Construction of the Antisense Eukaryotic Vector for Proliferating Cell Nuclear Antigen Gene and Its Expression in Bladder Cancer EJ Cell Line

To explore a novel strategy for antisense gene therapy of cancer,the coding sequence of hum an proliferating cell nuclear antigen(PCNA) c DNA was reversely inserted into the eukaryotic vector p L XSN by molecular cloning techniques and transferred into bladder cancer EJcells with li- posome. The PCNA expression in transferred cells was dynamically detected by immunofluo- rescence and RT- PCR techniques. Changes of proliferation activities of cancer cells were assayed by MTT colorim etric and cloning formation m ethods.In the experiment,the antisense eukaryotic vector was successfully constructed and nam ed as p L APSN.After transfection with it for1- 7 days,PCNA protein and m RNA levels in cancer cells were blocked by16 .74 % - 84 .2 1% (P< 0 .0 5 ) and2 3.2 7% - 86 .15 % (P<0 .0 5 ) respectively.The proliferation activities of transferred cells were inhibited by 2 7.91% - 6 2 .0 7% (P<0 .0 1) ,with cloning formation abilities being de- creased by 5 0 .81% (P<0 .0 1) . Itwas concluded that the in vitro proliferation activities of cancer cells could be effectively inhibited by blocking PCNA expression with antisense technique,which could serve as an ideal strategy for gene therapy of bladder cancer.

Proliferating cell nuclear antigen of Ulva prolifera is involved in the response to temperature stress

Ulva prolifera is the most common specie causative to green tide,and its growth is sensitive to temperature stress.However,the mechanisms of U.prolifera response to temperature stress remain elusive.In this study,high temperature(36℃)stimulus promoted the death of unformed cell wall protoplasts and delayed the division of formed cell wall protoplasts,while low-temperature(4℃)stimulus did not,suggesting that the mechanisms of the response of U.prolifera to high and low temperature stresses are different.Transcriptome results show that proliferation-related genes were differentially expressed under high and low-temperature stresses,especially the proliferating cell nuclear antigen(PCNA)and cyclins(CYCs).Subsequently,the interaction between PCNA and Cyclin A was confirmed by Co-immunoprecipitation,yeast two-hybrid,and so on.Furthermore,high-and low temperature stresses induced the expression of PCNA and Cyclin A in varying of degrees,and activated extracellular signal-regulated kinase(ERK)signal pathway.These results suggest,PCNA,Cyclin A,and ERK signal pathway played important roles in the resistance of U.prolifera to temperature stress.Interestingly,high-temperature stress induced an increase of miR-2916 in abundance,and exhibiting reverse expression of PCNA;and PCNA was target gene of miR-2916,suggesting that miR-2916 protected U.prolifera from high-temperature stress via post-transcriptionally regulation of PCNA.This study laid a foundation for understanding the function of PCNA and Cyclin A,moreover,it has a guiding significance to explore the mechanisms of the response to temperature stress from proliferation-related genes regulatory networks in U.prolifera.

Mast cell density and the context of clinicopathological parameters and expression of p185,estrogen receptor,and proliferating cell nuclear antigen in gastric carcinoma

AIM:To investigate the relationship between the mast cell density(MCD)and the context of clinicopathological parameters and expression of p185,estrogen receptor(ER), and proliferating cell nuclear antigen(PCNA)in gastric carcinoma. METHODS:Mast cell,p185,ER,and PCNA were detected using immunohistochemical S-P labeling method.Mast cell was counted in tissue of gastric carcinoma and regional lymph nodes respectively,and involved lymph nodes(ILN)were examined as usual. RESULTS:MCD was significantly related to both age and depth of penetration(x^2=4.688,P<0.05 for age and x^2=9.350, P<0.01 for depth of penetration)between MCD>21/0.03 mm^2 and MCD≤21/0.03 mm^2 in 100 patients;MCD in 1-6 ILN group patients was significantly higher than that in 7-15 ILN or>15 ILN group patients(u=6.881,8.055,P<0.01); There were significant differences intergroup in positive expression rate of p185,ER and PCNA between MCD>21/ 0.03 mm^2 and MCD≤21/0.03 mm^2 in 100 patients. CONCLUSION:Mast cell may have effect on inhibiting invasive growth of tumor,especially in the aged patients; The number of mast cells,in certain degree,may predicate the number of involved lymph nodes,which is valuable for assessment of prognosis;MCD was related to the expression of p185,ER,and PCNA in gastric carcinoma.It suggests that mast cell accumulation may inhibit the proliferation and the dissemination of the gastric carcinoma. INTRODUCTION Recently,many studies have reported on the association of mast cell with various tumorst.In several malignancies,mast cell has been found to correlate with growth,penetration and prognosis of tumor.Therefore,our study was undertaken to investigate the relationship between the mast cell density (MCD)and the context of clinicopathological parameters and expression of p 185,estrogen receptor(ER),and proliferating cell nuclear antigen(PCNA)in gastric carcinoma.

Clinical significance of expression of proliferating cell nuclear antigen and E-cadherin in gastric carcinoma

AIM to investigate the expression of proliferating cell nuclear antigen(p CNA)and E-cadherin in gastric carcinoma and to analyze their clinical significance.METHODS A total of 146 patients were selected for this study,including 38 patients with intestinal metaplasia,42with dysplasia,and 66 with primary gastric cancer.In addition,40 patients with normal gastric tissues were selected as controls.the expression of p CNA and E-cadherin was detected by immunohistochemistry.Differences in p CNA and the E-cadherin labeling indexes among normal gastric mucosa,intestinal metaplasia,dysplasia,and gastric carcinoma were compared.Subjects with normal gastric tissues were assigned to a normal group,while gastric cancer patients were assigned to a gastric cancer group.the difference in p CNA and E-cadherin expression between these two groups was compared.the relationship between expression of p CNA and E-cadherin and clinicopathological features was also explored in gastric cancer patients.furthermore,prognosis-related factors,as well as the expression of p CNA and E-cadherin,were analyzed in patients with gastric cancer to determine the 3-year survival of these patients.RESULTS the difference in p CNA and the E-cadherin labeling indexes among normal gastric mucosa,intestinal metaplasia,dysplasia,and gastric carcinoma was statistically significant(p<0.05).During the transition of normal gastric mucosa to gastric cancer,the p CNA labeling index gradually increased,while the E-cadherin labeling index gradually decreased(p<0.05).the p CNA labeling index was significantly higher and the E-cadherin labeling index was significantly lower in gastric cancer than in dysplasia(p<0.05).the expression of p CNA was significantly higher in the gastric cancer group than in the normal group,but E-cadherin was weaker(p<0.05).there was a negative correlation between the expression of p CNA and E-cadherin in gastric carcinoma(r=-0.741,p=0.000).p CNA expression differed significantly between gastric cancer patients with and without lymph node metastasis and between patients at different t stages.E-cadherin expression also differed significantly between gastric cancer patients with and without lymph node metastasis(p<0.05).High t stage and positive p CNA expression were risk factors for the prognosis of patients with gastric cancer(RR>1),while the positive expression of E-cadherin was a protective factor(RR<1).the sensitivity,specificity,and accuracy of p CNA positivity in predicting the 3-year survival of patients with gastric cancer were 93.33%,38.89%,and0.64,respectively;while these values for E-cadherin negativity were 80.0%,41.67%,and 0.59,respectively.When p CNA positivity and E-cadherin negativity were combined,the sensitivity,specificity,and accuracy were66.67%,66.67%,and 0.67,respectively.CONCLUSION Combined detection of p CNA and E-cadherin can improve the accuracy of assessing the prognosis of patients with gastric cancer.

Evaluation of germ-cell kinetics in infertile patients with proliferating cell nuclear antigen proliferating index

Aim: To explore the usefulness of proliferating cell nuclear antigen proliferating index (PCNA PI) in the pathologicaldiagnosis and treatment of male infertility. Methods: Testicular biopsy specimen obtained from 48 cases of male in-fertility and 2 normal controls were fixed and embedded. The sections were stained with anti-PCNA monoclonal anti-bodies or haematoxylin/eosin. Proliferating index (PI), expressed as the percentage of germ-cell nuclei positivelystained with PCNA antibody, was assessed from more than 20 seminiferous tubules or 600 germ-cells. Results: Theinfertile patients were divided into 4 groups: Group 1, normal spermatogenesis (14 cases); Group 2, hypospermato-genesis (16 cases); Group 3, germinal arrest (10 cases); Group 4, Sertoli cell only syndrome (8 cases). The PCNAPI of normal control testis was 86.5 % (mean value). Group 3 had a significantly lower PCNA PI (29.8 %) than nor-mal testis; Group 1 and 2 had similar Pis (82.3% and 82.3%, respectively) as the control testis. PI of the negativecontrol (Group 4) was 0 as no germ-cells were found. Conclusion: PCNA PI is useful for assessing germ-cell ki-netics, especially for pathological diagnosis of germinal arrest which is difficult to differentiate by routine HE stainingtechnique. In germinal arrest, there is a significantly lowered PCNA PI, which is an indication of DNA synthesis dete-rioration, suggesting the use of therapies be different from those for hypospermatogenesis. (Asian J Androl 2001 Mar;3: 63-66)

The Relationship of Expression of bcl-2, p53, and Proliferating Cell Nuclear Antigen (PCNA) to Cell Proliferation and Apoptosis in Renal Cell Carcinoma

To investigate the relationship of bcl-2, p53, proliferating cell nuclear antigen (PCNA) to cell proliferation, apoptosis and pathological parameters, the patterns of cell growth and turnover in renal cell carcinoma (RCC), formalin-fixed and paraffin-embedded tissue blocks from 34 patients with RCC were examined. Cell proliferation activity was detected by PCNA immunostaining and the proliferation index (PI) was expressed as a percentage of the PCNA-positive cells in the tumor cells. Apoptosis was detected by terminal deoxy- nucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), and the apoptotic index (AI) was expressed as a percentage of the TUNEL-positive cells in the tumor cells. Expressions of bcl-2 and p53 were assessed immunohistochemically. Our results showed that the PI ranged from 6.0 % to 24.0 % (median 12.3 %) and the AI from 2.0 % to 8.0 % (median 5.4 %) in RCC. The expression of the bcl-2 protein was demonstrated in 15 cases (44.1 %); the expression of the p53 protein, however, was seen in only 3 case. bcl-2 positivity was not associated with PI or AI or any pathological parameters. There were close associations between PI and tumor grade and stage, and a significant relationship between AI and the tumor grade of RCC. Our study suggests that bcl-2 positivity was not associated with PI or AI or any pathological parameters. There are close associations between PI and AI and tumor grade and stage of RCC. Active cell proliferation may be accompanied by frequent apoptosis in RCC.

The Relationship between Apoptosis and the Expression of Proliferating Cell Nuclear Antigen and the Clinical Stages in Gastric Carcinoma

The relationship between the apoptosis and the expression of proliferating cell nuclear antigen (PCNA) and the clinical stages in gastric cancers was studied. By using terminal deoxynucleotidyl transferase mediated nick end labelling (TUNEL) technique and PCNA immunohistochemical staining, the apoptosis and the expression of PCNA in tissue of gastric carcinoma were assayed in situ, the index of apoptosis (AI), index of PCNA (PI) and the rate of AI/PI were calculated. AI and PI in gastric cancer tissues were (6.5±3.7) % and (49.8±15.9) % respectively, and the rate of AI/PI was 0.13±0.05, which were obviously different from those of normal gastric mucosa in paragastric cancer ( P <0.01). With the advanced TNM stages of gastric carcinoma, the AI was decreased, PI was increased and the rate of AI/PI decreased in gastric carcinoma. There was significant difference in them between the gastric cancer tissues and normal gastric mucosa in pericarcinoma in TNM stage Ⅱ to Ⅳ ( P <0.05). It was suggested that the decreased apoptotic cells and the increased proliferating cells were obviously related to the tumor genesis and tumor progression in gastric carcinoma. The AI, PI and the rate of AI/PI would become the prognostic factors in advanced gastric carcinoma.

RELATIONSHIP BETWEEN PROLIFERATING CELL NUCLEARANTIGEN EXPRESSION AND ITS MALIGNANCY POTENTIAL IN COLORECTAL CARCINOMA

Objective: To study the relationship between proliferating cell nuclear antigen expression and its malignancy potential in colorectal carcinoma. Methods: Paraffin sections of 86 patients with advanced colorectal carcinoma were assessed by immunohistochemical study, using a mouse monoclonal antibody (pc-10, DAKO Co. USA) to check proliferating cell nuclear antigen (PCNA). To compare PCNA with conventional clinicopathologic factor, including p53 overexpression, tissue carcinoembnyonic antigen immunoreactivity pattern and flow cytometric DNA ploidy for assessing tumor malignancy potential. In addition, recurrence and survival of patients with advanced colorectal carcinoma after curative resection were analyzed in accordance with degree of PCNA expression. Results: PCNA-labeling index (PCNA-LI) increased significantly as the tumor stage advanced (p=0.0001). Strong correlations were observed between PCNA-LI and various pathologic parameters, including histologic differentiation (P=0.0027), lymphatic invasion (P=0.0001), vascular invasion (P=0.0001), lymph node metastasis (P=0.0001), and liver metastasis (P=0.0036). Mean PCNA-LI was also significantly higher in tumor with DNA aneuploidy (P=0.0006) and negative (P=0.01). Linear relationships were demonstrated between PCNA-LI and clinical outcomes; Recurrence rate was significantly greater in the group with higher than the mean PCNA-LI, who underwent curative resection (P<0.01), and three-year survival rates for curative cases with higher than the mean PCNA-LI were significantly poorer than those with lower than mean PCNA-LI (P<0.005). Conclusion: There were correlations between PCNA-LI and various pathologic parameters, PCNA-LI increased significantly as the tumor stage advanced in colorectal carcinoma, the rates of recurrence and death got higher as PCNA-LI increased after curative resection for colorectal carcinoma.

Cloning and characterization of proliferating cell nuclear antigen gene of Alexandrium catenella (Dinoflagellate) with respect to cell growth

Harmful algal blooms （HABs） have been affecting negatively the shellfish and aquaculture industries around the world. Though a lot of efforts have been made to disclose the changes of environmental factors involved and their effects on the HABs events, the molecular mechanism of this process remains unclear. To address this problem, proliferating cell nuclear antigen gene （pcna） was isolated and characterized from Alexandrium catenella. It showed high homology to those of other dinoflagellates （89% and 91% homology to Pfiesteria piscicid and Pyrocystis lunula, respectively）, and also 42%–43% homology to those of plant and animals. The expression level of pcna revealed by quantitative real time PCR was the lowest at the late lagging cell growth phase, increased to the highest at the late exponential phase, and then decreased at the stationary phase. Though the cell growth rate was also changing, no positive correlation between pcna expression level and cell growth rate was displayed throughout the whole cell growth stages （r 2 =0.024 6）. However, the pcna expression level had the similar trend with the change of cell growth rate throughout the whole growing process, e.g., from increasing at the earlier cell growth stage to decreasing at the following stages, though slightly lagging to the latter.

EXPRESSION OF P53 PROTEIN AND PROLIFERATING CELL NUCLEAR ANTIGEN IN HUMAN GESTATION TROPHOBLASTIC DISEASE

To study the relationship between p53 protein, proliferating cell nuclear antigen (PCNA) expression and benign or malignant gestational trophoblastic disease (MGTD). Methods: The histotomic sections of 48 patients with gestational trophoblastic disease and 24 patients of normal chorionic villi were stained using immunohistochemistry. The monoclonal antibodies were used to determine p53 protein and PCNA. Results: The frequency of p53 and PCNA positive expression were significantly different among the chorionic villi of normal pregnancy, hydratidiform mole (HM) and MGTD. But neither p53 nor PCNA has any relation with the clinical staging or metastasis of MGTD. Conclusion: Both P53 and PCNA are valuable in diagnosis of human gestational trophoblastic disease.

Proliferating cell nuclear antigen clamp associated factor,a potential proto-oncogene with increased expression in malignant gastrointestinal tumors

Gastrointestinal(GI)cancers,including malignancies in the gastrointestinal tract and accessory organs of digestion,represent the leading cause of death worldwide due to the poor prognosis of most GI cancers.An investigation into the potential molecular targets of prediction,diagnosis,prognosis,and therapy in GI cancers is urgently required.Proliferating cell nuclear antigen(PCNA)clamp associated factor(PCLAF),which plays an essential role in cell proliferation,apoptosis,and cell cycle regulation by binding to PCNA,is a potential molecular target of GI cancers as it contributes to a series of malignant properties,including tumorigenesis,epithelial-mesenchymal transition,migration,and invasion.Furthermore,PCLAF is an underlying plasma prediction target in colorectal cancer and liver cancer.In addition to GI cancers,PCLAF is also involved in other types of cancers and autoimmune diseases.Several pivotal pathways,including the Rb/E2F pathway,NF-κB pathway,and p53-p21 cascade,are implicated in PCLAF-mediated diseases.PCLAF also contributes to some diseases through dysregulation of the p53 pathway,WNT signal pathway,MEK/ERK pathway,and PI3K/AKT/mTOR signal cascade.This review mainly describes in detail the role of PCLAF in physiological status and GI cancers.The signaling pathways involved in PCLAF are also summarized.Suppression of the interaction of PCLAF/PCNA or the expression of PCLAF might be potential biological therapeutic strategies for GI cancers.

Abnormal expressions of proliferating cell nuclear antigen and P27 protein in brain glioma

Both proliferating cell nuclear antigen and P27 protein are important factors to regulate cell cycle. While, the combination of them can provide exactly objective markers to evaluate prognosis of patients with brain glioma needs to be further studied based on pathological level. OBJECTIVE： To observe the expressions of proliferating cell nuclear antigen and P27 protein in both injured and normal brain glioma tissues and analyze the effect of them on onset and development of brain glioma. DESIGN： Case contrast observation. SETTING： Department of Neurosurgery, the Second Affiliated Hospital of Xi＇an Jiaotong University. PARTICIPANTS： A total of 63 patients with brain glioma were selected from Department of Neurosurgery, the Second Affiliated Hospital of Xi＇an Jiaotong University from July 1996 to June 2000. There were 38 males and 25 females and their ages ranged from 23 to 71 years. Based on pathological classification and grading standards of brain glioma, patients were divided into grade I - II （n=30） and grade III- IV （n = 33）. All cases received one operation but no radiotherapy and chemiotherapy before operation. Sample tissues were collected from tumor parenchyma. Non-neoplastic brain tissues were collected from another 12 non-tumor subjects who received craniocerebral trauma infra-decompression and regarded as the control group. There were l0 males and 2 females and their ages ranged from 16 to 54 years. The experiment had got confirmed consent from local ethic committee and the collection was provided confirmed consent from patients and their relatives. All samples were restained with HE staining so as to diagnose as the brain glioma. While, all patients with brain glioma received radiotherapy after operation and their survival periods were followed up. METHODS： Primary lesion wax of brain glioma was cut into serial sections and stained with S-P immunohistochemical staining. Brown substance which was observed in tumor nucleus was regarded as the positive expressions of both proliferating cell nuclear antigen and P27 protein. Automatic imaging analytic system was used to quantitatively analyze staining results of tumor. MAIN OUTCOME MEASURES： To compare the expressions of proliferating cell nuclear antigen and P27 protein in brain glioma tissues and non-tumor brain tissues and investigate the effect of various sexes, ages, survival periods and severities on the expressions of them in brain tissues. RESULTS： There was no significant difference of sexes and ages in the expressions of proliferating cell nuclear antigen and P27 protein （P 〉 0.05）; however, the expressions of proliferating cell nuclear antigen and P27 protein were milder in non-tumor brain tissues than those in the brain glioma tissues （P 〈 0.05）. Expression of proliferating cell nuclear antigen in brain tissue of grade III- IV severity was stronger than that of grade I - II severity, and the expression in ≥ 5-year survival periods were also stronger than that in 〈 5-year survival periods （P 〈 0.05）. In addition, expression of P27 protein in brain tissue of grade III- IV severity was stronger than that of grade I - II severity, and the expression in ≥ 5-year survival periods were also stronger than that in 〈 5-year survival periods （P 〈 0.05）. CONCLUSION： Abnormal expressions of proliferating cell nuclear antigen and P27 protein in human brain glioma are closely related to onset, development and prognosis of tumor.

PROLIFERATING CELL NUCLEAR ANTIGEN(PCNA) IN OVARIAN CARCINOMA AND ITS RELATION TO LYMPH NODE METASTASIS AND PROGNOSIS

Objective: To investigate expression of proliferating cell nuclear antigen (PCNA) in ovarian epithelial cancer and its relation to lymph node metastasis, outcome of second look laparotomy (SLL) and prognosis Methods: Monoclonal antibody PC10 was used to stain PCNA in archival paraffin embedded tissues Results: PC10 immunostaining was performed successfully in all 74 primary and 31 intraperitoneal metastatic tumors The expression levels of PCNA were significantly increased in 31 metastatic tumors compared with their primary tumor from the same patients (7 94 vs 6 89, P=0 042) The expression levels was more elevated in bilateral than in unilateral ovarian cancer, but it was not associated with lymph node metastasis, clinical stage, histological grade and subtype In 28 patients with stage III ovarian cancer undergone SLL, the mean immunoreactive score (IRS) of PCNA of the primary tumor was significantly higher in patients with negative SLL than in those with positive SLL (7 59 vs 6 10, P =0 03) Since chemotherapy was performed following surgical debulking, negative SLL more frequently seen in patients with high PCNA expression might suggest better chemotherapeutic sensitivity due to higher proliferation fraction of tumor cell Univariate analysis of survival indicated that the overall survival was inversely associated with the level of PCNA expression, while multivariate analysis with Cox's model showed that independent prognostic factors were the residual tumor after primary debulking ( P<0 001 ) and clinical stage ( P <0 05), followed by PCNA expression( P =0 09) Conclusion: The expression of PCNA may beuseful in predicting the patients' prognosis, but is not correlated with lymph node metastasis

Effects of UVB on the expression of proliferating cell nuclear antigen (PCNA) in dinoflagellate Prorocentrum donghaiense Lu

PCNA has been considered as a useful marker for the estimation of growth rates of marine phytoplankton at the species level. Since dinoflagellates are noted for having many prokaryotic features in that they are the only eukaryotes to have permanently condensed chromosomes as well as lacking histones and a nucleosome, the sensitivity to UVB radiation and the validity of PCNA as a maker of growth rate in dinoflagellate need to be evaluated. Prorocentrum donghaiense Lu was investigated to valuate the UVB sensitivity in relation to cellular and molecular aspects of PCNA as a growth indicator. The effects of UVB radiation on PCNA were studied using the methods of western blots technology and whole-cell immunoflurescence with one mono-antibody. UVB changed the cell morphology, halted the growth and increased the cell size, even caused cell death to a certain extent after treatment with UVB radiation in P. donghaiense. Compared with the control, treating the algal cultures in exponential phases with UVB radiation for 24 h caused chromatin release and increases in protein levels of PCNA.

The Effect of the LysoPC-induced Endothelial Cell Conditioned Medium on Proliferating Cell Nuclear Antigen Expression of the Calf Thoracic Aorta Smooth Muscle Cells

In order to study the effect of and mechanism of lysophosphatidylcholine (LysoPC) on proliferation of the calf thoracic aorta smooth muscle cells (ASMCs), the ASMCs were used to observe the effects of LysoPC induced endothelial cell conditioned medium on the DNA content and proliferating cell nuclear antigen (PCNA) expression in the calf thoracic ASMCs by flow cytometry and Western Blot technique. It was found that LysoPC induced endothelial cell conditioned medium could significantly promote PCNA expression of the calf ASMCs, induce the converting of ASMCs from G 0 /G 1 phase to S phase of DNA synthesis, and increase the tyrosine phosphorylation protein expression. Tyrosine protein kinase inhibitor (TPKi) RG50864 could obviously inhibit proliferation of LysoPC induced ASMCs in a dose dependence manner. The results indicated that the effect of LysoPC promoting the proliferation of ASMCs is partly evoked by endothelial cell derived growth factors such as PDGF and so on.

Clinical Significance of p53, Proliferating Cell Nuclear Antige and bcl-2 Expression in Bladder Transitional Cell Carcinoma

Objective: To correlate the frequency of p53 mutations, bcl-2 expression and the proliferation status (proliferating cell nuclear antigen, PCNA) in patients with bladder cancer with cell proliferation, apoptosis and their clinico-pathologic ?ndings. Methods: Para?n-embedded sections from 39 super?cial (T1G1-G3) and 23 invasive (T2-T4a G3 N0M0) primary transitional cell carcinomas (TCC) in the bladder were investigated immunohistochemically for p53, bcl-2 and PCNA. The median follow-up was 37 months; 24 had recurrences. The proliferation index (PI) was expressed as a percentage of the PCNA-positive cells in the tumor cells. Apoptosis was detected by terminal deoxy-nucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), and the apoptotic index (AI) was expressed as a percentage of the TUNEL- positive tumor cells. Results: P53 mutation was identi?ed in 50 patients (80.6%). The mutation was most common in tumors of grade 3 (91.3%) as compared to grade 2 (78.5%) and grade 1 (72.7%, P<0.05). Stage pT2 tumors had a higher frequency of p53 mutation (95.7%) as compared to pTa-1 tumors (74.3%, P<0.01). Only 14 tumors (22.5%) expressed bcl-2; grade 3 tumors expressed bcl-2 signi?cantly more frequently (P<0.05); there was no correlation between bcl-2 and tumor stage. There was no interrelation between p53 mutation and bcl-2 expression (P>0.05). The PI ranged from 17.2% to 41.8% (median 22.4%) and the AI from 1.9% to 3.5% (median 2.9%) in bladder cancer. Statistical analyses revealed a close associations between PI, AI and tumor grade and stage of bladder cancer. Conclusion: P53 mutation correlates with invasion. P53 and PCNA overexpression may o?er valuable additional prognostic information in bladder tumors. With the progression of the tumor grade, cell proliferation may be accompanied by frequent apoptosis in bladder cancer, but the PI increased much more than the AI.

Influence of norcantharidin on proliferation,proliferation-related gene proteins prolifera-ting cell nuclear antigen and Ki-67 of human gallbladder carcinoma GBC-SD cells

BACKGROUND: Gallbladder carcinoma is a highly lethal and aggressive disease with early metastasis, strong invasion and poor prognosis. Most patients with this disease are at the advanced and un-resectable stage and should be consi- dered for palliative treatment such as chemotherapy and ra- diotherapy. Unfortunately, reports of chemotherapy and radiotherapy for gallbladder carcinoma are disappointing. We investigated the influence of norcantharidin (NCTD) on proliferation, proliferation-related gene proteins PCNA and Ki-67 of human gallbladder carcinoma GBC-SD cells in vitro. METHODS: GBC-SD cell lines of human gallbladder carci- noma were cultured by the cell culture technique. The ex- periment was divided into NCTD group and control group. The tetrazolium-based colorimetric assay was used to evaluate cell growth. The streptavidin-biotin complex method was used to determine the expressions of prolifera- tion-related gene proteins PCNA and Ki-67 of human gall- bladder carcinoma GBC-SD cells. RESULTS: NCTD inhibited the growth and proliferation of GBC-SD cells from 10 mg/L or after 6 hours in a dose- and time-dependent manner, with the IC50 value of 56.18 μg/ ml at 48 hours. After treatment with NCTD, the expression of PCNA (0.932 ±0.031 vs. 0.318 ±0.023, P<0.001) and Ki-67 (0.964 ±0.092 vs. 0.297 ±0.018, P<0.001) proteins were decreased significantly. CONCLUSION: NCTD inhibits the proliferation of human gallbladder carcinoma GBC-SD cells in vitro and the expres- sion of their proliferation-related gene proteins PCNA and Ki-67.

EXPRESSIONS OF P_(53), PROLIFERATING CELL NUCLEAR ANITIGEN,BCL-2 PROTEIN AND THEIR SIGNIFICANCE IN SALIVARY ADENOID CYSTIC CARCINOMA

Objective To study the effects of P53, PCNA, Bc1-2 protein and their relationship in salivary adenoid cystic carclnoma(SACC). Methods These protelns were examlned by lmmunohistochemistry. Results overexpressions of Ps, and PCNA were revealed in ACC samples, they were higher than those in (polymorphous adenomas) PA, but expression of Bc1-2 protein was not different between ACC and PA. In 3 subtypes of ACC, expressions of 3 proteins were different. Conciusion Mutations of P53, Bc1-2 may be involed in the occurrence of SACC, expression of PCNA and mutation of P53 may coexist in the development of the SACC.

Effect of aloe-emodin on expression of proliferating cell nuclear antigen of vascular smooth muscle cells in culture after arterial injury

Objective To observe the effect of aloe-emodin on the expression of proliferating cell nuclear antigen (PCNA) in smooth muscle cells (SMCs) after arterial injury and study the molecular mechanism of inhibition of aloe-emodin on SMC proliferation.Methods Deendothelialization was performed at the abdominal aorta in Japanese white rabbits using a 3F Fogarty arterial embolectomy catheter. 48 hours later, the medium of abdominal aorta was isolated and primary SMCs culture was performed. Cells were synchronized to G0 by serum starvation, then aloe-emodin at a concentration of 20?μg/ml was added to the culture medium containing 10%［v/v］ fetal calf serum. Vehicle was also added to the medium as a control. After 18 hours, the expression of PCNA at the level of mRNA and protein were examined using techniques of RT/PCR, Western blotting and inmmunocytochemistry respectively. Results Compared with the control group, the expression of PCNA mRNA and protein was prominently decreased after addition of aloe-emodin. Conclusion The inhibition of aloe-emodin on SMCs proliferation may be caused by inhibiting the expression of the PCNA gene.

Effect of pre-moxibustion on apoptosis and proliferation of gastric mucosa cells

AIM： TO observe the effects of pre-moxibustion on apoptosis and proliferation of gastric mucosal cell in rats with stress-induced ulcer, and to analyze the relationship between those effects and the expression of heat-shock protein 70 （HSP70）. METHODS： Sixty healthy Sprague Dawley rats were randomly assigned into four groups, namely group A, B, C and D. The animal model of stress ulcer was established by water immersion and restraint stress. The rats in group A, B, and D served as the restraint, model, and non-acupoint controls, respectively, while those in group C received moxibustion at Zusanli and Uangmen points. Immunohistochemical methodology was used to detect the expression of HSP70, apoptosis index （AI, × 10^-6/μm^2） and proliferation index （PCNA-LI, × 10^-6/μm^2）. The mucosal expression of transforming growth factor α （TGF-α） was detected by radioimmunoassay. RESULTS： IVloxibustion at Zusanli and Liangmen points significantly decreased the gastric injury and the apoptosis of gastric mucosal cells, while markedly increased the mucosal expression of TGF-α and HSP70 as well as the proliferation of gastric mucosal cells. Compared with group A, ulcer index （UI） （26.8 ± 9.8 vs 12.0 ± 5.9, P 〈 0.01）, AI （9.6 ± 4.2 vs 4.4 ± 2.6, P 〈 0.05） and expression of HSP70 （9.6 ± 4.2 vs 4.4 ± 2.6, P 〈 0.05） were significantly increased, but the content of TGF-α （104.7 ± 51.2 pg/mL vs 254.0 ± 86.9 pg/mL, P 〈 0.01） and PCNA-LI （6.9 ± 4.7 vs 14.9 ± 4.6, P 〈 0.05） were significantly decreased in group B. However, ulcer index values （UI） and AI were obviously lower in group C compared to groups B and D （14.1 ± 5.4 vs 26.8 ± 9.8 and 26.2 ± 7.7, P 〈 0.01, 3.0 ± 1.6 vs 9.6 ± 4.2 and 8.2 ± 5.2, P 〈0.05, respectively）, but content of TGF-α （237.0 ± 72.6 pg/mL vs 104.7 ± 51.2 pg/mL and 154.1 ± 61.3 pg/mL, P 〈0.01） and expression of HSP70 （0.13 ± 0.03 vs 0.08 ± 0.06 and 0.06 ± 0.04, P 〈 0.05） were higher in group C. Furthermore, the PCNA-LI was significantly higher in group C than in group B （21.6 ± 4.1 vs 6.9 ± 4.7, P 〈 0.01）. CONCLUSION： Moxibustion at Zusanli and Liangmen points has a protective effect on rats gastric mucosa in stress-induced gastric ulcer, which is closely related to its actions in promoting synthesis of TGF-α and proliferation of gastric mucosal cells, suppressing gastric mucosal cell apoptosis, and up-regulating HSP70 expression.