Proliferating Cell Nuclear Antigen Labeling Research Articles

Susceptibility of liver proliferative lesions in heterozygous p53 deficient CBA mice to various carcinogens.

To investigate the liver tumorigenic sensitivity to various carcinogens in heterozygous p53 deficient [p53 (+/-)] CBA mice and their wild-type littermates [p53 (+/+) mice], 71 p53 (+/-) and 74 p53 (+/+) CBA mice (male, 6-12 weeks of age) were given diet containing 4,000 or 0 ppm flumequine (FL) for 26 weeks or a single intraperitoneal injection of 5 mg/kg body weights dimethylnitrosamine (DMN) at start of the study in Exp. 1, diet containing 6,000 or 0 ppm di(2-ethylhexyl)-phthalate (DEHP) for 26 weeks in Exp. 2, or diet containing 12,000, 6,000 or 0 ppm phenolphthalein (PhP) for 26 weeks in Exp. 3. All surviving animals of these groups were killed after completion of treatment of the test substances for 26 weeks. In the FL groups, the incidences of hepatocellular altered foci in p53 (+/-) mice, the multiplicities of those in p53 (+/-) and p53 (+/+) mice were significantly increased as compared to the corresponding control groups. The incidences and multiplicities of altered foci in the DMN groups were higher than those in the corresponding control groups in p53 (+/-) and p53 (+/+) mice, but no significant differences were indicated between the groups. There were no significant differences in the incidences, multiplicities and proliferating cell nuclear antigen labeling indices of altered foci in the FL or DMN groups between p53 (+/-) and p53 (+/+) mice. There were no significant differences in the incidences and multiplicities of altered foci between the DEHP or PhP and control groups. The present results suggest that p53 gene knocked out heterozygously does not enhance the chemical hepatocarcinogenesis in CBA mice.

Exogenously Administered HGF Activator Augments Liver Regeneration through the Production of Biologically Active HGF

Hepatocyte growth factor (HGF) plays a crucial role in the recovery of injured liver. Liver functions are mostly impaired in patients with liver diseases including cirrhosis. However, a significant amount of inactive HGF precursor (proHGF) is reported in the plasma of these patients. proHGF is proteolytically converted to an active form (mature HGF) by HGF-activator. Thus conversion of proHGF into mature HGF presumably contributes to the recovery of liver functions. In this study, rats with a partial hepatectomy were used, as proHGF is accumulated in the remnant liver. Recombinant human HGF-activator was administered via the portal vein to investigate the effect on molecular forms of HGF and its biological signaling. rhHGF-activator promptly converted proHGF into mature HGF, reaching maximal levels at 5–10 min after the injection, while the decreased proHGF was quickly recovered to the initial levels in the liver. The HGF receptor/c-Met was found to be autophosphorylated in the liver treated with rhHGF-activator. Further, the proliferating cell nuclear antigen labeling index and the liver regeneration rate were significantly higher in rhHGF-activator group than in control animals. These results indicate that exogenously administered HGF-activator produces a biologically active HGF from its precursor form and increases the potential for liver regeneration in vivo.

Pathological characteristics, PCNA labeling index and DNA index in prognostic evaluation of patients with moderately differentiated hepatocellular carcinoma.

To study the relationship between prognosis and pathological characteristics, proliferating cell nuclear antigen labeling index (PCNA-LI) and DNA index (DI) in patients with moderately differentiated hepatocellular carcinoma(HCC). 51 cases of moderately differentiated HCC were analyzed with respect to the relation between their clinical follow-up data and pathological characteristics. Meanwhile, PCNA-LI of HCC cells was detected by immunohistochemistry assay and DI was measured by Feulgen staining and automatic image analysis technique. Patients with a single tumor nodule, less than 5 cm in diameter, no tumor emboli, no daughter nodules and necrosis had relatively better prognosis; patients with euploidy HCC had better prognosis than those with aneuploidy; among the aneuploidy patients those with DI<1.5 had better prognosis than the cases with DI>1.5; The higher the PCNA-LI, the worse would be the prognosis. The increase in DI was correlated with the increase in PCNA-LI, and both of them were correlated with the pathological changes of the tumor. A composite analysis of the pathological characteristics of tumor tissue, DI and PCNA-LI might be useful in predicting the prognosis of HCC patients.

口腔領域に発生した軟部肉腫の臨床病理学的検討

Because soft tissue sarcoma rarely arises in the oral cavity, there are few reports on this lesion in the head and neck region. Eight cases of soft tissue sarcoma treated at our department during the past two decades were studied clinicopathologically. Some small lesions were clinically given a diagnosis of benign tumor at presentation, followed by excisional biopsy. It was difficult to achieve an exact pathologic diagnosis from the biopsy specimens. The definitive diagnosis based on the resected material differed from the original diagnosis in some cases. Six of the eight cases were subjected to immunostaining to analyze their cytologic characteristics. Alteration of the p53 tumor suppressor gene was detected in 4 cases, and loss of p16 was noted in 5 cases. The average proliferation cell nuclear antigen labeling index of the soft tissue sarcomas was 31.3%, higher than the value for oral squamous cell carcinoma. All soft tissue sarcomas expressed metalloprotease (MMP)-2 and MMP-9 and showed low intratumoral microvessel density. These results suggest that soft tissue sarcomas have high proliferation potential and aggressive growth without angiogenesis.

Promoting the effects of intravesical instillation of saline on bladder lesion development in rats pre-treated with BBN.

At present, immunotherapeutic agents such as bacillus Calmette-Guerin (BCG) and anti-tumor chemotherapeutic agents in saline are used intravesically in patients with bladder carcinoma. However, of greater significance is the possibility that the saline vehicle may itself promote carcinoma development in the bladder. The potential promoting effects of intravesical instillation of saline were assessed in female F344 rats. The animals were divided into 3 groups, all of which received 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in their drinking water for the first 10 weeks. They were then maintained without further treatment (group 1) or received intravesical instillations of 0.3 mL of saline or distilled water once a week for 6 weeks, 15 weeks after the end of the BBN treatment (groups 2 and 3). At 32 weeks, all the animals were killed and examined immunohistochemically with proliferating cell nuclear antigen (PCNA) antibody, as well as by routine histopathologic examination. Both the incidence and the number of bladder carcinomas were higher in the animals that received instillations of saline than in those who did not receive the instillations. Significant increases in tumor size were also noted for the saline-treated groups, although this was not linked with the PCNA labeling index. The results indicate that saline is a promoter of urinary bladder carcinogenesis either because of the catheterization or the fluid itself.

Adenovirus-mediated suicide-gene therapy using the herpes simplex virus thymidine kinase gene in cell and animal models of human prostate cancer: changes in tumour cell proliferative activity.

To determine the feasibility and efficacy of suicide-gene therapy using adenovirus (Ad)-mediated herpes simplex virus thymidine kinase (HSV-TK) and the prodrug acyclovir, and to evaluate changes in the biological phenotype for tumour cell proliferative activity after suicide-gene therapy in animal models of human prostate cancer. Using a replication-defective adenoviral vector (cytomegalovirus, CMV) containing the beta-galactosidase gene (Ad-CMV-beta-gal) as a control and Ad-CMV-TK as the therapeutic vector under the transcriptional control of the CMV promoter, transduction efficiency was assessed in vitro by infecting LNCaP and PC-3 androgen-dependent and independent human prostate cancer cells with Ad-CMV-beta-gal, and using X-gal staining. The TK activity in prostate cancer cells infected with Ad-CMV-TK was determined by measuring TK-mediated [3H]-gancyclovir phosphorylation. The sensitivity of LNCaP and PC-3 cells to Ad-CMV-TK in vitro was determined after infection with the therapeutic vector with or without acyclovir. The inhibition of PC-3 tumour growth in vivo induced by the Ad-CMV-TK/acyclovir suicide-gene system was assessed in separate and controlled experiments using human prostate cancer mouse models. Ki-67 proliferative antigen and proliferating cell nuclear antigen (PCNA), both useful proliferative indices, were evaluated using immunohistochemical staining (MIB-1 monoclonal antibody and monoclonal anti-PCNA antibody) in formalin-fixed, paraffin-embedded tissues from gene therapy-treated and control animals. The mean TK activity was significantly higher in LNCaP and PC-3 cells infected with Ad-CMV-TK than in cells infected with Ad-CMV-beta-gal, used as a control (P < 0.05). The growth of human prostate cancer cells with Ad-CMV-TK was significantly inhibited by adding acyclovir in vitro (P < 0.05). In the in vivo experiments using the PC-3 human prostate cancer mouse model, tumour volume and growth was lower in mice treated with Ad-CMV-TK/acyclovir than in those treated with Ad-CMV-TK only, acyclovir only or untreated (controls) (P < 0.05). Histochemical staining of tumour tissues showed that Ad-CMV-TK/acyclovir destroyed PC-3 tumours through tumour cell death and apoptosis, with local lymphatic infiltration. The mean PCNA labelling index in prostate cancer cells of mice treated with Ad-CMV-TK/acyclovir was significantly lower than that in untreated controls (P < 0.05, Mann-Whitney U-test). The Ki-67 labelling index in prostate cancer cells of mice treated with Ad-CMV-TK/acyclovir was also lower than that in untreated controls (P < 0.05, Student's t-test). Adenovirus-mediated suicide-gene therapy using the HSV-TK gene decreased the proliferative activity of PC-3 human prostatic cancer cells in vivo. Adenovirus-mediated suicide-gene therapy using an HSV-TK/acyclovir system provided effective therapy in an experimental human prostate cancer mouse model, by significantly inhibiting tumour growth and decreasing the proliferative activity of human prostate cancer cells. Such therapy could be developed as a novel method for treating patients with androgen-independent prostate cancer.

Assessment of proliferative activity of canine dermal mast cells by bromodeoxyuridine and proliferating cell nuclear antigen labelling.

Cutaneous mast cells from skin biopsies of three healthy dogs and three dogs with atopic dermatitis were assessed for their proliferative potential using bromodeoxyuridine and proliferating cell nuclear antigen labelling. Mast cells isolated from the skin of two healthy dogs were also studied using bromodeoxyuridine labelling. Mast cells in skin biopsy specimens and mast cells isolated from the skin of healthy dogs did not incorporate bromodeoxyuridine. Two mast cells expressing proliferating cell nuclear antigen were seen around two superficial vessels in the dermis of one atopic dog. Epidermal cells, glandular epithelial cells, fibroblasts and endothelial cells incorporated bromodeoxyuridine and showed positive staining for proliferating cell nuclear antigen. These results suggest that canine mature mast cells do not proliferate in the dermis.

Changes in functional capacity after portal branch ligation in rats.

Preoperative portal branch embolization is performed to increase residual liver lobe volume so that posthepatectomy liver insufficiency associated with major hepatic resection can be prevented. Although it is essential to evaluate functional liver capacity after portal branch embolization. the timing of hepatectomy from this viewpoint remains unelucidated. The aim of this study was to clarify the optimal timing of hepatectomy after portal branch embolization. We evaluated the functional capacity of the liver after portal branch ligation (PBL) in rats, and studied the correlation between morphological and functional changes. Morphological changes were evaluated on the basis of the liver lobe weight ratio, and the proliferating cell nuclear antigen labeling index. Functional changes were evaluated by the indocyanine green disappearance rate and serum aminopyrine clearance as functional capacity. Although the total liver weight ratio remained unchanged after PBL, the unembolized lobe ratio increased significantly. Although functional capacity was minimal 2 days after PBL, it had recovered to the preoperative value by 8 days after PBL. The functional capacity of the liver after PBL was minimal around the cellular proliferative period but recovered thereafter. These findings suggest that major hepatectomy after portal branch embolization should be perfomed following the cellular proliferative period of the unembolized lobe.

Blood transfusion causes deterioration in liver regeneration after partial hepatectomy in rats.

This study investigated the effects of blood transfusion on liver regeneration and function after hepatectomy in rats. Inbred male Sprague-Dawley rats underwent a sham operation or a 70% hepatectomy (PHx) and were randomly divided into seven groups according to transfusion type: groups I and II underwent a sham operation and received saline (I) or whole blood (II). Groups III to VII underwent PHx with saline (III), whole blood (IV), irradiated/leukocyte-depleted whole blood (V), plasma (VI), or autologous blood (VII). The liver regeneration rate, proliferating cell nuclear antigen (PCNA) labeling index, serum aspartate aminotransferase, alanine aminotransferase, purine nucleoside phosphorylase (PNP) activity, hepatocyte growth factor (HGF), and activated transforming growth factor beta1 (TGF-beta(1)) were measured 6 and 24 h and 5 days after PHx. The liver regeneration rate and PCNA labeling index were lower in groups IV and V than in the other groups. Serum liver enzymes 6 h after PHx were worst in groups IV and V. PNP activity increased most in group IV, 6 and 24 h after PHx. The HGF values 6 h after PHx in all the transfused groups were lower than in group III. The activated TGF-beta(1) level 6 h after surgery was highest in group IV. Whole blood or irradiated/leukocyte-depleted whole blood impaired liver regeneration after PHx, probably through the production of activated TGF-beta(1) and HGF outside the liver, and plasma or autologous blood reduced the deleterious effects.

Up-regulation by IGF-I of proliferating cell nuclear antigen and Bcl-2 protein expression in human uterine leiomyoma cells.

IGF-I has been reported to play a role in regulating proliferation of human leiomyoma cells. There is, however, little evidence to suggest that IGF-I inhibits apoptosis in the leiomyoma cells. The present study was conducted to elucidate whether IGF-I affects apoptosis and Bcl-2 protein expression, an apoptosis-inhibiting gene product, in cultured leiomyoma cells. In addition, we examined the effect of IGF-I on proliferating cell nuclear antigen (PCNA) expression in cultured leiomyoma cells. Isolated human leiomyoma cells were subcultured in phenol red-free DMEM supplemented with 10% FBS for 120 h and then stepped down to serum-free conditions for an additional 72 h in the absence or presence of graded concentrations of IGF-I (1.0, 10, and 100 ng/ml). The effects of IGF-I on Bcl-2 protein and PCNA expression in cultured leiomyoma cells were assessed by Western immunoblot analysis and immunocytochemical staining, whereas the effects of IGF-I on the cell viability and apoptosis of the cultured cells were determined by 3-(4,5-dimethylatriazol-2-yl)-2,5diphenyltetrasodium bromide (MTT) assay and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling assay, respectively. Immunocytochemical staining demonstrated that IGF-I treatment resulted in the increase in PCNA labeling index in cultured leiomyoma cells in a dose-dependent manner. Immunoblot analysis of proteins extracted from the cultured leiomyoma cells revealed that the addition of IGF-I (10 and 100 ng/ml) significantly increased the expression of 35-kDa immunoreactive PCNA and 26-kDa Bcl-2 protein, compared with those in control cultures. Cell survival and proliferation of cultured leiomyoma cells, assessed by MTT assay, was significantly augmented by IGF-I treatment, compared with those of control cultures. Terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling assay showed that the apoptosis-positive rate of leiomyoma cells treated with IGF-I was significantly decreased, compared with that in control cultures. The present results suggest that IGF-I plays crucial roles in leiomyoma cell growth, not only in promoting the proliferative potential by up-regulation of PCNA expression but also in down-regulating apoptosis by up-regulation of Bcl-2 protein expression in leiomyoma cells.

High rates of hepatocellular carcinoma in cirrhotic patients with high liver cell proliferative activity.

The prevalence, risk factors, and clinical significance of high liver cell proliferative activity were investigated in 208 well-compensated cirrhotic patients (150 men; 50 years; 135 with chronic hepatitis C) who had been under prospective surveillance for hepatocellular carcinoma (HCC) with annual abdominal ultrasound (US) and serum alpha-fetoprotein (AFP) determination. Immunostaining for proliferating cell nuclear antigen (PCNA) was employed to assess liver cell proliferative activity in formalin-fixed, paraffin-embedded liver specimens. The percentage of reactive nuclei was calculated by a computer-assisted image analysis system. The overall PCNA labeling index (LI) ranged from 0.1% to 12.5% (mean, 2.1%), being significantly higher in the 50 patients who developed HCC during 88 +/- 42 months of follow-up than in the 158 patients who remained cancer-free (3.6% +/- 2.4% vs. 1.6% +/- 1.5%; P <.0001). By receiver operating curve (ROC), a 2.0% cut-off value of PCNA-LI discriminated between patients at high and low risk for developing cancer. By multivariate analysis, high histologic grading scores and gender were associated to PCNA LI >2.0%. The yearly incidence of HCC was 5.2% for the 80 patients with PCNA-LI >2.0% compared with 1.1% for the 128 with low PCNA-LI (relative risk, 4.90; 95% CI, 2.63-9.55). By multivariate analysis, PCNA-LI >2.0% was the strongest independent predictor of cancer (hazard ratio, 5.49; 95% CI, 2.90-10.37). Overall, survival was significantly lower in patients with high liver cell proliferative activity rates than in those with low proliferative rates (10% vs. 75%; P <.0001). In conclusion, development of HCC in patients with compensated cirrhosis seems to be reliably predicted by liver cell proliferation status.

Immunohistochemical study of p53, c-erbB-2, and PCNA in barrett's esophagus with dysplasia and adenocarcinoma arising from experimental acid or alkaline reflux model.

An immunohistochemical study of p53, c-erbB-2, and proliferating cell nuclear antigen (PCNA) in Barrett's esophagus with dysplasia and adenocarcinoma, arising from experimental acid or alkaline reflux, was performed in dogs. Cardiectomy was performed in group A (n = 26) as an acid reflux model, and total gastrectomy was performed in group B (n = 24) as an alkaline reflux model. After surgery, the esophageal mucosa was observed and biopsied endoscopically every 3 months over a period of 6 years. Immunohistochemical staining of p53. c-erbB-2, and PCNA was performed, using biopsied specimens. In group A, Barrett's esophagus developed in 14 of the 26 dogs. Low-grade dysplasia occurred in 5 of the 26 dogs, and in 1 of these 5 dogs, it developed into high-grade dysplasia. In this animal, adenocarcinoma arose 63 months after the operation. In group B, Barrett's esophagus developed in 10 of the 24 dogs. Low-grade dysplasia was observed in 4 of the 24 dogs. In 1 of these 4 dogs, the dysplasia became high-grade and adenocarcinoma occurred 66 months after the operation. In group A, PCNA was positive in adenocarcinoma; the PCNA labeling index (LI) was 58. c-erbB-2 and p53 were negative in all animals in group A. In group B, PCNA was positive in Barrett's esophagus with high-grade dysplasia and adenocarcinoma; the PCNA LI was 77. p53 was positive in adenocarcinoma. c-erbB-2 was negative in adenocarcinoma. CONCLUSIONS; The results of this study provided evidence of the dysplasia-carcinoma sequence arising from alkaline reflux, as well as from acid reflux. To the best of our knowledge, this is the first report of the use of an alkaline reflux model and a 6-year study using dogs to observe the course of Barrett's esophagus.

Association between Shear Stress, Angiogenesis, and VEGF in Skeletal Muscles In Vivo

ABSTRACTObjective: To investigate the hypothesis that capillary proliferation in skeletal muscles, induced by a long‐term increase in blood flow which elevates capillary shear stress, is associated with capillary expression of vascular endothelial growth factor (VEGF).Methods: Adult rats received prazosin in drinking water (∼2 mg per day) or had extensor digitorum longus (EDL) muscles stimulated by implanted electrodes for up to 14 days. At intervals, serial frozen sections of EDL were stained for alkaline phosphatase to identify capillaries, proliferating cell nuclear antigen (PCNA), and VEGF‐A protein. Shear stress was estimated from capillary red blood cell velocities and diameters, measured by direct observation of epiilluminated EDL.Results: Chronic stimulation and prazosin treatment both increased capillary: fiber ratio by ∼40% after 14 days. In stimulated muscles, the percentage of capillaries positively stained for VEGF increased within 3 to 4 days, while the density of PCNA‐positive capillaries had increased 20‐fold after 2 days. With prazosin, VEGF‐positive capillaries increased after 2 and 4 days, accompanied by a threefold increase in PCNA. By 14 days, PCNA labeling and VEGF were still high in stimulated muscles, but no longer different from controls with prazosin. After 3 to 4 days of treatment, capillary shear stress in resting muscle was 57% higher than in controls as a result of stimulation, but 4 times higher with prazosin.Conclusions: Higher capillary shear stress with prazosin than with stimulation may upregulate VEGF expression in the early stages of treatment. Greater proliferation of capillaries preceding a higher proportion of VEGF‐positive capillaries in stimulated muscles, in the presence of a modest increase in shear stress, suggests that angiogenesis was initiated by other factors in addition to shear stress.

Proliferating cell nuclear antigen labeling index and p53 expression predict outcome for breast cancer patients with four or more lymph node metastases.

In general, extensive lymph node metastasis indicates poor prognosis in breast cancer. Nevertheless, some patients with this condition survive for a long time without recurrent disease. This study involved 112 breast cancer patients with four or more lymph node metastases and without distant metastasis who underwent surgery. p53 expression was associated with shorter overall and disease-free survival. High PCNA labeling index was associated with shorter overall disease-free survival. p53-negative and low-PCNA patients survived much longer than p53-positive and high-PCNA patients. This study showed that p53-negative or low-PCNA index patients have relatively good prognoses despite extensive lymph node metastasis.

Expression of androgen receptor in meningiomas.

In order to investigate the expression of androgen receptor (AR) in meningiomas and its relation to tumor proliferative potential, we examined the expression of AR and proliferating cell nuclear antigen (PCNA) by avidine-biotin complex immunohistochemistry in 39 cases of meningiomas. Of the 39 cases of meningiomas, 20(51%) showed positive AR immunoreactivity. The AR expression positivity rates were 31% (6/19) in benign meningiomas, 58% (7/12) in atypical meningiomas, 87.5% (7/8) in malignant meningiomas, respectively. In addition to the tumor cells, cells of microvascular endothelial proliferation were frequently AR positive. Malignant meningiomas had a significantly higher percentage of AR positive cells compared with atypical and benign meningiomas (P < 0.05). The mean proliferating cell nuclear antigen labeling index (PCNA LI) was significantly higher in the malignant meningiomas when compared with atypical meningiomas (P < 0.05) and benign meningiomas (P < 0.05). AR positive meningiomas had higher PCNA LI than AR negative meningiomas (P < 0.05). The expression of AR in tumor tissues was significantly related with PCNA LI. These data indicated that AR in the meningiomas was correlated with histological grade and AR might participate in the growth of these tumors and tumor angiogenesis. The measurement of AR in these tumors may indirectly represent tumor growth potential.

Frequent p53 mutation in brain (fetal)-type glycogen phosphorylase positive foci adjacent to human ‘de novo’ colorectal carcinomas

‘de novo’ carcinogenesis has been advocated besides ‘adenoma carcinoma sequence’ as another dominant pathway leading to colorectal carcinoma. Our recent study has demonstrated that the distribution of brain (fetal)-type glycogen phosphorylase (BGP) positive foci (BGP foci) has a close relationship with the location of ‘de novo’ carcinoma. The aims of the present study are to investigate genetic alteration in the BGP foci and to characterize them in the ‘de novo’ carcinogenesis. 17 colorectal carcinomas without any adenoma component expressing both immunoreactive p53 and BGP protein were selected from 96 resected specimens from our previous study. Further investigations to examine the proliferating cell nuclear antigen (PCNA)-labelling index, and the p53 and the codon 12 of K-ras mutation using the polymerase chain reaction-single strand conformation polymorphism were performed in the BGP foci, BGP negative mucosa and carcinoma. The BGP foci were observed sporadically in the transitional mucosa adjacent to the carcinoma in all cases. The PCNA labelling index in the BGP foci was significantly higher than that in the BGP negative mucosa (P< 0.001). p53 mutations were observed in 8 carcinomas, but no K-ras mutation was detected. Interestingly, although none of the overexpressions of p53 protein was detected immunohistochemically in the BGP positive foci, the p53 gene frequently (41.2% of the BGP foci tested) mutated in spite of no K-ras mutation. The present study demonstrates potentially premalignant foci in the colorectal transitional mucosa with frequent p53 gene mutation. It is suggested that BGP foci are promising candidates for the further investigation of ‘de novo’ colorectal carcinogenesis. © 2001Cancer Research Campaign http://www.bjcancer.com

Prognostic value of hepatocyte proliferative activity after transjugular intrahepatic portosystemic shunt

OBJECTIVES: Previous data indicated that the proliferating cell nuclear antigen–labeling index (PCNA-LI) reflects the liver functional reserve in human liver cirrhosis. The aim of the study was to evaluate the hepatocyte proliferative activity as a marker for the outcome of patients after transjugular intrahepatic portosystemic shunt (TIPS). METHODS: Twenty-eight consecutive patients were electively treated with TIPS for recurrent variceal bleeding (n = 14), refractory ascites (n = 12), or hydrothorax (n = 2). PCNA immunostaining was analyzed on methanol-fixed, paraffin-embedded liver biopsies. RESULTS: After TIPS, six patients died within the first 3 months, eight other patients died later, two were transplanted, and 12 were alive at the time of analysis. Early death occurred in patients with refractory ascites (5/12) and/or in Child C patients (3/6). Among the evaluated variables, there was a statistical trend for the PCNA-LI to be lower in patients who died early after TIPS than in those having long term survival (1.55% vs 2.65%, p = 0.07). After TIPS insertion, the probability of remaining alive during the first 6 months of follow-up was significantly higher in patients with a preprocedural PCNA-LI > 2.9%. CONCLUSIONS: The PCNA-LI measured on liver biopsy before the TIPS procedure might be a pre-TIPS marker to discriminate those patients for whom TIPS is likely to be beneficial.

Expression of PDGF and its receptor as well as their relationship to proliferating activity and apoptosis of meningiomas in human meningiomas

In this study, we detected the expression of platelet-derived growth factor (PDGF) and its receptor in 61 human meningiomas by immunohistochemistry and in situ hybridisation. The results showed that almost all the 61 meningiomas expressed PDGFBB and PDGF β receptor and the positive rate of PDGFAA was 49%. Only two meningiomas expressed PDGF α receptor. The positive rate and the immunostaining intensity of PDGFBB and PDGF β receptor were higher in atypical meningiomas than in benign types. There was no significant difference between the different types of benign meningiomas. The expression of PDGFB chain mRNA was coincident with that of PDGFB chain protein. There was no co rrelation between the expression of PDGFAA and the types or grades of meningiomas. The correlation between overexpression of PDGFBB/R β and tumour grade provides a useful parameter in evaluating the prognosis of patients with meningiomas. The proliferative activity of meningiomas was evaluated by the proliferating cell nuclear antigen labelling index (PCNA LI). In the 61 meningiomas, t he average PCNA LI (%) was 1.8±1.3, 1.9±1.3, 1.7±0.8 and 11.6±5.3 (in fibrous, meningothelial, transitional and atypical meningiomas, respectively). Statistic analysis shows that the PCNA LI is higher in atypical meningiomas than that in benign types, and there was no significant difference between the different ty pes of benign meningiomas. The expression of PDGFBB and PDGF β receptor was significant enhanced in ascending order from low PCNA LI meningiomas to high ones. This result suggested that PDGFBB/R β autocrine loop may stimulate the growth of meningiomas. In this study, we also detected the cell apoptosis of meningiomas by terminal deoxynucleitidyl transferase mediated dUTP nick end labelling (TUNEL) method. The average apoptosis labelling index (%) was 0.11±0.05, 0.08±0.04, 0.09±0.03 and 0.35±0.15 in fibrous, meningothelial, transitional and atypical meningiomas respectively. The apoptosis labelling index was higher in atypical meningiomas than that in benign types. There was a positive correlation between the apoptosis labelling index and PC NA LI of meningioma. When the positive rate of PDGFBB and/or PDGF β receptor was higher in meningioma, the apoptotic cells was also increased. In conclusion, the overexpression of PDGFBB and its relevant receptor PDGFR β in meningiomas was correlated with grade of miningiomas and the proliferative activity of meningiomas; PDGFBB/R β autocrine loop may play an critical role in the pathogenesis of meningiomas.

Vascular endothelial growth factor secreted by replicating hepatocytes induces sinusoidal endothelial cell proliferation during regeneration after partial hepatectomy in rats

Background : The aim of this study was to investigate regulatory mechanisms of sinusoidal endothelial cell (SEC) proliferation after hepatectomy in rats. Methods : We investigated expressions of vascular endothelial cell growth factor (VEGF) and its receptors, flt-1 and KDR/flk-1, in regenerating liver after 70% hepatectomy. Proliferation of both hepatocytes and SECs was also monitored by evaluating the proliferating cell nuclear antigen (PCNA) labeling index. Furthermore, VEGF production by cultured hepatocytes isolated at different times after hepatectomy was measured in vitro. Results : The expression of VEGF mRNA was increased markedly between 48 and 72 h after hepatectomy, and thereafter decreasing gradually. The immunohistochemical staining revealed that expression of VEGF started to increase 24 h after hepatectomy, with a peak at 72 h, and the majority of the VEGF-positive cells were hepatocytes located in periportal areas. Meanwhile, expression of flt-1 and KDR/flk-1 was observed along the sinusoids even before hepatectomy, but was increased between 72 and 120 h. Furthermore, VEGF production by cultured hepatocytes isolated 72 h after hepatectomy was significantly increased. The PCNA labeling index of the SECs exhibited a delayed and slower regenerative response in comparison to the hepatocytes, reaching a peak at 72 h. Conclusions : These data strongly suggest that VEGF secreted by proliferating hepatocytes may represent an important stimulator of SEC proliferation.

Clinicopathological and prognostic significance of thymidine phosphorylase and proliferating cell nuclear antigen in gastric carcinoma

We have reported that thymidine phosphorylase (dThdPase) is identical to platelet derived-endothelial cell growth factor and that it has an angiogenic activity in vitro and in human carcinoma tissues as well as gastric carcinoma. Recently, we revealed that dThdPase may have an another function(s) besides angiogenesis in vitro and in human solid tumors. Using immunohistochemistry, we examined retrospectively whether the expression of dThdPase was correlated with tumor growth, comparing it with the proliferating cell nuclear antigen labeling index (PCNA LI) and examining their prognostic significance in 116 patients with gastric carcinoma. A direct correlation of these two factors was observed ( R=0.659, P<0.001). A multivariate Cox regression analysis showed that both dThdPase positivity and PCNA LI were independent prognostic factors, as were depth of invasion and lymph node metastasis. Furthermore, the patients with dThdPase-positive/high PCNA LI tumors had the worst prognoses. The combination of dThdPase and PCNA expression is a better tool for predicting the prognosis of patients with gastric carcinoma than the expression of either of them alone. These results raise the possibility that dThdPase may have a function(s) involved in tumor growth besides angiogenesis.