Expression Levels Of Proliferating Cell Nuclear Antigen Research Articles

Abstract 2956: Prognostic value of PCNA expression in salivary gland cancer in consideration of different histological subtypes

Abstract Background: The expression of the cell proliferation-related marker Proliferating Cell Nuclear Antigen (PCNA) has shown to be a significant prognostic indicator in various human cancers. Its role in malignant tumors of the major salivary glands remains unclear. Methods: We analysed the expression of PCNA in 159 patients with parotid gland cancer (12 different histological subtypes) by means of immunohistochemistry. Patients were divided into two groups due to the median PCNA expression level. The experimental findings were correlated with clinicopathological and survival parameters. The mean follow-up time was 56.6 months. Results: Cytoplasmic expression of PCNA was found in 89% of cases. A high cytoplasmic expression of PCNA was found in 47% of the examined tumors. High cytoplasmic PCNA expression was an independent prognostic indicator for a significant poorer 5-year disease-free survival rate (50.6% versus 73.8%, p=0.036) as well as overall survival rate (57.0% versus 78.8%, p=0.006). Conclusion: The correlation between cytoplasmic PCNA expression and survival probabilities of salivary gland cancer might make this an effective tool in patient follow-up, prognosis and therapy in future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2956. doi:10.1158/1538-7445.AM2011-2956

Comparative proteomic analysis of anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-transformed and normal human bronchial epithelial G0/G1 cells

In the present study, we investigated the proteomic profiling of anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (anti-BPDE)-transformed human bronchial epithelial cell line (16HBE-C) and its parental cell line (16HBE) G0/G1 cells. Differential analysis of proteomic profiling indicated that 67 polypeptides were down-regulated and 77 polypeptides were up-regulated in 16HBE-C G0/G1 cells compared to 16HBE G0/G1 cells. Then 16 differentially expressed protein spots were analyzed with Q-TOF MS/MS. Of these spots, 3 down-regulated polypeptides were identified as sorcin, small ubiquitin-related modifier 2 precursor and eukaryotic translation initiation factor 5A-1, and 9 up-regulated polypeptides were identified as calmodulin, myosin light polypeptide 6, eukaryotic translation initiation factor 6, proliferating cell nuclear antigen (PCNA), tumor protein D52 (TPD52), superoxide dismutase [Cu-Zn], prohibitin, nuclear protein Hcc-1 and vimentin. These proteins are involved in cell proliferation, protein synthesis, signal transduction and carcinogenesis. Western blotting analysis verified the increased expression levels of PCNA and TPD52 in 16HBE-C G0/G1 cells. Based on the clues from proteomic analysis, the migration and invasion capabilities of 16HBE-C and 16HBE cells were tested. The results indicated that 16HBE-C cells showed much higher migration and invasion capabilities than 16HBE cells, and moreover, the suppression of TPD52 by RNAi resulted in significant decrease of migration and invasion capabilities of 16HBE-C cells. These results will be valuable for further investigating and understanding the mechanisms underlying BaP-induced carcinogenesis.

Preventive effect of JTE-522, a selective cyclooxygenase-2 inhibitor, on DEN-induced hepatocarcinogenesis in rats

Background Chemopreventive effect of a selective cyclooxygenase-2 (COX-2) inhibitor JTE-522 on diethylnitrosamine (DEN)-induced hepatocarcinogenesis was evaluated in Wistar rats. Methods Animals in the control group (G1) were injected with phosphate buffered saline (PBS), those in hepatocellular carcinoma (HCC) group (G2) were injected with DEN with regular foods for 14 weeks, and those in the treatment groups were injected with DEN for 14 weeks fed with JTE-522 for 7 (G3) and 14 weeks (G4), respectively. Proliferation and precancerous lesions were evaluated by expression levels of proliferating cell nuclear antigen (PCNA) and glutathione S-transferase-P (GST-P), respectively by immunohistochemistry and Western blot analysis. Apoptosis and oxidative stress were evaluated by TdT-mediated dUTP-biotin nick-end labeling (TUNEL) and 8-hydroxy-2’-deoxyguanosine (8-OHdG) staining, respectively. Results After 14 weeks of the treatment, HCC was developed in G2, G3, and G4 showing no significant differences in gross appearance and histology of the liver among the three groups. There were no significant differences in the expression levels of PCNA and numbers of TUNEL and 8-OHdG positive cells in the liver among the three groups. However, GST-P positive area was significantly suppressed in G3 and G4 compared to G2. Conclusion Our data revealed that JTE-522 had a modest inhibitory effect on hepatocarcinogenesis in rats in a manner independent of induction of apoptosis and inhibition of oxidative stress.

Analysis of risk factors for recurrence after the resection of sacral chordoma combined with embolization

Background context Although several authors have already reported on the high local recurrence rate of sacral chordomas after surgical resection, there are no reports on the risk factors for recurrence after resection when combined with preoperative tumor-related blood vessel embolism by digital subtraction angiography (DSA) technique. Purpose To investigate the factors related to the continuous disease-free survival time (CDFS) after the resection of sacral chordomas combined with embolization. Study design/setting Retrospective review of the signs, images, and immunohistochemical data of patients with sacral chordomas treated with an initial operation combined with transcatheter arterial embolization. Patient sample Twenty-two patients with sacral chordomas received initial resection combined with transcatheter arterial embolization. Outcome measures Recurrence, proliferating cell nuclear antigen (PCNA) expression, basic fibroblast growth factor (bFGF) expression, CDFS. Methods All cases were selected and followed for an average of 39.2 months. The roles of gender, age, tumor size, tumor location, surgical method, radiation therapy, PCNA expression, and bFGF expression in local recurrence were analyzed using the log-rank test. Results Sacral chordomas recurred in eight of 22 cases. The CDFS was significantly greater in tumors located below S3 as compared with those above S3. When evaluating PCNA and bFGF expression levels, the CDFS was greater in low expressions rather than high expressions. It was determined that the surgical method used was of prognostic significance to the CDFS. Conclusions Higher tumor location and higher expressions of PCNA and bFGF will lead to a shorter CDFS. Resecting the tumor as completely as possible will decrease the chances of local recurrence of sacral chordomas.

Effects of mesenchymal stem cell transplantation on growth of liver cancer: experiment with rats

To evaluate the effects of mesenchymal stem cell (MSC) transplantation on the growth of liver cancer. MSCs were isolated from the bone marrows of SD rats. Walker-256 cancer cells were isolated from the cancerous ascites of rat and cultured. Forty-five SD rats were randomly divided into 3 equal groups: mixed transplantation group undergoing laparotomy and transplantation of cancer cells mixed with MSCs into the liver, MSC IV transplantation group undergoing injection of MSCs into the caudal vein, and control group undergoing only MSC transplantation into the liver. MR imaging was performed s at days 3, 6, 9 and 12 after modeling to measure the maximum cross section area of the tumor. At day 12 the rats were killed after MR imaging with their livers taken out to undergo HE staining and pathological examination. Immunohistochemistry was used to detect the expression of vascular endothelial cell growth factors (VEGF), nm23 gene, a tumor metastasis inhibiting gene, and proliferating cell nuclear antigen (PCNA), a nuclear polypeptide necessary in the DNA synthesis. No significant evidence of tumor formation was detected by MRI at days 3 and 6 after modeling in all rats and tumor nodules were observed since day 9. The maximum cross section areas of tumor of the mixed transplantation group and MSC IV transplantation group were significantly larger than that of the control group at days 9 and 12 (F = 4.21, P < 0.05; F = 8.52, P < 0.01). Immunohistochemistry showed that VEGF expression levels of the two study groups were both significantly higher than that of the control group (F = 9.58, P < 0.01), while the nm23 gene expression levels of the 2 study groups were both significantly lower than that of the control group (F = 4.61, P < 0.05). The PCNA expression level of the mixed transplantation group was significantly higher than that of the control group (d'((1, 0.05)) = 0.34, d'((1, 0.01)) = 0.63, P < 0.05), however, there was no significant difference in the PCNA expression level between the MSCs IV transplantation group and the control group (d'((1, 0.05)) = 0.32, d'((1, 0.01)) = 0.48, P > 0.05). There was no significant difference in the tumor apoptotic index between the 2 study groups and the control group (F = 1.25, P > 0.05). MSC transplantation increases the expression of VEGF and PCNA, while decreases the expression of nm23 gene in cancer cells, thus favoring the tumor growth.

Cloning of proliferating cell nuclear antigen gene from the dinoflagellate Prorocentrum donghaiense and monitoring its expression profiles by real-time RT-PCR

The growth rate of marine phytoplankton at the species level is an important parameter for estimating a phytoplankton population in the field. Therefore, monitoring this rate may have potential to be useful for providing early warnings for red tide blooms. We investigated the proliferating cell nuclear antigen (PCNA) gene of the red tide species Prorocentrumdonghaiense Lu et Goebel to study the relationship between its expression level and the growth rate. The gene was isolated and the full cDNA sequence encoding PCNA was obtained. It consisted of 1,057 base pairs (bp), comprising 81 bp of the 5′ untranslated region (UTR) and 196 bp of the 3′ UTR, and had an open reading frame (ORF) of 780 bp, encoding 259 amino acid residues. The coding region was highly conserved compared with other organisms PCNA gene. Using real-time PCR, we found that the average of PCNA transcript, normalized to per cell basis, decreased obviously from 26.12 ± 3.04 copies in the exponential and transition phase to 5.95 ± 0.79 copies in the stationary phase. However, in the decline phase, the copies of PCNA transcription per cell is below one copy. Furthermore, the expression level of PCNA changed consistently with the growth rate as assessed by the relative quantitation method. These results indicate that PCNA has a potential as a molecular marker of growth phase and rate for P. donghaiense. This may help in forecasting blooms, allowing for effective control measures to be implemented in a timely fashion.

Maternal administration of betamethasone inhibits proliferation induced by fetal tracheal occlusion in the nitrofen rat model for congenital diaphragmatic hernia: a placebo-controlled study

Fetal tracheal occlusion (TO) is offered to fetuses with severe pulmonary hypoplasia due to congenital diaphragmatic hernia (CDH). TO induces lung growth, but even when performed minimally invasive, there is a risk for iatrogenic preterm delivery. Whenever this is anticipated, maternal glucocorticoids (GC) may be given to enhance lung maturation. The pulmonary effects of GC in fetuses with CDH that underwent TO are yet poorly defined. Therefore, we conducted a placebo-controlled study in the nitrofen (NF) rat model for CDH. Pregnant rats were gavage fed NF or olive oil (OO) on ED9.5. At ED19.0, fetuses were either assigned to TO or left untouched. Maternal betamethasone (BM) or saline (PLAC) was administered on ED20. Necropsy was done on ED21.5 to obtain lung-to-body-weight ratio (LBWR), and perform quantitative RT-PCR and fluorescent immunostaining for Ki-67 and proliferating cell nuclear antigen (PCNA) in fetal lungs. CDH fetuses had a lower LBWR than normal fetuses, but comparable pulmonary PCNA and Ki-67 expression levels. TO increased LBWR, irrespective of maternal BM or PLAC. However, BM but not PLAC inhibited proliferation in TO and unoperated fetuses. Rats with NF-induced CDH have hypoplastic lungs with normal proliferation indices. TO triggers proliferation, an effect countered by BM.

Combating endometriosis by blocking proteasome and nuclear factor- B pathways

The objective of this study is to investigate the effect of pyrrolidine dithiocarbamate [PDTC; a nuclear factor-kappaB (NF-kappaB) inhibitor] and bortezomib (Velcade; a proteasome inhibitor) on the development of experimental endometriotic implants in rats. Endometriosis was surgically induced in 30 rats using the method of Vernon and Wilson. Three weeks later the viability and volume of the implants were recorded and classified. Afterwards, rats were put into three groups with equal numbers. The groups were labelled as the control, the PDTC and the bortezomib groups. Seven days after treatment, a third laparotomy was done and the volume of implants was measured again. The animals were then sacrificed, and the implants were stained with Ki67, proliferating cell nuclear antigen (PCNA), CD34, CD31 and Masson's trichrome histochemical staining. In 80% of the implanted rats, vesicles at the suture region were observed, and the rats graded according to average vesicle diameter (D) as: Grade 1 (no vesicle, 20% of rats), Grade 2 (D < 2 mm, 33.3% of rats), Grade 3 (2 mm<D > 4.5 mm, 26.7% of rats) and Grade 4 (D > 4.5 mm, 20% of rats). After treatment with PDTC or bortezomib, these percentages were decreased for Grades 3 and 4, and increased in Grade 1. The post-treatment implant volumes were decreased in the PDTC and bortezomib groups (P < 0.002 and P < 0.001), and slightly increased in the control group (P = 0.279). In the PDTC and bortezomib groups, CD34, CD31, PCNA and Ki67 expression levels were similar but were significantly reduced compared with the control group. PDTC and bortezomib may represent a novel therapeutic strategy for treatment of endometriosis.

Differential response of human fetal smooth muscle cells from arterial duct to retinoid acid

The aim of the present study was to understand the role of retinoic acid (RA) in the development of isolated patent ductus arteriosus and the features of arterial duct-derived vascular smooth muscle cells (VSMC). The VSMC were isolated, and the biological characteristics and the response to RA were investigated in the arterial duct, aorta, and pulmonary artery VSMC from 6 human embryonic samples. Western blotting, immunostaining, and cell-based ELISA were employed to analyze the proliferation regulation of VSMC. The VSMC from the arterial duct expressed proliferating cell nuclear antigen (PCNA) at a significantly lower rate than those from the aorta and pulmonary artery, but expressed a higher level of Bax and Bcl-2. The expression level of PCNA or Bcl-2 was associated with the embryonic age. The effects of RA on the VSMC from the arterial duct were quite different from those from the aorta and pulmonary artery. In arterial duct VSMC, RA stimulated PCNA expression, but such stimulation could be suppressed by CD2366, an antagonist of nuclear retinoid receptor activation. In aorta or pulmonary artery VSMC, the expression response of PCNA to RA was insignificant. The ratio of Bax/Bcl-2 decreased in arterial duct VSMC after RA treatment due to the significant inhibition of Bax expression. The VSMC from the arterial duct possessed distinct biological behaviors. RA might be important in the development of ductus arteriosus VSMC.

The role of histamine in human mammary carcinogenesis: H3 and H4 receptors as potential therapeutic targets for breast cancer treatment.

There is increasing evidence that describes a histamine role in normal and cancer cell proliferation. To better understand the importance of histamine in breast cancer development, the expression of histamine H3 (H3R) and H4 (H4R) receptors and their association with proliferating cell nuclear antigen (PCNA), histidine decarboxylase (HDC) and histamine content were explored in mammary biopsies. Additionally, we investigated whether H3R and H4R were implicated in the biological responses triggered by histamine in MDA-MB-231 breast cancer cells. The expression levels of H3R, H4R, PCNA, HDC and histamine content were determined by immunohistochemistry in 40 benign and malignant lesions. MDA-MB-231 cells proliferation (clonogenic assay and BrdU incorporation) and cell cycle distribution (flow cytometry) were evaluated upon treatment with histamine, H3R and H4R agonists and antagonists. Apoptosis was determined by Annexin staining and TUNEL assay. Cell migration was assessed by transwell system. Results indicate that H3R was detected in 67% (10/15) of benign lesions and in almost all carcinomas (24/25), being the level of its expression significantly higher in carcinomas (P=0.0016). The non-tumoral breast tissue surrounding carcinomas revealed a lower H3R expression compared to the tumor cells. Only 13% (2/15) of the benign lesions expressed H4R compared to 44% (11/25) of the carcinomas. Interestingly, H3R expression was correlated in carcinomas with the expression of HDC and PCNA (P

Epidermal growth factor receptor and proliferating cell nuclear antigen in astrocytomas

The involvement of various growth factors, growth factor receptors and proliferative markers in the molecular pathogenesis of astrocytic neoplasms are being studied extensively. Epidermal Growth Factor Receptor (EGFR) gene overexpression occurs in nearly 50% of cases of glioblastoma. Since EGFR and proliferating cell nuclear antigen (PCNA) are involved in mitogenic signal transduction and cellular proliferation pathway, we have studied the correlation between the expression of EGFR and PCNA labeling index in astrocytic tumors. We investigated the immunohistochemical expression of EGFR and PCNA using the appropriate monoclonal antibodies in 40 cases of astrocytic tumors of which 21 cases were glioblastoma, eight cases were Grade III or anaplastic astrocytomas and six cases were Grade II or diffuse astrocytomas and five cases were Grade I or pilocytic astrocytomas. Both the EGFR expression and PCNA labeling index increase with increasing grades of astrocytomas with a significantly high percentage of cells showing positive staining for both EGFR and PCNA in GBM and Grade III astrocytomas compared to Grade II astrocytomas. The expression levels of both EGFR and PCNA were low in Grade I or pilocytic astrocytomas. A significant correlation was found between EGFR overexpression and PCNA labeling index in Grade III and Grade II astrocytomas and glioblastoma. These suggest that the tumor proliferation, at least in higher grades of astrocytomas is dependent in some measure on EGF and EGFR-related signaling pathways.

Tumor doubling time and local immune response to hepatic metastases from colorectal cancer

A number of studies have investigated the role of tumor-infiltrating lymphocytes in cancer, yet the local immune response to hepatic colorectal cancer metastasis remains unclear. As the tumor doubling time (DT) of hepatic colorectal cancer metastases is a good index of tumor growth, we examined the correlation between tumor DT and the local immune response by phenotype in hepatic colorectal cancer metastases. Tumor DT and local immune response were examined in 20 patients with hepatic colorectal cancer metastases by analyzing tumor-infiltrating lymphocytes using flow cytometry or immunohistochemical studies. Tumor proliferative activity was also investigated by determining the expression levels of Ki-67 and proliferating cell nuclear antigen (PCNA). Locally abundant populations of CD83(+) dendritic cells (DCs) and CD8(+) T cells were positively related to longer tumor DT (P < 0.05), as were abundant CD8(+) T cells having interferon-gamma-producing potentials (P < 0.05). There was no significant correlation between tumor cell expression levels of Ki-67 or PCNA and tumor DT. Longer DT tumors have increased local populations of CD8(+) T cells and CD83(+) DCs even in hepatic colorectal cancer metastases.

Sequential assessment of the intrahepatic expression of epidermal growth factor and transforming growth factor-β1 in hepatofibrogenesis of a rat cirrhosis model

Responses of the liver to chronic injury include inflammation, regeneration and fibrosis, which finally lead to cirrhosis. The cause of liver cirrhosis appears to be impaired proliferative capability of hepatocytes caused by continuous hepatic damage, and subsequent accumulation of extracellular matrix produced by hepatic stellate cells (HSCs). Epidermal growth factor (EGF) and transforming growth factor-beta1 (TGF-beta1) play a crucial role in hepatocyte proliferation and hepatofibrogenesis, respectively. However, sequential analyses of the intrahepatic expression of EGF and TGF-beta1 in the course of cirrhosis development have not been examined fully. In the present study, liver cirrhosis was produced in rats by intraperitoneal administration of dimethylnitrosamine (DMN), and intrahepatic mRNA expression levels of proliferating cell nuclear antigen (PCNA), EGF and TGF-beta1 were quantitatively estimated by a real-time reverse transcription-polymerase chain reaction method. Histological and semiquantitative densitometric examination of liver sections revealed that the accumulation of extracellular matrix components was increased according to the period of DMN treatment. Histological examination of liver sections of rats treated with DMN for 4 and 6 weeks revealed pre-cirrhosis and cirrhosis, respectively. Intrahepatic mRNA expression levels of PCNA and EGF correlated well. Expression levels of both molecules were increased significantly during the course of cirrhosis development, but decreased significantly at the time of complete cirrhosis manifestation. In contrast, intrahepatic TGF-beta1 expression was increased significantly according to the period of DMN treatment, and reached a peak at the time of cirrhosis manifestation. These results suggest that proliferative capability of hepatocytes was impaired by continuous liver damage due, in part, to the decrease of a hepatocyte mitogen EGF, and that increased intrahepatic TGF-beta1 activated HSCs to retrieve space lost by hepatocyte destruction, resulting in complete cirrhosis manifestation.

Expression of Glucose Transporter-1, Hexokinase-II, Proliferating Cell Nuclear Antigen and Survival of Patients with Pancreatic Cancer

Objectives: 18F-fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET) has been shown to be useful in diagnosis and staging of pancreatic cancer. However, the prognostic value of FDG-PET remains controversial. The aim of this study was to evaluate relations between the factors suggested to be related to the FDG accumulation in tumor tissue, such as glucose transporter-1 (GLUT-1), hexokinase type-II (HK-II), proliferating cell nuclear antigen (PCNA), and survival of pancreatic cancer patients. Methods: Histological specimen of pancreatic cancer obtained from seventy-four consecutive patients were evaluated for the expression of GLUT-1, HK-II, and PCNA by visual analysis of immunohistochemical staining of paraffin sections from the tumor specimens using anti-GLUT-1, anti-HK-II, and anti-PCNA antibody, respectively. The percentages of cells strongly expressing GLUT-1, HK-II and PCNA were scored on a 5-point scale (1 = 0–20 percent, 2 = 20–40 percent, 3 = 40–60 percent, 4 = 60–80 percent, 5 = 80–100 percent). After initial treatment, each patient was followed-up and survival time was recorded. Median survival curves of the patients with different levels of GLUT-1, HK-II, and PCNA expression were evaluated using the Kaplan-Meier method. Statistical significance of the differences in survival was calculated with the log rank test. Results: Median survival of examined patients showed no relation with the levels of GLUT-1 expression, while patients with low expression of HK-II (HK-II index < 3) had significantly shorter survival than those with higher expression of HK-II (HK-II index ≥ 3) (6.5 ± 4.1 versus 12.9 ± 22.4 months, respectively, p < 0.05). Median survival of examined patients also showed significant relations with the levels of PCNA expression. Patients with low expression of PCNA (PCNA index < 4) had significantly longer survival than those with higher expression of PCNA (PCNA index ≥ 4) (11.9 ± 20.1 versus, 5.8 ± 10.8 months, respectively, p < 0.01): Conclusions: Our results showed that the expression of GLUT-1 had no prognostic value in the examined patients with pancreatic cancer. On the other hand, high levels of HK-II expression and low levels of PCNA expression may allow accurate identification of the patient with longer survival who may benefit from intensive anticancer treatment.

Alveolar Cell Senescence in Patients with Pulmonary Emphysema

The prevalence of chronic obstructive pulmonary disease (COPD) is age-dependent, suggesting an intimate relationship between the pathogenesis of COPD and aging. In this study we investigated whether the senescence of alveolar epithelial and endothelial cells is accelerated in emphysematous lungs. Samples of lung tissue were obtained from patients with emphysema, asymptomatic smokers, and asymptomatic nonsmokers. Paraffin-embedded lung tissue sections were evaluated for cellular senescence by quantitative fluorescence in situ hybridization to assess telomere shortening, and by immunohistochemistry to assess the expression of senescence-associated cyclin-dependent kinase inhibitors. Tissue sections were also immunostained for proliferating cell nuclear antigen (PCNA), surfactant protein A, and CD31. The patients with emphysema had significantly higher percentages of type II cells positive for p16INK4a and p21CIP1/WAF1/Sdi1 than the asymptomatic smokers and nonsmokers. They had also significantly higher percentages of endothelial cells positive for p16INK4a than the asymptomatic smokers and nonsmokers, and higher percentages of endothelial cells positive for p21CIP1/WAF1/Sdi1 than the asymptomatic nonsmokers. Telomere length in alveolar type II cells and endothelial cells was significantly shorter in the patients with emphysema than in the asymptomatic nonsmokers. The level of p16INK4a expression was negatively correlated with the level of PCNA expression. The level of alveolar cell senescence was positively correlated with airflow limitation. These results suggest that the senescence of alveolar epithelial and endothelial cells is accelerated in patients with emphysema. Cellular senescence may explain the abnormal cell turnover that promotes the loss of alveolar cells in emphysematous lungs.

Influence of prostaglandin A2 on Bax, Bcl-2 and PCNA expression in MCF-7 cells

The effects of 20 microg/mL exogenous prostaglandin A(2) (PGA(2)) were determined on Bax, Bcl-2 and proliferating cell nuclear antigen (PCNA) expression levels in MCF-7 cells. Flow cytometric analysis indicated a pronounced increase in the S phase and a decrease in the G(1) phase, whereas a significant increase in the DNA content preceding the G(0)/G(1) peak was also observed after 48 h of exposure to PGA(2). Confirmation of apoptosis was determined after 12 h, 36 h and 48 h of PGA(2) exposure employing the mitosensor reagent that detects potential changes in the mitochondrial membrane. Twenty-eight percent of PGA(2)-exposed cells were in apoptosis when compared to the 7.1% vehicle-treated cells after 48 h. PGA(2) exposure led to statistically significant increase (1.25-fold) over vehicle-treated controls in Bax expression levels. Decreases in Bcl-2 (0.79-fold), as well as PCNA (0.69-fold) expression levels over vehicle-treated controls were observed. The Bax/Bcl-2 ratio for PGA(2)-exposed cells was 2.7. The present study suggests that an accumulation in the S phase, a decrease in expression levels of PCNA, as well as an altered ratio in favor of Bax, could lead to the induction of apoptosis in these cells.

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.

Acid fibroblast growth factor reduces rat intestinal mucosal damage caused by ischemia-reperfusion insult

To detect the effects of acid fibroblast growth factor (aFGF) on apoptosis and proliferation of intestinal epithelial cells in differentiation or proliferation status to explore the protective mechanisms of aFGF. Wistar rats were randomly divided into sham-operated control group (C, n=6), intestinal ischemia group (I, n=6), aFGF treatment group (A, n=48) and intestinal ischemia-reperfusion group (R, n=48). Apoptosis of intestinal mucosal cells was determined with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) technique. Proliferating cell nuclear antigen (PCNA) protein expression and distribution were detected with immunohistochemical method. Plasma levels of D-lactate were determined with modified Brandts method. In A group, administration of exogenous aFGF could improve intestinal histological structure and decrease plasma D-lactate levels at 2-12 h after the reperfusion compared with R group. The apoptotic rates and PCNA protein expressions were not increased until 2 h after reperfusion and were maximal at 12 h. After reperfusion for 2-12 h, the apoptotic rates were gradually augmented along the length of jejunal crypt-villus units. Administration of aFGF could significantly reduce the apoptotic response at 2-12 h after reperfusion (P<0.05). Apoptosis rates in villus and crypt epithelial cells in A group at 12 h after reperfusion were (62.5+/-5.5)% and (73.2+/-18.6)% of those in R group, respectively. Treatment of aFGF could apparently induce protein expression of PCNA in intestinal mucosal cells of A group compared with R group during 2-12 h after reperfusion (P<0.05). There were approximately 1.3- and 1.5-times increments of PCNA expression levels in villus and crypt cells in A group at 12 h after reperfusion compared with R group, respectively. Intestinal I/R insult could lead to histological structure change and apoptotic rate increment. The protective effects of aFGF against ischemia/reperfusion in rat intestinal mucosa might be partially due to its ability to inhibit ischemia/reperfusion-induced apoptosis and to promote cell proliferation of crypt cells and villus epithelial cells.

Mouse skin tumor promotion by sodium arsenate is associated with enhanced PCNA expression

Drinking water contamination by arsenicals remains a major public health problem in many parts of the world more particularly in India and Bangladesh. Despite arsenic being a health hazard and implicated in human carcinogenesis, the experimental evidence available is much limited even now and the mechanisms involved during carcinogenesis and tumor promotions are not clear. Accordingly, in this study, we have studied the tumor promoter effects of sodium arsenate on mouse skin tumor promoter model system using 9,10-dimethyl-1,2-benzanthracene (DMBA) as a initiating carcinogen. Our studies showed development of papillomas on mice skin treated with only DMBA. However, mice treated with DMBA on skin and administered arsenate (As) in drinking water showed development of well differentiated squamous cell carcinomas. Further, both by immunohistochemistry and western blotting analysis studies higher levels of proliferating cell nuclear antigen (PCNA) was observed in mice treated with DMBA plus arsenate compared to only DMBA treated group. PCNA is known to be associated with S phase and DNA replication of the cell cycle. The plain controls and arsenate controls did not show significant difference either in tumor development or in PCNA levels. The present study demonstrates mouse skin tumor promoting effect of arsenate which seems to be associated with abnormal cell proliferation as indicated by higher levels of PCNA expression.

Role of differential and cell type-specific expression of cell cycle regulatory proteins in mediating progressive glomerular injury in human IgA nephropathy

The activities of cell cycle regulatory proteins have been reported to be associated with the development of pathological lesions in glomerulonephritis. To assess the cellular mechanisms underlying the mesangial cell proliferation and glomerulosclerosis in progressive human IgA nephropathy (IgAN), we examined the expression of E2F1, Rb, c-Myc, proliferating cell nuclear antigen (PCNA), cyclins (D1, E and A), cyclin-dependent kinase 2 (CDK2) and CDK inhibitors (p21waf1, p27kip1, 57kip2 and p16ink4a) by immunohistochemistry in renal biopsy specimens. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was also performed to detect the presence of apoptosis. In total, 51 cases of IgAN were categorized into four subgroups according to histological severity. A dramatic upregulation of E2F1 expression in mesangial cells was identified in proliferating glomeruli, which correlated well with the proliferation index. High endogenous expression of p27kip1 and p57kip2 by podocytes in normal glomeruli and glomeruli with minor lesions was observed to decrease in proliferating and sclerosing glomeruli; this pattern displayed a strong inverse correlation with the mean glomerulosclerosis score and the index of glomerular lesion. Increased apoptotic activity was identified in progressive glomerular lesions of advanced IgAN, which correlated with the proliferative activity in these lesions as assessed by total expression levels of PCNA and CDK2 in glomeruli, E2F1 expression levels in the mesangium, cyclin D1 expression levels in endothelium and the c-Myc glomerular staining score. Our results suggest that the onset and magnitude of mesangial cell proliferation and glomerulosclerosis is associated with the upregulation of E2F1 by mesangial cells and the downregulation of p27kip1 and p57kip2 by glomerular epithelial cells. The cell type-specific and coordinated regulation of proliferative and proapoptotic activities of cell cycle regulatory proteins may play an important role in mediating progressive glomerular injury in human IgAN.