Proliferating Cell Nucleus Antigen Research Articles

Cellular behavior and extracellular matrix turnover in bovine annulus fibrosus cells under hydrostatic pressure and deviatoric strain.

Intervertebral disc herniation is a common spinal disorder that is often treated with discectomy when conservative measures fail. To devise therapeutic strategies for tears in the annulus fibrosus (AF), the regenerative capability of AF cells under spinal loading needs to be addressed. We hypothesized that the compressive loading associated with deformation in AF cells reduces synthetic and degradative activities in extracellular matrix and cell proliferation. We evaluated expression of key matrix molecules and cell proliferation by RT-PCR and immunohistochemistry by inner and outer bovine AF cells incubated under hydrostatic pressure (HP), arc-bending strain (Strain), and combined HP and Strain (HP/Strain) mimicking spinal loading. Inner AF cells showed significantly increased levels of aggrecan core protein, chondroitin sulfate N-acetylgalactosaminyltransferase-1, and tissue inhibitor of metalloproteinases-2 by 6 days under HP (p < 0.05), with a tendency toward increased matrix metalloproteinase-13. Outer AF cells demonstrated a significant decline in collagen type-2 under Strain and HP/Strain (p < 0.05) and a tendency toward suppression of collagen type-1 and elastin expression compared to HP and unloaded control. On the other hand, proliferating cell nucleus antigen increased significantly under Strain and HP/Strain in inner AF and declined under unloaded and HP in outer AF (p < 0.05). Immunohistology findings supported reductions in gene expressions of matrix molecules. Thus, changes in HP/Strain in AF appear to diminish synthetic and degradative activities while increasing cell proliferation. To promote regeneration, continuous overloading should be avoided, as it converts the synthetic activity to a state in which tissue repair is limited.

Role of putrescine in ovary and embryo development in fruit bat Cynopterus sphinx during embryonic diapause.

The aim of this study was to evaluate the effect of putrescine on ovarian activity and the rate of embryonic development in Cynopterus sphinx during delayed development. The result showed the presence of a rate-limiting enzyme, ornithine decarboxylase-1, in both ovary and utero-embryonic unit of C. sphinx suggests a synthesis of putrescine in these sites. The corpus luteum showed increased, whereas utero-embryonic unit showed decreased production of putrescine during delayed development as compared with the normal development. The bat treated in vivo with putrescine during delayed development showed increase in progesterone and estradiol synthesis, correlated with increased expression of luteinizing hormone receptor, steroidogenic acute receptor protein, and 3β-hydroxysteroid dehydrogenase through extracellular signal-regulated kinase (ERK1/2)-mediated pathway in the ovary; but showed increase in the weight and expression of progesterone receptor (PR), B-cell lymphoma 2, proliferating cell nucleus antigen, and vascular endothelial growth factor proteins in utero-embryonic unit. The in vitro treatment of putrescine showed stimulatory whereas treatment with an inhibitor of putrescine, 2-difluoromethylornithine caused an inhibitory effect on ovarian progesterone synthesis and cell proliferation, and cell survival in the utero-embryonic unit. In conclusion, the putrescine showed two separate roles during embryonic diapause, high concentration of putrescine in the ovary may support corpus luteum and basal synthesis of progesterone, whereas a low level of putrescine causes retarded embryonic development by inhibiting cell proliferation in the utero-embryonic unit. The bat treated with putrescine either directly promotes cell proliferation, cell survival, and angiogenic activities or acts indirectly increasing PR on utero-embryonic unit thereby activating development in delayed embryo in C. sphinx.

Correlation between HSD17B4 expression in rat liver cancer tissues and inflammation or proliferation.

Pathogenesis and progression of liver cancer are correlated with inflammatory response and estrogen level. 17β-estradiol dehydrogenase IV (HSD17B4) is highly expressed in human liver cancer tissues. HSD17B4 participates in liver cancer cell proliferation via suppressing estradiol (E2) activity. This study generated a rat liver cancer model, on which the correlations between HSD17B4 and tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), proliferating cell nucleus antigen (PCNA) expression were analyzed. Male Sprague Dawley (SD) rats were randomly assigned into control and model group (N=30). Diethylnitrosamine was used to induce liver cancer in a rat model. HE staining was used to observe liver injury whilst ELISA was used to measure serum TNF-α and IL-6 levels. The level of serum E2 was quantified by radioimmunoassay. Serum liver function indexes were measured by automatic biochemical analyzer. Protein expressions of HSD17B4, p-Akt, p-ERK and PCNA were measured by Western blot. The inflammatory infiltration and necrosis of hepatocytes were shown in model group by HE staining, along with aggravated liver indexes. Significantly high phosphorylation level of Akt and ERK, along with the increase of HSD17B3 and PCNA expressions, was found in model group (p<0.05 compared to control group). Serum E2 level was statistically decreased, whilst TNF-α and IL-6 were up-regulated (p<0.05). HSD17B4 was positively correlated with TNF-α, IL-6 and PCNA expressions (r=0.68, 0.62 and 0.56, p<0.05). HSD17B4 is over-expressed in rat liver cancer tissues. Its expression was positively correlated with TNF-α, IL-6 and PCNA levels, and probably participates in liver cancer cell proliferation via ERK and Akt signal pathway.

Effect of leech on VSMCs in early atherosclerosis rats via p38MAPK signaling pathway

To explore the effect of leech on proliferation and apoptosis of vascular smooth muscle cells(VSMCs) in early atherosclerosis rats via p38MAPK signaling pathway and investigate its possible mechanism. Biochemical analyzer was used to examine the regulation of leech on levels of triglycerides(TG), total cholesterol(TC), low-density lipoprotein(LDL-C), and high-density lipoprotein(HDL-C) in blood lipid of rats. The expression of transforming growth factor-beta 1(TGF-β1) in serum was detected by ELISA. Immunological histological chemistry (IHC) was taken to measure the expression levels of proliferating cell nucleus antigen(PCNA) and cell apoptosis proteinase-3(Caspase-3), while the protein expression levels of MKK3, p38 and C-myc were detected by Western blot. In addition, hematoxylin and eosin(HE) staining was used to observe the morphological change of thoracic aortas. The results showed that leech decreased the levels of TC, LDL-C obviously and increased HDL-C, suppressed the expression levels of TGF-β1 and PCNA, up-regulated Caspase-3, down-regulated the expression levels of MKK3, p38, and C-myc protein. HE staining indicated that it could inhibit intimal thickening and reduce plaque formation. The above results indicated that leech may affect the protein expression of the p38MAPK signaling pathway to inhibit proliferation and promote the apoptosis of VSMCs via reducing blood lipid levels and suppressing TGF-β1, aiming at inhibiting intimal thickening and reducing plaque formation, tand then slowing down the process of early atherosclerosis.

Suture cartilage formation pattern varies with different expansive forces

Midpalatal suture expansion could induce osteogenesis to correct maxillary insufficiency; cartilage formation could also be induced, and lower-magnitude forces might generate a preferable response pattern. In this study, we aimed for an enhanced understanding of the cartilage formatting effects of expansion. Thirty 6-week-old male C57BL/6 mice were randomly and evenly assigned to 3 groups; the animals in each group received a sustained suture expansion at 0, 10, and 20g, respectively. Ten additional mice were fed the same as the baseline controls and received no expansion. After 7days, the animals were killed; coronal paraffin sections were stained using toluidine blue and safranin-O. The proliferating cell nucleus antigen,the nuclear antigen Ki-67, alkaline phosphatase, and matrix metalloproteinase 13 expressions were visualized with immunohistochemistry. All data were analyzed statistically, and the differences were considered significant at P<0.05. Compared with the control, the cartilage matrix volume was significantly increased by the 20-g expansion, showing increased cartilage matrix and hypertrophic chondrocytes with the highest matrix metalloproteinase 13 expression. The 10-g expansion formed condensed proliferating chondrocyte masses, within which the highest percentages of proliferating cell nucleus antigen and Ki-67 positive cells were present. The 10-g and the 20-g expansions equally intensified the alkaline phosphatase expression. The lower expansion (10g) promoted chondrocyte proliferation and induced a more preferable suture cartilage response pattern compared with the higher expansion (20g), which just increased the cartilage matrix production.

Abstract LB-178: A novel chemopreventive molecular mechanism of selenium: p53-mediated DNA repair

Abstract Selenium is an essential trace element which has been considered as chemopreventive agent against cancer, due to its antioxidant activity. Among several selenium compounds, selenomethionine (SeMet) has significant protective effect toward various genotoxic stresses via stimulating DNA repair process. However, its mechanism is still not well elucidated. In this study, we examined the mechanism of SeMet in terms of p53-mediated base excision repair (BER). Our data showed that the amount of DNA damage was rapidly decreased in the presence of SeMet when methyl methanesulfonate (MMS), a BER-inducing agent, was treated in the cells. In addition, the removal of apurinic/apyrimidinic sites was significantly enhanced in p53 wild type cells in response to SeMet. Furthermore, we observed the Gadd45a, known to involve BER as one of the p53 downstream genes, was accumulated by SeMet in p53 wild-type RKO cells. Indeed the interaction of BER-mediated repair proteins including PCNA (proliferating cell nucleus antigen) and APE/Ref-1 with Gadd45a was notably decreased by SeMet in p53 siRNA-treated cells. In in vivo study, the frequency and size of polyp in AOM/DSS model as an animal model for colitis-related carcinogenesis was decreased in response to SeMet. Moreover, proteins related with DNA repair including Trx (thioredoxin), p53 and Gadd45a were increased in SeMet treated AOM/DSS model. Those results suggested that BER activity might be dependent on wild-type p53 under the modulation of protein complexes with Gadd45a and repair protein including PCNA and APE/Ref-1 as a distinct chemopreventive mechanism of SeMet. Our study might provide an important evidence to develop the chemopreventive strategies against various oxidative stresses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-178. doi:1538-7445.AM2012-LB-178

Abstract B73: A novel chemopreventive molecular mechanism of selenium: p53-mediated DNA repair

Abstract Selenium is an essential trace element which has been considered as chemopreventive agent against cancer, due to its antioxidant activity. Among several selenium compounds, selenomethionine (SeMet) has significant protective effect toward various genotoxic stresses via stimulating DNA repair process. However, its mechanism is still not well elucidated. In this study, we examined the mechanism of SeMet in terms of p53-mediated base excision repair (BER). Our data showed that the amount of DNA damage was rapidly decreased in the presence of SeMet when methyl methanesulfonate (MMS), a BER-inducing agent, was treated in the cells. In addition, the removal of apurinic/apyrimidinic sites was significantly enhanced in p53-wild type cells in response to SeMet. Furthermore, we observed the Gadd45a, known to involve BER as one of the p53 downstream genes, was accumulated by SeMet in p53-wild type RKO cells. Indeed the interaction of BER-mediated repair proteins including PCNA (proliferating cell nucleus antigen) and APE/Ref-1 with Gadd45a was notably decreased by SeMet in p53 siRNA-treated cells. In in vivo study, the frequency and size of polyp in AOM/DSS model as an animal model for colitis-related carcinogenesis was decreased in response to SeMet. Moreover, proteins related with DNA repair including Trx (thioredoxin), p53 and Gadd45a were increased in SeMet treated AOM/DSS model. Those results suggested that BER activity might be dependent on wild type p53 under the modulation of protein complexes with Gadd45a and repair protein including PCNA and APE/Ref-1 as a distinct chemopreventive mechanism of SeMet. Our study might provide an important evidence to develop the chemopreventive strategies against various oxidative stresses. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B73.

Pseudoceramide-Containing Physiological Lipid Mixture Reduces Adverse Effects of Topical Steroids

PurposeVarious therapeutic approaches have been suggested for preventing or reducing the adverse effects of topical glucocorticoids, including skin barrier impairment. Previously, we have shown that impairment of skin barrier function by the highest potency topical glucocorticoid, clobetasol 17-propinate (CP), can be partially prevented by co-application of a physiological lipid mixture containing pseudoceramide, free fatty acids, and cholesterol (multi-lamellar emulsion [MLE]). Skin atrophic effects of CP were also partially reduced by MLE. In this study, the preventive effects of MLE on the lowest potency topical glucocorticoid, hydrocortisone (HC), were investigated using animal models.MethodsAnti-inflammatory activity of topical HC was evaluated using a 12-O-tetradecanoylphobol-13-acetate-induced skin edema model. Topical steroid induced adverse effects were evaluated using hairless mouse.ResultsThe results showed that the anti-inflammatory activity was not altered by co-application of either MLE or hydrobase. However, co-application of MLE and 1.0% HC showed less impairment in the epidermal permeability barrier function, skin hydration, and skin surface pH compared with hydrobase. Stratum corneum integrity, evaluated by measuring trans-epidermal water loss after repeated tape stripping, showed less damage with MLE co-application. Long-term application of topical HC induced skin atrophy, measured by a reduction in skinfold and epidermal thickness and in the number of epidermal proliferating cell nucleus antigen (PCNA)-positive keratinocytes. Co-application of MLE did not affect the skinfold or epidermal thickness, but the number of PCNA-positive keratinocytes was less decreased with MLE use.ConclusionsThese results suggest that co-application of MLE is effective in reducing the local adverse effects of low-potency topical glucocorticoids and supports the therapeutic efficacy of physiological lipid mixtures on skin barrier function.

Shenmai Injection (参麦注射液) inhibiting the extracellular signal regulated kinase-induced human airway smooth muscle proliferation in asthma

To investigate the relationship between the proliferation of sensitized human airway smooth muscle cells (HASMCs) and the expression of extracellular signal regulated kinase (ERK) and the effect of Shenmai Injection (SMI) on HASMCs. The HASMCs cultured in vitro were divided into three groups: (1) control group; (2) sensitized group: containing 10% asthmatic serum; (3) SMI group: further divided into three different concentration subgroups interferred with 10 microL/mL, 50 microL/mL, and 100 microL/mL SMI, respectively. The proliferation of HASMCs was detected using MTT method, the expression of proliferating cell nucleus antigen (PCNA) in HASMCs was detected using immunocytochemical staining, and the expression of phosphoration-ERK1/2 (p-ERK1/2) protein was detected using Western-blot. After passive sensitization,: the optical density value (A A(490) value) of HASMCs was significantly increased from 0.366+/-0.086 to 0.839+/- 0.168 (P<0.05). In addition, the expression of PCNA was significantly increased from 28.7%+/-5.9% in the control group to 69.8%+/-7.5% in the sensitized group (P<0.05). At the same time, the expression of p-ERK1/2 in passively sensitized HASMCs was significantly increased compared with the control group (all P<0.05). After application of 10 microL/mL, 50 microL/mL, and 100 microL/mL SMI to the cultured media of passively sensitized group, the A(570) value was significantly decreased from 0.839+/-0.168 to 0.612+/-0.100, 0.412+/-0.092, and 0.339+/-0.077, respectively (P<0.05). Moreover, the expression of PCNA was significantly decreased from 69.8%+/-7.5% to 57.8%+/-6.2%, 40.7%+/-5.4%, and 26.1%+/-5.2%, respectively. At the same time, the expression of p-ERK1/2 in each SMI group was significantly decreased compared with the sensitized group (all P<0.05). ERK signal transduction pathway may be involved in the airway remodeling in asthma. The expression of ERK can be inhibited by SMI in a dose-dependent manner, thus preventing the proliferation of HASMCs.

Crystal Structure of the Human Rad9–Hus1–Rad1 Clamp

Three evolutionarily conserved proteins, Rad9, Hus1, and Rad1, form a heterotrimeric 9–1–1 complex that plays critical roles in cellular responses to DNA damage by activating checkpoints and by recruiting DNA repair enzymes to DNA lesions. We have determined the crystal structure of the human Rad9 (residues 1–272)–Hus1–Rad1 complex at 2.5 Å resolution. The 9 1–272–1–1 complex forms a closed ring, with each subunit having a similar structure. Despite its high level of similarity to proliferating cell nucleus antigen in terms of overall structure, the 9 1–272–1–1 complex exhibits notable differences in local structures, including interdomain connecting loops, H2 and H3 helices, and loops in the vicinity of the helices of each subunit. These local structural variations provide several unique features to the 9–1–1 heterotrimeric complex—including structures of intermolecular interfaces and the inner surface around the central hole, and different electrostatic potentials at and near the interdomain connecting loops of each 9–1–1 subunit—compared to the proliferating cell nucleus antigen trimer. We propose that these structural features allow the 9–1–1 complex to bind to a damaged DNA during checkpoint control and to serve as a platform for base excision repair. We also show that the 9 1–272–1–1 complex, but not the full-length 9–1–1 complex, forms a stable complex with the 5′ recessed DNA, suggesting that the C-terminal tail of Rad9 is involved in the regulation of the 9–1–1 complex in DNA binding.

Effects of calcium-activated chloride channels on proliferation of pulmonary artery smooth muscle cells in rats under chronic hypoxic condition

Objective To investigate the effects of calcium-activated chloride (ClCa) channels on proliferation of pulmonary artery smooth muscle cells(PASMCs) in rats under chronic hypoxic condition. Methods The cultured PASMCs were placed under normoxic and chronic hypoxic conditions:The cells were observed by light and electron microscope; The cell cycles were observed by flow-cytometry; Immunocytochemistry staining was used to detect the expressions of PCNA, c-fos and c-jun of PASMCs; Cytoplasmic free Ca 2+ concentration ([Ca 2+] i) in PASMCs was investigated by fluorescent quantitation using fluorospectrophotometer. Results The PASMCs were contractile phenotype under normoxic conditions. Observation by transmission electron microscope: In kytoplasm of contractile phenotype cells, myofilament bundles were abundant and the content of cell organs such as Golgi's bodies were rare. The PASMCs were synthetic phenotype under chronic hypoxic condition. There were increased free ribosomes, dilated rough endoplasmic reticulums, highly developed Golgi complexes, decreased or disappeared thick filaments and dense body in kytoplasm of synthetic phenotype cells. After NFA and IAA-94, the situations were reversed The number of S+G 2M PASMCs were significantly increased in chronic hypoxic condition; The NFA and IAA-94 were shown to significantly decrease them from (28.6 ± 1.0)% to (16.0 ± 1.6)% and the number of G 0G 1 PASMCs significantly increased from (71.4 ± 1.9)% to (83.9 ± 1.6)% ( P < 0.01). In chronic hypoxic conditions, the expression of proliferating cell nucleus antigen was significantly increased; The NFA and IAA-94 were shown to significantly decrease it from (81 ± 6)% to (27 ± 7)%( P < 0.01). The expression of c-fos and c-jun were significantly increased in chronic hypoxic conditions; The NFA and IAA-94 were shown to significantly decrease them from 0.15 ± 0.02, 0.32 ± 0.05 to 0.05 ± 0.01, 0.12 ± 0.05, respectively ( P < 0.01); Under chronic hypoxic conditions, [Ca 2+] i was increased; The NFA and IAA-94 decreased it from (281.8 ± 16.5)nmol/L to (117.7±15.4)nmol/L( P < 0.01). Conclusion Hypoxia initiated the change of PASMCs from contractile to synthetic phenotype and increased proliferation of PASMCs. NFA and IAA-94 depressed cell proliferation by blocking ClCa channels in hypoxic condition. These may play an important role in proliferation of PASMCs under chronic hypoxic conditions.

Experimental Study on the Estrogen-Like Effect of Boric Acid

There are now considerable evidences that boric acid has reproductive and developmental toxicity, but it is uncertain whether such toxicity is caused by estrogen-like effect. Our objective is to determine the estrogen-like effect of boric acid. Proliferation assay of MCF-7 human breast cancer cells, uterotrophic assay, measure assay of the estradiol (E2), proliferation assay of mucous membrane cells, and assay of estrogen receptor were conducted in this study. Boric acid could increase the weight of uterus of ovariectomized SD rats and the height of epithelium cells of mucous membrane, enhance the expression of the proliferating cell nucleus antigen, and reduce the density of estrogen receptors. However, boric acid could not affect the level of estradiol in serum and stimulate the proliferation of MCF-7 human breast cancer cells. In this study, boric acid exhibited the estrogen-like effect in vivo.

Treatment results of oral verrucous carcinoma and its biological behavior

The biologic behavior of and optimal treatment for oral verrucous carcinoma (VC) remain controversial. We analyzed the clinicopathological characteristics of 12 patients with oral VC. Immunohistochemical techniques were used to evaluate p53 protein, CD44 variant 9, and proliferating cell nucleus antigen. The TNM classification (UICC, 1997) was T1 in 1 patient, T2 in 3, T3 in 4, and T4 in 4. All patients were classified as N0M0. Four patients were treated by surgery alone and 8 by surgery after chemotherapy, radiotherapy, or both. After surgery, two patients had primary recurrence of disease. Immunohistochemically, the proliferative activity of tumor cells as evaluated by proliferating cell nuclear antigen labeling index and p53 protein expression was similar in VC and well-differentiated squamous cell carcinoma. However, CD44 varient 9 expression was positive in 8 of 10 VC, suggesting that oral VC is associated with a low risk of lymph node metastasis. Positive CD44 variant 9 expression by most oral VCs, indicating a low risk of cervical lymph node metastasis, suggests that most cases can be controlled by surgical intervention.

Induced epidermal cell turnover in the seawater‐adapted guppy,poecilia reticulata

The turnover of epidermal cells after seawater adaptation of the freshwater fish was studied in the guppy (Poecilia reticulata) by means of proliferating cell nucleus antigen (PCNA) immunocytochemistry and transmission electron microscopy. The number of PCNA‐immunoreactiye cells in the epidermis of the seawater‐adapted guppies, which becomes thinner than that in the freshwater, generally increases four times as much. Degeneration of filament‐containing cells by necrosis or apoptosis occurs mainly in epidermal cells. Apoptotic filament‐containing cells seem to be shed into the water in the environment instead of phagocytosis by adjacent macrophages. The apoptotic chloride cell has a highly condensed cytoplasm and the lumen of tubular system is distended. The apoptotic mucous cell, which has an electron‐dense cytoplasm, is characterized by the presence of a large multivesicular body of different electron densities. Macrophages contain many electron‐dense lysosomal bodies and large vesicles filled with cellu...