in Hela cells were analyzed by western blot and radioimmunoassay. Both HF and FP inhibited proliferation and induced apoptosis in HeLa cells via induction of PCNA/p21 expression, cleaved caspase-3/poly (ADP-ribose) polymerase (PARP)-1, elevation of cAMP levels, and cell cycle arrest with accumulation of cells in the G0/G1 fraction. The effects of FP were more potent than those of HF. The interactions of FP with Ca2+-calmodulin (CaM) and Ca2+-CaM-phosphodiesterase (PDE)1 were explored by electrospray ionization-mass spectrometry and fluorescence spectra. FP, but not HF, formed non-covalent complexes with Ca2+-CaM-PDE1, indicating that FP is an inhibitor of PDE1, and resulting in elevated cellular cAMP levels. It is possible that the elevated cAMP levels inhibit growth and induce apoptosis in Hela cells through induction of p21 and cleaved caspase-3/PARP-1 expression, and causing down-regulation of PCNA and cell cycle arrest with accumulation of cells in the G0/G1 and G2/M fractions. In conclusion, FP was shown to be a Ca2+-CaM-PDE inhibitor, which might account for its underlying anti-cancer mechanism in HeLa cells. These observations clearly demonstrate the special roles of phosphorylated flavonoids in biological processes, and suggest that FP might represent a potential new drug for the therapy of human cervical carcinoma.

Methylene blue photodynamic therapy in malignant melanoma decreases expression of proliferating cell nuclear antigen and heparanases

Malignant melanoma (MM) is a very aggressive tumour. Although surgical excision of MM in the early stages has a very good prognosis, it often fails to completely inhibit tumour progression. Methylene blue photodynamic therapy (MB-PDT) is a technique that induces tissue damage by reactive oxygen species (ROS). To investigate the efficacy of and potential use of MB-PDT in restraining the aggressiveness of MM by analysing levels of proliferating cell nuclear antigen (PCNA) and heparanase (HPSE, a molecular marker of cell invasion) in a mouse model. Expression of PCNA and two HPSE isoforms were analysed using immunohistochemistry (IHC) after MB-PDT in mice. Tumour volume and weight were also measured. Two treatments with MB-PDT promoted a decrease of 99% decrease in tumour volume and 75% in tumour weight compared with untreated mice (P < 0.05). Using IHC, a decrease in expression of 75% for PCNA and 95% for both HPSE isoforms (P < 0.05) was found. MB-PDT is a cheap and efficient method of decreasing MM volume and thus disease progression. This reduction is mediated by downregulation of PCNA and heparanases.

Vitamin D inhibits proliferation of human uterine leiomyoma cells via catechol- O-methyltransferase

To evaluate the effects and mechanisms of action of vitamin D on human uterine leiomyoma (HuLM) cell proliferation in vitro. Laboratory study. University hospitals. Not applicable. Not applicable. HuLM cells were treated with 1,25-dihydroxyvitamin D3 (vitamin D), and cell proliferation was assayed by the methylthiazolyl tetrazolium technique. proliferating cell nuclear antigen (PCNA), BCL-2, BCL-w, cyclin-dependent kinase (CDK) 1, and catechol-O-methyltransferase (COMT) protein levels were analyzed by Western blotting. COMT mRNA and enzyme activity were assayed by quantitative reverse-transcription polymerase chain reaction (RT-PCR) and high-performance liquid chromatography analysis, respectively. The role of COMT was evaluated in stable HuLM cells by silencing COMT expression. Vitamin D inhibited the growth of HuLM cells by 47±0.03% at 1 μM and by 38±0.02% at 0.1 μM compared with control cells at 120 hours of treatment. Vitamin D inhibited extracellular signal-regulated kinase activation and down-regulated the expression of BCL-2, BCL-w, CDK1, and PCNA. Western blot, RT-PCR, and enzyme assay of COMT demonstrated inhibitory effects of vitamin D on COMT expression and enzyme activity. Silencing endogenous COMT expression abolished vitamin D-mediated inhibition of HuLM cell proliferation. Vitamin D inhibits growth of HuLM cells through the down-regulation of PCNA, CDK1, and BCL-2 and suppresses COMT expression and activity in HuLM cells. Thus, hypovitaminosis D appears to be a risk factor for uterine fibroids.

Nitrofen suppresses cell proliferation and promotes mitochondria-mediated apoptosis in type II pneumocytes

To characterize the molecular mechanisms of nitrofen-induced pulmonary hypoplasia. After administration of nitrofen to cultured type II A549 pneumocytes, cell proliferation and DNA synthesis were investigated by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetry, colony formation assay, flow cytometry and [3H]-thymidine incorporation assay. Apoptosis was measured by terminal transferase-mediated dUTP nick-end-labeling, acridine orange-ethidium bromide staining and flow cytometry. Expression of proliferating cell nuclear antigen (PCNA) and apoptosis-related genes was assayed by immunofluorescence, RT-PCR and Western blot. Nitrofen inhibited the cell proliferation of A549 cells in a dose- and time-dependent manner, accompanied by downregulation of PCNA. As a result, the DNA synthesis of nitrofentreated A549 cells decreased, while cell cycle was arrested at G0/G1 phase. Moreover, nitrofen induced apoptosis of A549 cells, which was not abolished by Z-Val-Ala- Asp(OCH3)- fluoromethylketone. In addition, nitrofen decreased the expression of Bcl-x( L), but not of Bcl-2, Bax, and Bak, resulting in a loss of mitochondrial membrane potential and the nuclear translocation of apoptosis-inducing factor (AIF). Meanwhile, nitrofen strongly activated the p38 mitogen-activated protein kinase (p38-MAPK). Pretreatment of cells with SB203580 (5 micromol/L) blocked nitrofen-induced phosphorylation of p38-MAPK and abolished nitrofen-induced AIF translocation and apoptosis in A549 cells. Nitrofen suppresses the proliferation of cultured type II pneumocytes accompanied by the downregulation of PCNA, and induces mitochondria-mediated apoptosis involving the activation of p38-MAPK.

Short-interfering RNA-mediated silencing of proliferating cell nuclear antigen inhibit proliferation and induce apoptosis in HeLa cells

Proliferating cell nuclear antigen (PCNA) is an important protein for DNA polymerase delta in the nucleus, and shown to have a fundamental role in cellular proliferation. It is overexpressed to support cell growth in cervical carcinoma. To study its role in stress response, we design and use short hairpin RNA (shRNA) to inhibit PCNA expression in HeLa cells and validate its effect on cell proliferation. In this study, three PCNA-shRNA expression vectors are constructed and introduced into HeLa cells, and the cell cycle is analyzed by flow cytometry. Apoptotic cell is detected by single cell gel electrophoresis assay (comet assay), and caspase cleavage is studied also. Expression of PCNA is assessed by real-time reverse transcription-polymerase chain reaction and Western blot analysis. Upon transient transfection with plasmid encoding shRNA, it is found that expression of PCNA decreased in shRNA-transfected cells, downregulation of PCNA inhibit cell growth and induce apoptosis in HeLa cells. PCNA downregulation also increase cell population in the G0-G1 phase. In conclusion, our findings demonstrate that shRNA can inhibit the DNA replication and induce apoptosis in HeLa cells effectively and, therefore, could be used as a new potential anticancer tool for therapy of human cervical carcinoma.

Potential Predictive Markers for Proliferative Capacity of Cultured Human Articular Chondrocytes: PCNA and p21

The purpose of this study was to investigate age-related changes in the proliferative ability of human articular chondrocytes in culture. In addition, the possible markers for the proliferative capacity of chondrocytes were examined. Chondrocytes obtained from human articular cartilages of young (under 40 years) or old (over 60 years) individuals were expanded until their growth was arrested. The number of cells and the type II collagen phenotype were determined together with the expression levels of proliferating cell nuclear antigen (PCNA) and p21(WAF1/CIP) along with the passages of cultured chondrocytes. The results showed that young chondrocytes had higher proliferative capacity and viability than old chondrocytes. The growth arrest and the cessation in the expression of type II collagen were accompanied by down-regulation of PCNA and up-regulation of p21(WAF1/CIP) levels in both young and old chondrocytes. Notably, the expression levels of PCNA and p21(WAF1/CIP) along with the passages were correlated inversely to each other and showed distinct patterns between young and old chondrocytes. These results suggest that senescence of human articular chondrocytes leads to the decrease in the proliferative capacity and phenotypic stability. In addition, PCNA and p21 could be molecular markers that represent the status of these age-related properties of human articular chondrocytes in vitro.

Human cytomegalovirus inhibits proliferation of duct epithelial cells of human salivary gland

To investigate the effects of human cytomegalovirus (HCMV) on the proliferation of duct epithelial cells of human salivary gland (HSG). The expression of proliferating cell nuclear antigen (PCNA) and p53 were studied in 11 cases of parotid cytomegalic inclusive disease (PCID) using immunohistochemical staining method. The effects of human cytomegalovirus (HCMV) on the proliferation of HSG were investigated by MTT method in vitro. The expression of PCNA in HSG infected by HCMV was examined using immunocytochemical staining and Western blotting. PCNA was expressed weakly in most of megalic inclusion cells which were positive for HCMV, while all the megalic inclusion cells were p53 negative in all 11 cases of PCID. HCMV inhibited proliferation of HSG in vitro in a time dependent and dose dependent manner. Down-regulation of PCNA was shown in infected cells. HCMV inhibits proliferation of HSG and down-regulation of PCNA may be an expression of the inhibition.