Proliferating Cell Nuclear Antigen Antigen Research Articles

Influence of Ki-67 and proliferating cell nuclear antigen expression on the biological behavior of ameloblastomas

Background: Ameloblastomas, the second-most common odontogenic tumors, are locally aggressive benign lesions that derive from cellular components of the enamel organ. Objective: The objective of the study is to assess the possible influence of cell proliferation markers Ki-67 and proliferating cell nuclear antigen (PCNA) on the biological behavior of ameloblastomas. Materials and Methods: A PubMed database search through February 2018, using the following medical subject headings (MeSH) terms were performed: “ameloblastoma” and (“Ki 67 antigen” or “proliferating cell nuclear antigen”). Selection Criteria: Studies with findings on cell proliferation markers in ameloblastoma. There were no restrictions regarding language or date of publication. Data Analysis: The data were analyzed using statistical software RevMan 5.3 (The Cochrane Collaboration, Oxford, UK). For continuous outcomes, the estimates of effects of an intervention were expressed as mean differences using the inverse variance method together with 95% confidence intervals. Results: Fourteen studies on cell proliferation markers in ameloblastoma were included in this meta-analysis. A greater significant Ki-67 expression in recurrent ameloblastomas (P < 0.01), in follicular type of solid/multicystic ameloblastomas (P < 0.05) and maxilla located ameloblastomas (P < 0.05) was found. In contrast, no significant influence of Ki-67 expression was observed in the following parameters: clinical type of ameloblastoma, histological type of unicystic ameloblastoma, age, gender, or size of the lesion. Finally, PCNA expression had no significant influence in any case. Conclusions: Ki-67 but not PCNA antigens seem to have an influence on the biological behavior of ameloblastomas.

Inhibitors of c-Jun phosphorylation impede ovine primordial follicle activation

Is the c-Jun-N-terminal kinase (JNK) pathway implicated in primordial follicle activation? Culture of ovine ovarian cortex in the presence of two different c-Jun phosphorylation inhibitors impeded pre-antral follicle activation. Despite its importance for fertility preservation therapies, the mechanisms of primordial follicle activation are poorly understood. Amongst different signalling pathways potentially involved, the JNK pathway has been previously shown to be essential for cell cycle progression and pre-antral follicle development in mice. Ovine ovarian cortex pieces were cultured with varying concentrations of SP600125, JNK inhibitor VIII or anti-Mullerian hormone (AMH) in the presence of FSH for 9 days. Follicular morphometry and immunohistochemistry for proliferating cell nuclear antigen (PCNA), apoptosis and follicle activation (Foxo3a) were assessed. Inhibition of primordial follicle activation occurred in the presence of SP600125, JNK inhibitor VIII and AMH when compared with controls (all P < 0.05) after 2 days of culture. However, only in the highest concentrations used was the inhibition of activation associated with induction of follicular apoptosis (P < 0.05). In growing follicles, PCNA antigen expression was reduced when the JNK inhibitors or AMH were used (P < 0.05 versus control), indicating reduced proliferation of the somatic compartment. Although we evaluated the effects of inhibition of c-Jun phosphorylation on primordial follicle development, we did not determine the cellular targets and mechanism of action of the inhibitors. These results are the first to implicate the JNK pathway in primordial follicle activation and could have significant consequences for the successful development of fertility preservation strategies and our understanding of primordial follicle activation. n/a. Dr Michael J. Bertoldo and the laboratories involved in the present study were supported by a grant from 'Région Centre' (CRYOVAIRE, Grant number #320000268). There are no conflicts of interest to declare.

P-0188 Chemopreventive Potential of 22β-hydroxyoleanonic Acid in experimental Colon Cancer model: Expression of Transcription Factors

ABSTRACT Introduction Triterpenoids have shown tremendous potential to be developed as chemopreventive agent against number of cancers. 22β-Hydroxyoleanonic acid is a semisynthetic triterpenoid, which has been synthesized from pentacyclic triterpenoid Lantadene A. We have determined the chemopreventive effect of 22β-hydroxyoleanonic acid in colon cancer, using in vitro and in vivo models. Methods The 22β-hydroxyoleanonic acid was synthesized from pentacyclic triterpenoid Lantadene A. This synthesis is a part of our Lantadene A pharmacophore optimization programme. The structure of 22β-hydroxyoleanonic acid was identified by spectral and elemental analysis. The ability to induce apoptosis was determined by its in vitro antiradical activity, cytotoxic studies using human colon adenocarcinoma and normal monkey kidney cell lines, and the expression of β-catenin and proliferating cell nuclear antigen (PCNA) in human colon cancer cell lines (COLO 320 DM). The chemopreventive potential of 22β-hydroxyoleanonic acid in colon carcinogenesis was assessed by injecting 1,2-dimethylhydrazine (DMH, 20 mg/kg b.w.) into male Wistar rats and supplementing this with 22β-hydroxyoleanonic acid throughout the experimental period of 20 weeks at 5, 10, and 20 mg/kg b.w. Further, effect of 22β-hydroxyoleanonic acid on various transcription factors was studied by ELISA and immunohistochemical localization Results 22β-Hydroxyoleanonic acid induced significant dose-dependent growth inhibition of COLO 320 DM cells (IC50 32.2 µM), induced apoptosis by scavenging reactive oxygen species, and suppressed the expression of β-catenin and PCNA antigens in human colon cancer cells. 22β-Hydroxyoleanonic acid supplementation reduced the number of aberrant crypt and crypt multiplicity in DMH-initiated rats in a dose-dependent manner with no toxic effects. Significant increase in the protein levels of c-jun, p65, and p53 by ELISA were observed in DMH treated mice tumors whereas less expression was observed in 22β-hydroxyoleanonic acid treated tumors. Further, immunohistochemical localization of transcription factors was studied which also showed less localization of c-jun, p65, and p53 in 22β-hydroxyoleanonic acid treated tumors as compared to localization in DMH treated tumors. Conclusion The study showed that 22β-hydroxyoleanonic acid showed potential chemopreventive activity and may be linked to the deregulation of above molecular targets which warrants further studies.

Assessment of bone marrow angiogenesis using F VIII-related antigen and its relationship to proliferating cell nuclear antigen (PCNA) in multiple myeloma.

Background: Multiple myeloma (MM) is a malignant clonal expansion of plasma cells. Previous studies had demonstrated that both bone marrow angiogenesis as measured by microvessel density (MVD) and PCNA were increased in variety of malignant disorders, including multiple myeloma.Objective: Assessment of angiogenesis in bone marrow of MM patients by using immunohistochemical stain to measure microvessel density (MVD) using factor VIII –related antigen and to evaluate the expression of PCNA antigen in plasma cells of MM bone marrow sections . Also to find the correlation between these two parameters and between them and the percentage of plasma cells infiltration in bone marrow of MM.Patients, materials and methods: This retrospective study was conducted from May 2007 to Jaunary 2008 on 37 bone marrow biopsies diagnosed as multiple myeloma, along with 10 age matched control subjects who had reactive plasmacytosis of less than 10% in their bone marrow. The cases were retrieved from recording archive files of Department of Pathology in the Teaching Laboratory of Medical City Hospital, laboratory of Al- Yarmouk hospital and AL-Yarmouk National Center of Haematology. Three sections were taken from each formalin fixed paraffin embedded bone marrow trephine biopsy of MM and of control cases. One representative section was stained with Hematoxylin and Eosin (H&amp;E), while the other two sections were stained immunohistochemically for factor VIII-related antigen as an endothelial cell marker, and for PCNA as an indicator of the proliferative state of myeloma cells All stained sectionswere examined by light microscopy and the mean vessel density (MVD) was estimated and was used for statistical analysis. For immunostained PCNA cells, the labeling index scoring system of Alves et al was adopted for estimating the percentage of positive nuclear staining.Results: This study revealed that there was a significant increase in bone marrow angiogenesis and in the expression of PCNA in plasma cells of patients with multiple myeloma compared to control cases. Moreover this study showed that PCNA correlated significantly with bone marrow microvessel density and both parameters correlated significantly with the percent of plasma cell in the bone marrow of patient with multiple myeloma.Conclusion : Both PCNA and angiogenesis as expressed by MVD were increased in multiple myeloma and both of them correlated with the percentage of plasma cell infiltration which reflected the activity of the disease .Moreover the proliferative activity of plasma cell as represented by PCNA expression was closely related to angiogenic activity , thus it can be proposed that both markers reflect the disease activity and they may provide additional information when included in the initial evaluation of myeloma bone marrow biopsies .

Fascaplysin exert anti-tumor effects through apoptotic and anti-angiogenesis pathways in sarcoma mice model

Previous studies indicated that fascaplysin derived from marine sponge can induce tumor cell death by apoptosis and possesses anti-angiogenesis activity. In order to verify these two effects in animal model and to identify action mechanisms, we established a sarcoma mice model, and treated mice with fascaplysin for 10 days. The tumor tissues were examined morphologically and immunohistochemically. The differential gene expression was also investigated by mRNA array. Fascaplysin treatment resulted in a significant suppression of tumor growth. Typical apoptotic phenomena were observed by transmission electron microscope and histological detection. Tissue sections were stained with monoclonal antibody directed to proliferating cell nuclear antigen (PCNA) and CD31. The decreased PCNA and CD31 antigen staining indicate the reduction of tumor cell proliferation and tumor vasculature property of fascaplysin in vivo. Microarrays were used to examine the gene expression profiles of tumors on CapitalBio mouse genome oligo array. The regulated genes analyzed from the expression level showed overlapping gene ontology (GO) categories and pathway mapping. Our findings indicate that cell cycle arrest, apoptosis, regulation of actin cytoskeleton, and cell adhesion all play important roles in the onset of fascaplysin. Detailed analysis by real time PCR of key genes confirmed the experimental results of microarrays. From these findings, it can be considered that fascaplysin can inhibit the growth of S180 cell implanted tumor, and the action mechanisms may involve in apoptosis, anti-angiogenesis, or cell cycle arrest.

Chemopreventive potential of beta-Sitosterol in experimental colon cancer model--an in vitro and In vivo study.

BackgroundAsclepias curassavica Linn. is a traditional medicinal plant used by tribal people in the western ghats, India, to treat piles, gonorrhoea, roundworm infestation and abdominal tumours. We have determined the protective effect of β-sitosterol isolated from A. curassavica in colon cancer, using in vitro and in vivo models.MethodsThe active molecule was isolated, based upon bioassay guided fractionation, and identified as β-sitosterol on spectral evidence. The ability to induce apoptosis was determined by its in vitro antiradical activity, cytotoxic studies using human colon adenocarcinoma and normal monkey kidney cell lines, and the expression of β-catenin and proliferating cell nuclear antigen (PCNA) in human colon cancer cell lines (COLO 320 DM). The chemopreventive potential of β-sitosterol in colon carcinogenesis was assessed by injecting 1,2-dimethylhydrazine (DMH, 20 mg/kg b.w.) into male Wistar rats and supplementing this with β-sitosterol throughout the experimental period of 16 weeks at 5, 10, and 20 mg/kg b.w.Resultsβ-sitosterol induced significant dose-dependent growth inhibition of COLO 320 DM cells (IC50 266.2 μM), induced apoptosis by scavenging reactive oxygen species, and suppressed the expression of β-catenin and PCNA antigens in human colon cancer cells. β-sitosterol supplementation reduced the number of aberrant crypt and crypt multiplicity in DMH-initiated rats in a dose-dependent manner with no toxic effects.ConclusionWe found doses of 10-20 mg/kg b.w. β-sitosterol to be effective for future in vivo studies. β-sitosterol had chemopreventive potential by virtue of its radical quenching ability in vitro, with minimal toxicity to normal cells. It also attenuated β-catenin and PCNA expression, making it a potential anticancer drug for colon carcinogenesis.

Relationship between epithelial-immunologic cells transdifferentiation and pseudoepitheliomatous granuloma lesion

Inappropriate treatment at early stage of wound could result in the formation of pseudoepitheliomatous granuloma (PEG). The correlation of abnormal transdifferentiation of epithelial cells to immunologic cells and the occurrence of PEG lesion was investigated. Morphological change of epithelial tissue was observed with histopathology in 11 specimens of PEG lesions and 6 specimens of normal skins from PEG edge (PEG-N) from 11 patients with damaged skin. The expression characteristics and distribution of pan-cytokeratin (CKp), IV type collagen, laminin (LM), epithelial cadherin (E-Cad), beta-catenin (beta-Cat), focal adhesion kinase (FAK), stem cell factor (SCF) and its receptor-c-Kit, proliferating cell nuclear antigen(PCNA), and cluster of differentiation-14 (CD14), CD68 and mast cell tryptase (MCT) in PEG were detected with the immunohistochemical and the indirect immunofluorescent double-staining. In comparison with PEG-N, epithelial tissue take on squamous metaplasia, and stroma was infiltrated with intensive microvessels and inflammatory cells in the PEG lesion. Poor epithelial basal layer constitution, basal polarization, and migration of basal cells to stroma could be observed. In the ultrastructure, the loose intercellular junction of basal cells and the increased nucleus/cytoplasm ratio and intercellular space could be observed, neonatal monocytoid cells and macrophages and mast cells as a exuviate-like manner brooded from cytoplasm of original epithelial cells and basement membrane. protein expression of CKp and E-Cad by basal cells was significantly decreased, and the IV type collagen and LM protein could not be found in basement membrane of identical locus. By contrast, the immunoreactivity of beta-Cat and FAK was apparently increased. In addition, CD14(+) monocytes, CD68(+) macrophages, MCT(+) mast cells and CD68(+)/MCT(+) cells with various size, and these cells of stronger immuno-staining of SCF, c-Kit and PCNA antigen could be found in epithelial tissue and stroma. Epithelial cells in PEG related to wound are characteristized by transdifferentiation of epithelial cells to immunologic cells, wich may be associated with local infectious and inflammatory reaction, ultimately resulting in enhancement the ratio of beta-Cat/E-Cad signal and activation SCF-c-Kit signal pathway. The phenomena of transdifferentiation epithelial cells in the PEG lesion will help to recognize of the neoplatic immune and trauma repair mechanism.

Immunohistochemical Analysis of Cell Proliferation and Suppression of Ameloblastoma with Special Reference to Plexiform and Follicular Ameloblastoma

To understand proliferation and suppression in ameloblastoma that exhibit locally invasive growth and recur clinically, the expression of proliferating cell nuclear antigen (PCNA), Ki-67 antigen, topoisomerase IIα, p53 and p21 proteins were analyzed with immunohistochemistry. Sections of archival ameloblastoma tissues were used (16 plexiform and 14 follicular types). Each antigen was labeled by the polymer method with optimal antigen retrieval. The plexiform type exhibited higher expression of PCNA, Ki-67 and topoisomerase IIα antigens than the follicular type. Expression of PCNA correlated significantly with that of the Ki-67 antigen. The plexiform type also exhibited greater expression of p53 and p21 proteins than the follicular type. Expression of p53 protein correlated significantly with that of p21, PCNA, Ki-67 antigen and topoisomerase IIα. Expression of p21 protein did not correlate with that of the proliferation-related antigens. These findings suggest that the plexiform ameloblastoma has higher proliferating activity and malignant potentiality as neoplastic cells than the follicular ameloblastoma.

Multiparametric flow cytometric analysis in a breast cancer cell line (MCF-7).

To investigate whether multiparameter flow cytometric analysis of solid tumor specimens, including gynecologic tumors, which were stained triply with phycoerythrin (PE), fluorescein isothiocyanate (FITC) and propidium iodide (PI), can be performed simultaneously without interference from normal diploid cell populations and spectral overlap on a standard flow cytometer. MCF-7 breast cancer cell lines and heterogeneous cell populations mixed with MCF-7 cells and human peripheral blood lymphocytes (PBL) were fixed with 1% paraformaldehyde and permeabilized with 100% methanol. Cytokeratin and several proliferation-associated cellular antigens (proliferating cell nuclear antigen (PCNA), p53, c-erbB/2 and c-myc) were labeled with PE and FITC, which was followed by DNA staining using PI. These labeled cells were measured on a standard FACScan flow cytometer equipped with a 488 nm single laser. The coefficient of variation (CV) of the G0G1 peak of MCF-7 cells was 4.3 and the cell cycle phase fractions of G0G1, S and G2M were 44.9, 45.9 and 9.2%, respectively. Fluorescein isothiocyanate, PE and PI fluorescences were detected without interference. The MCF-7 cells expressed cytokeratin, PCNA, p53, c-erbB/2 and c-myc antigen. In the heterogeneous population of MCF-7 cells mixed with PBL, two cellular populations were clearly separated into diploid PBL and aneuploid MCF-7 cells without interference. The CV of G0G1 peak of PBL was 2.3 and the G0G1, S and G2M phase fractions were 85.5, 2.7 and 11.8%, respectively. The DNA index of MCF-7 cells was 1.7, which indicated that the MCF-7 cell line was composed of tumor cells with aneuploid DNA. The CV of the G0G1 peak of the MCF-7 cells was 4.2, and the cell cycle phase fractions were 47.5% for G0G1, 42.3% for S, and 10.2% for G2M. The MCF-7 cells expressed cytokeratin, but the PBL did not. Multiparameter flow cytometer analysis was useful to determine DNA ploidy status, phase fraction of the cell cycle and expression of cellular antigens and selective cytokeratin expression allowed epithelial originated tumor cells to be differentiated from normal stromal cells. This analysis could be performed without interference of spectral overlaps of fluorochromes using software-based algorithmic compensation of spectral overlaps. Thus, this method offers new possibilities for multiparameter flow cytometric analysis and its use should be extended to future studies of the diagnosis, treatment and prediction of prognosis of the neoplasm.

Fas antigen expression and outcome of oral squamous cell carcinoma

Paraffin sections of biopsy specimens obtained from 46 patients with oral squamous cell carcinomas were stained with both anti-peptide antibody against human Fas antigen and monoclonal mouse antibody against human proliferating cell nuclear antigen (PCNA). The patients received chemotherapy with a combination of carboplatin and peplomycin sulfate or mitomycin C and peplomycin sulfate before surgery. The relation between the expression of Fas antigen and the clinical features of each case was examined. The correlation between PCNA and Fas antigen expression was also studied. The mean PCNA labeling index of the 22 Fas-negative cases was 46.9%, which was significantly higher than that of the 24 Fas-positive cases (39.5%). Strong correlations were found between the expression of Fas antigen and the response to chemotherapy, tumor recurrence, and survival. The Fas-negative group had only a minor response to chemotherapy and a poor outcome, whereas the Fas-positive group had a better response to chemotherapy and a good outcome. Although lymph node metastasis was significantly related to survival, there was no correlation between Fas antigen expression and lymph node metastasis. The Kaplan-Meier survival curve of patients positive for Fas antigen was significantly better than that of patients negative for Fas antigen. Our results suggest that Fas antigen expression is an independent predictor of outcome whose usefulness should be evaluated in prospective studies.

Clinical relevance of p53 index and expression of proliferating cell nuclear antigen and Ki-67 in gastric cancer.

The prognostic value of the immunohistochemical expression of p53 protein, proliferating-cell nuclear antigen (PCNA) and Ki-67 antigen was evaluated in a series of 116 stage I-II gastric cancer patients. The staining for p53 protein (staining frequency and intensity) in malignant cells was expressed as a p53 index. Similarly, the staining frequency and intensity for PCNA and Ki-67 were evaluated. The p53 index was independent of the stage and differentiation grade, but significantly related to DNA ploidy, S-phase fraction and mitotic activity. A high p53 index was a sign of inferior survival, compared to a low or intermediate index. p53-negative tumours were also associated with poor survival. In a multivariate analysis, only the depth of tumour infiltration and the presence of nodal metastases were independent prognostic factors in stage I-II gastric cancer. PCNA expression and Ki-67 antigen expression were not related to the stage, ploidy, proliferative activity or p53 expression, and they had no impact on survival. The results indicate that p53 protein expression may be of prognostic significance in gastric cancer, while PCNA and Ki-67 antigen expression have no predictive value.

Cellular apoptosis and proliferation in experimental renal fibrosis.

The progression of chronic renal failure (CRF) is associated with the progressive deletion of renal cells along with the fibrosis of the kidney. We have studied the role of programmed cell death (apoptosis) in the progression of experimental CRF and renal scarring. The sub-total (5/6th) nephrectomy (SNx) model of CRF was studied in adult male Wistar rats, with renal tissue collected from experimental and control animals on days 7, 15, 30, 60, 90, and 120 post SNx (n = 6 per group). These were examined for morphological signs of apoptosis by light and electron microscopy. Further, we stained the nuclear chromatin by the acridine orange fluorescent method and detected signs of DNA cleavage by endonucleases via the principal of TUNEL staining (ApopTag). Rates of cellular proliferation were measured simultaneously by immunohistochemical staining for the proliferating cell nuclear antigen (PCNA). In addition, cell division was monitored by counting of morphologically mitotic motifs detectable by light microscopy. Progressive renal insufficiency associated with glomerulosclerosis and tubulointerstitial fibrosis took place in the majority of SNx rats. In these animals, we noted a marked and progressive increase in the number of apoptotic glomerular, tubular as well as interstitial cells. The most significant apoptotic changes were seen in the tubules of remnant kidneys peaking at day 120 post-SNx. At this stage, the increase in apoptosis compared to controls was 10.33+/-2.67 (M+/-SEM) fold for glomerular cells (P< or =0.006), 26.20+/-4.56 fold for tubular cells (P < 0.0001) and 4.66+/-0.81 fold for interstitial cells (P< or =0.001). Parallel changes in the number of PCNA positive renal cells were observed. Maximal PCNA staining was seen at day 120 when the increase with respect to controls was 14.00+/-4.93 fold (P< or = 0.05) for glomerular cells, 60.01+/-12.20 fold (P< or =0.05) for tubular cells and 28.59+/-4.45 fold (P< or = 0.05) for interstitial cells. As expected, the number of cells undergoing division and detectable by conventional light microscopy was lower at any time point to those expressing PCNA. We also observed a close correlation between the severity of tubular atrophy and tubulointerstitial fibrosis with the rate of tubular apoptosis (r=0.970, R2 =0.941, P< or =0.001). We have shown a time-dependent increase in apoptosis and PCNA antigen positive staining in the sub-total nephrectomy model of chronic renal failure correlating with the progression of renal fibrosis. PCNA staining did not match analysis for mitosis and was considered to overestimate the number of proliferating cells in the tissue. With this reservation in mind and taking into account the relative time-frames in vivo of apoptosis and proliferation; apoptosis potentially outweighs proliferation by a factor of 2 8-fold, when examined over the same time period. Consequently, even small changes in the finite numbers of apoptotic cells become highly significant. Our results have shown the definite role of apoptosis within progression of renal damage and highlighted how it may contribute to the progression of tubular atrophy and play a role in the pathogenesis of tubulo-interstitial scarring.

Autoantibodies recognizing proteins copurified with PCNA in patients with connective tissue diseases.

Proliferating cell nuclear antigen (PCNA), one of the target antigen recognized by lupus sera, has been reported to be present as a subnuclear multi-peptide complex. But autoantibodies reacting with components of PCNA complex are poorly understood. To study the specificity of those autoantibodies, immunoreactivities of autoimmune sera against purified PCNA antigen were studied. PCNA antigens were purified from rabbit thymus extract by affinity column using murine monoclonal antibodies (mAbs) to PCNA, TOB7, TO17 and TO30. Immunoreactivities of autoimmune sera against purified PCNA were analyzed by WB. PCNA antigen purified by serum AK predominantly showed a 34 kD band specific for PCNA in SDS-PAGE. When antigens were purified by anti-PCNA mAb TOB7 and TO30 which are known to be targeting different epitopes on PCNA antigen, SDS-PAGE analysis showed various mol. wt of proteins in addition to the 34 kD PCNA while both AK and mAbs reacted only with 34 kD PCNA in WB. In WB using PCNA purified by TOB7, various immunoreactivities were observed at 150, 66, 58, 48, 45, 37, 32 and 16 kDa in sera from patients with connective tissue diseases. These results suggested that many of the proteins copurified with PCNA were also targets of autoimmune responses and these autoantibody expression may be induced through antigen-driven mechanisms.

Serum hepatocyte growth factor levels in primary biliary cirrhosis

Recent studies have shown that serum levels of hepatocyte growth factor, a potent mitogen for hepatocytes, are increased in patients with fulminant liver failure and with advanced stages of liver cirrhosis. Retrospective study. Serum levels of hepatocyte growth factor (HGF) were determined in 26 patients with advanced stages of liver cirrhosis by means of an enzyme-linked immunosorbent assay. In 23 of these patients, liver biopsies were obtained before treatment with ursodeoxycholic acid and investigated for hepatocyte expression of the proliferating cell nuclear antigen (PCNA) and the Ki-67 antigen by immunocytochemical methods. In 25 of the 26 patients, serum HGF levels did not differ from those of patients with histologically normal livers. Levels did not vary between the different stages of primary biliary cirrhosis but increased moderately when re-evaluated after a period of 24 +/- 4.9 months or 54 +/- 10.7 months. An abnormally increased serum HGF level was observed in only one patient, who had end-stage liver cirrhosis before liver transplantation. Both PCNA and Ki-67 antigen labelling indices were significantly higher at all stages of primary biliary cirrhosis than in normal liver, indicating increased hepatocyte proliferation, but the labelling indices did not correlate with the HGF concentrations in serum. Serum HGF levels in almost all patients with primary biliary cirrhosis were within normal limits despite increased hepatocyte proliferation. The results support the hypothesis that HGF serum levels may reflect liver dysfunction rather than active hepatocyte proliferation.

Evaluation of proliferation parameters in in vivo bromodeoxyuridine labelled lung cancers.

In a series of 44 bronchial biopsies from patients suspected of having endobronchial lung carcinoma, the validity of proliferating cell nuclear antigen (PCNA) and Ki67 antigen as proliferative indicators was evaluated in ethanol fixed, paraffin embedded tissue. The percentages of cells positive for these markers were compared to the in vivo bromodeoxyuridine (BrdU) labelling index. A good correlation was found between PCNA immunoreactivity and BrdU labelling index, while Ki67-antigen expression showed a significant relation with BrdU labelling index and with PCNA expression. All three parameters showed a trend towards similar values for the individual cases. Based on the fact that Ki67 antigen is expressed in all cycling cells, whereas replicon-associated PCNA and BrdU only reflect the S-phase fraction, the differences between Ki67-antigen scores on the one hand and BrdU and PCNA scores on the other were smaller than expected. In order to determine the degree of concordance between immunohistochemically and flow cytometrically detected proliferation variables, BrdU incorporation was measured using both methods in duplicate bronchial specimens. Discrepancies in labelling indices were observed predominantly in DNA diploid samples, with consistently lower values in the flow cytometrically analysed specimens. In tumour specimens with an aneuploid DNA content, flow cytometric determination of proliferative activity yielded results similar to those obtained by tissue section examination. We conclude that the scores for PCNA and Ki67 antigen, immunohistochemically detected in ethanol fixed, paraffin embedded tissue reflect functional proliferative activity.

Cell kinetics in human breast cancer: comparison between the prognostic value of the cytofluorimetric S-phase fraction and that of the antibodies to Ki-67 and PCNA antigens detected by immunocytochemistry.

The determination of cell proliferation is one of the more widely used tools for assessing prognosis. However, additional research in this field is warranted because today there are several methodological procedures available for monitoring cell kinetics and it has still not been established which is the most reliable marker of proliferation and which possesses the greatest prognostic value. We performed this study in a series of primary invasive breast cancers to compare the prognostic value of S-phase fraction (SPF) by flow cytometry, the most widely used method for detecting proliferation at present, with that of antibodies to Ki-67 and PC-10 to proliferating-cell nuclear antigen (PCNA) detected by immunocytochemical methods. A significant linear relationship was observed only between SPF and Ki-67. In univariate analysis SPF and Ki-67 values, nodal status, histological grading and peritumoral lymphatic-vessel invasion were significant predictors of relapse-free survival (RFS). As far as overall survival (OS) is concerned, only SPF, Ki-67 and nodal status were significantly associated with the risk of death. PCNA had no prognostic value for either RFS or OS. In multivariate analysis only SPF and nodal status retained a significant and independent prognostic value. Neither the cell-kinetics parameters assessed by immunocytochemistry (i.e. Ki-67 and PCNA) nor histological grading were independent prognosticators. In conclusion, our results provide evidence that the determination of SPF by flow cytometry was the strongest cell-kinetics marker used to assess prognosis in this series of breast cancers. However, different and novel markers of cell kinetics need to be compared in larger series in order to identify the best one.

Correlation between PCNA and AgNOR scores in non-Hodgkin's lymphomas using sequential staining technique.

To investigate the relation between the numbers of interphase silver stained nucleolar organiser regions (AgNORs) and immunolabelling with the monoclonal antibody PC10, which demonstrates proliferating nuclei by reacting with proliferating cell nuclear antigen (PCNA). The established sequential technique for the demonstration of interphase AgNORs and of antigens in paraffin wax sections was applied to a small series of non-Hodgkin's lymphomas (NHL) of both low and high grade type, together with specimens of palatine tonsil. The numbers of PCNA positive cells were counted in all specimens; in the tonsils the cells were counted in both follicle centres and in the interfollicular areas. The numbers of AgNORs in both PCNA positive and PCNA negative nuclei were then counted. Lower numbers of PCNA positive cells were found in the low grade than the high grade NHL (18-28.4% and 34.2-51.3%, respectively). This was reflected in the two areas of the palatine tonsils, the counts being higher in the follicle centre nuclei than in those in the interfollicular compartments. In general, the numbers of AgNORs were higher in the PCNA positive nuclei than in those lacking the antigen; however, a consistent finding was that relatively high AgNOR scores were observed in PCNA negative nuclei, especially in the high grade lymphomas. In the tonsils, however, the AgNOR counts were much lower in the nuclei lacking PCNA than in those containing it. The results obtained in the lymphomas were rather unexpected as, in general, previous studies have shown a close direct or indirect relation between AgNOR scores and proliferating cell counts. An explanation for these findings may be that the argyrophil proteins associated with proliferating cells remain in the nucleus in detectable form longer than the PCNA antigen, at least in neoplastic tissue.

Detection of A 140 KDA nucleolar protein with an anti-PCNA autoantibody

To assess the numbers and types of PCNA (proliferating cell nuclear antigen) species, immunoprecipitation studies on HeLa cell, nuclear and nucleolar extracts were performed. A 140 KDa protein from HeLa nucleoli was immunoprecipitated by an autoantibody (E.B.) previously used to detect the proliferating cell nuclear antigens (PCNA). The 140 KDa protein was also detected in the nuclear extract of colon carcinioma cells (Ω) labeled in vitro with 125I-Bolton Hunter reagent. When the growth of the colon carcinoma cells was inhibited by 1% N,N-dimethylformamide for two weeks, the 140 KDa protein was not detected which suggests this protein is associated with cell growth. Several studies have indicated there are nucleolar antigens associated with various stages of the cell cycle. These antigens are referred to as PCNA (Proliferating Cell Nuclear Antigen), SS-B/La (Tan, 1982; Deng et al. , 1981) and RZ-2 (Zweig et al. , 1984). The PCNA antigens were detected in nucleoli of tumor and normal growing cells but not in normal resting tissues (Chan et al. , 1983; Smetana et al. , 1983). Using the immunofluorescence technique, the PCNA antigen was found in nucleoplasm and localized to the nucleoli during the G 1/S phase of the cell cycle (Chan et al. , 1983; Smetana et al. , 1983; Miyachi et al. , 1978; Takasaki et al. , 1981). It was enriched in the nucleoli of rapidly growing tumor cells (Chan et al. , 1983). Several proteins which reacted with the PCNA antibody have been described (Chan et al. , 1983; Yoshinari et al. , 1984; Mathews et al. , 1984). We previously reported that one such protein with a molecular weight of 40 KDa and a pI of 5.0 was immunoprecipitated from total HeLa cell extract (Chan et al. , 1983). Another antigen with molecular weight 33 KDa was partially purified from rabbit thymus extract by Yoshinari et al. , 1984. Mathews et al. (1984) reported that the 35 KDa antigen is related to “cyclin” (Bravo et al. , 1981). The present study was designed to identify these antigens in isolated nucleoli. A 140 KDa protein was detected in nucleoli of HeLa and colon carcinoma cells.