MDM4 overexpression contributes to synoviocyte proliferation in patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease with features of inflammatory cell infiltration, synovial cell invasive proliferation, and ultimately, irreversible joint destruction. It has been reported that the p53 pathway is involved in RA pathogenesis. MDM4/MDMX is a major negative regulator of p53. To determine whether MDM4 contributes to RA pathogenesis, MDM4 mRNA and protein expression were assessed in fibroblast-like synoviocytes (FLS) by real-time PCR, western blotting, and in synovial tissues by immunohistochemistry. Furthermore, MDM4 was knocked down and overexpressed by lentivirus-mediated expression, and the proliferative capacity of FLS was determined by MTS assay. We found that cultured FLS from RA and osteoarthritis (OA) patients exhibited higher levels of MDM4 mRNA and protein expression than those from trauma controls. MDM4 protein was highly expressed in the synovial lining and sublining cells from both types of arthritis. Finally, MDM4 knockdown inhibited the proliferation of RA FLS by enhancing functional p53 levels while MDM4 overexpression promoted the growth of RA FLS by inhibiting p53 effects. Taken together, our results suggest that the abundant expression of MDM4 in FLS may contribute to the hyperplasia phenotype of RA synovial tissues.     

Apoptosis resistance in pigmented villonodular synovitis

OBJECTIVE: Pigmented villonodular synovitis (PVNS) is a proliferative lesion originating from synovial tissue with a locally aggressive behaviour. We analysed the pathogenetic role of apoptosis resistance for sustained cell proliferation in PVNS. METHODS: The expression of bcl-2, p53 and Ki-67 was examined in 80 cases of PVNS using immunohistochemistry. In 43 of these cases, DNA content and distribution of cell-cycle phases were investigated by flow cytometry. Additionally, 10 cases of PVNS were analysed by multi-parametric flow cytometry for expression of p53, caspase3, and bcl-2 and by TUNEL to detect DNA fragmentation. RESULTS: No apoptotic cell fractions were detected in any investigated cases. Expression of bcl-2 was found in 84% of cases (up to 6.5% of cells) and was significantly associated with DNA-fragmentation observed by TUNEL (p=0.037). Orthologous p53 expression was observed in 37% of cases. The level of p53 expression correlated with the proliferative activity and the expression of both caspase3 (p=0.017) and bcl-2 (p=0.0013). (No statistically significant correlations between expression of bcl-2, p53, caspase3, DNA fragmentation or proliferative index and age, sex of patients, disease recurrence, growth pattern or size of lesion were found). CONCLUSION: Apoptosis resistance is a critical event in the progression of PVNS and may contribute to the survival of the proliferating synovial cells in PVNS and to the permanent slow progression of these lesions.     

Expression of Ki-67, p53 and p63 proteins in keratocyst odontogenic tumours: an immunohistochemical study

Aim To investigate the immunohistochemical expression of Ki-67, p53 and p63 in Keratocyst Odontogenic Tumours (KOTs) in order to contribute to the biological profile of this tumor. Methods Immunohistochemical technique was performed using the EnVision™ System in 37 cases of KOTs. Results Ki-67- and p53-immunostained cells were mainly located in the suprabasal layers. p63-positive cells were found throughout the lining cystic epithelium. No difference in the immunostaining for these proteins was observed between primary and recurrent KOTs (Ki-67: P = 0.5591; p53: P = 0.9847; p63: P = 0.9127), or between KOTs associated with Nevoid Basal Cell Carcinoma Syndrome (NBCCS) and sporadic KOTs (Ki-67: P = 0.7013; p53: P = 0.3197; p63: P = 0.2427). Conclusions It is possible that biological behavior of KOTs may be related to suprabasal proliferative compartment in the cystic epithelium as observed by high levels of Ki-67, p53 and p63. In addition, p63 immunostaining may represent immaturity of keratinocytes in KOTs, and suggests that this protein may participate in the regulation of epithelial cell differentiation. Taken together, these data may favor tumorigenesis on KOTs.     

Expression and function of cell cycle proteins in rheumatoid arthritis synovial tissue

Rheumatoid Arthritis (RA) is a chronic disease characterised by synovial lining hyperplasia and progressive destruction of joint tissues. Experimental data suggests that abnormal alterations in the expression of proteins involved in maintaining homeostatic control of the cell cycle is involved in disease progression in RA. By contributing to the overgrowth of synovial tissue, factors such as dysregulated proliferation or reduced apoptosis of cells can directly influence the pathological outcome of RA.     

A cell-cycle independent role for p21 in regulating synovial fibroblast migration in rheumatoid arthritis

Rheumatoid arthritis (RA) is characterized by synovial hyperplasia and destruction of cartilage and bone. The fibroblast-like synoviocyte (FLS) population is central to the development of pannus by migrating into cartilage and bone. We demonstrated previously that expression of the cell cycle inhibitor p21 is significantly reduced in RA synovial lining, particularly in the FLS. The aim of this study was to determine whether reduced expression of p21 in FLS could alter the migratory behavior of these cells. FLS were isolated from mice deficient in p21 (p21^(-/-)) and were examined with respect to growth and migration. p21^(-/-) and wild-type (WT) FLS were compared with respect to migration towards chemoattractants found in RA synovial fluid in the presence and absence of cell cycle inhibitors. Restoration of p21 expression was accomplished using adenoviral infection. As anticipated from the loss of a cell cycle inhibitor, p21^(-/-) FLS grow more rapidly than WT FLS. In examining migration towards biologically relevant RA synovial fluid, p21^(-/-) FLS display a marked increase (3.1-fold; p < 0.05) in migration compared to WT cells. Moreover, this effect is independent of the cell cycle since chemical inhibitors that block the cell cycle have no effect on migration. In contrast, p21 is required to repress migration as restoration of p21 expression in p21^(-/-) FLS reverses this effect. Taken together, these data suggest that p21 plays a novel role in normal FLS, namely to repress migration. Loss of p21 expression that occurs in RA FLS may contribute to excessive invasion and subsequent joint destruction.     

NK4 inhibits the proliferation and induces apoptosis of human rheumatoid arthritis synovial cells

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by proliferation and insufficient apoptosis of synovial cells. NK4 is a hepatocyte growth factor antagonist and is implicated in cell proliferation, viability, and apoptosis of many tumour cells. This study aimed to investigate the role of NK4 in the regulation of human RA synovial cell proliferation and apoptosis. Fibroblast‐like synoviocytes (FLSs) isolated from RA patients and MH7A synovial cells were subjected to MTT, flow cytometry, and Western blot analysis. We found that NK4 suppressed cell proliferation through cell cycle arrest at the G0/G1 phase and induced apoptosis in RA synovial cells. Furthermore, NK4 altered the expression of cell cycle and apoptosis‐related proteins such as cyclin D1, cyclin B1, PCNA, p21, p53, Bcl‐2, Bax, cleaved caspase‐9, and cleaved caspase‐3. Additionally, NK4 reduced the phosphorylation level of NF‐κB p65 and upregulated the expression of sirt1, but did not change the levels of p38 and p‐p38 in RA‐FLS and MH7A cells. In conclusion, NK4 inhibits the proliferation and induces apoptosis of human RA synovial cells. NK4 is a promising therapeutic target for RA. We demonstrated that NK4 inhibited cell proliferation by inducing apoptosis and arresting cell cycle in RA‐FLS and MH7A cells. The apoptotic effects of NK4 may be mediated in part by decreasing Bcl‐2 protein level, increasing Bax and caspase 3 protein levels, and inhibiting NF‐κB signalling in RA‐FLS and MH7A cells. These findings reveal potential mechanism underlying the role of NK4 in RA synovial cells and suggest that NK4 is a promising agent for RA treatment.     

Transition of healthy to diseased synovial tissue in rheumatoid arthritis is associated with gain of mesenchymal/fibrotic characteristics

The healthy synovial lining layer consists of a single cell layer that regulates the transport between the joint cavity and the surrounding tissue. It has been suggested that abnormalities such as somatic mutations in the p53 tumor-suppressor gene contribute to synovial hyperplasia and invasion in rheumatoid arthritis (RA). In this study, expression of epithelial markers on healthy and diseased synovial lining tissue was examined. In addition, we investigated whether a regulated process, resembling epithelial to mesenchymal transition (EMT)/fibrosis, could be responsible for the altered phenotype of the synovial lining layer in RA. Synovial tissue from healthy subjects and RA patients was obtained during arthroscopy. To detect signs of EMT, expression of E-cadherin (epithelial marker), collagen type IV (indicator of the presence of a basement membrane) and alpha-smooth muscle actin (alpha-sma; a myofibroblast marker) was investigated on frozen tissue sections using immunohistochemistry. Fibroblast-like synoviocytes (FLSs) from healthy subjects were isolated and subjected to stimulation with synovial fluid (SF) from two RA patients and to transforming growth factor (TGF)-beta. To detect whether EMT/fibrotic markers were increased, expression of collagen type I, alpha-sma and telopeptide lysylhydroxylase (TLH) was measured by real time PCR. Expression of E-cadherin and collagen type IV was found in healthy and arthritic synovial tissue. Expression of alpha-sma was only found in the synovial lining layer of RA patients. Stimulation of healthy FLSs with SF resulted in an upregulation of alpha-sma and TLH mRNA. Collagen type I and TLH mRNA were upregulated after stimulation with TGF-beta. Addition of bone morphogenetic protein (BMP)-7 to healthy FLS stimulated with SF inhibited the expression of alpha-sma mRNA. The finding that E-cadherin and collagen type IV are expressed in the lining layer of healthy and arthritic synovium indicates that these lining cells display an epithelial-like phenotype. In addition, the presence of alpha-sma in the synovial lining layer of RA patients and induction of fibrotic markers in healthy FLSs by SF from RA patients indicate that a regulated process comparable to EMT might cause the alteration in phenotype of RA FLSs. Therefore, BMP-7 may represent a promising agent to counteract the transition imposed on synoviocytes in the RA joint.     

Angiogenesis and p53 and H-ras mutations in pancreatic ductal adenocarcinoma

OBJECTIVE: To evaluate the correlation of angiogenesis and p53 and H-ras mutations with prognostic factors and proliferative activity assessed with Ki-67 protein expression by studying archival tissues from 24 patients with primary pancreatic ductal adenocarcinoma. STUDY DESIGN: Vascular structures were labeled immunohistochemically using factor VIII-related antigen. Vascular surface density (VSD) and microvessel number (NVES) were assessed by stereology. The tissues were also analyzed with the immunohistochemical method for the expression of proteins, including p53, H-ras and Ki-67. RESULTS: Statistical analysis revealed that tumors with greater NVES and VSD values significantly correlated with occurrence of metastases, higher proliferative activity, poorer histologic differentiation and greater tumor size. p53 Mutations were found in 11 cases (45.8%). However, only three cases (12.5%), all negative for p53 mutations, showed H-ras mutations. p53 Mutation-positive tumors exhibited a statistically significant correlation with occurrence of metastases and higher proliferative activity, whereas H-ras mutations did not show such a correlation. CONCLUSION: Angiogenesis might have a role in predicting prognosis in pancreatic carcinomas, and p53 mutations might be acquired in later stages associated with metastatic progression and higher proliferative activity. Although H-ras mutations were rare in the present study, they might play a role in a different carcinogenic pathway excluding p53 mutations.     

Effects of paclitaxel on cultured synovial cells from patients with rheumatoid arthritis

BACKGROUND: Proliferation of synovial cells is considered to play a key role in rheumatoid arthritis (RA). Using paclitaxel, a unique antineoplastic agent known to suppress collagen-induced arthritis, we conducted an in vitro study of cell kinetics on cultured synovial cells from patients with RA. METHODS: Alterations of the cell cycle of cultured fibroblast-like synovial cells (FLSs) from patients with RA were studied using flow cytometry and laser scanning cytometry. Apoptosis and accumulation of cyclin concerning effects of paclitaxel were detected. RESULTS: Paclitaxel induced arrest of the cell cycle at G2/M phase and apoptosis in FLSs. The late stage of apoptosis was determined by the positivity of terminal deoxynucleotidyl transferase assay. Morphological observation by combined usage of both annexin V and propidium iodide on FLSs on a slide glass showed early apoptotic changes in detail. FLSs arrested at G2/M phase showed marked accumulation of cyclin B1. The effects of paclitaxel decreased on FLSs, which diminished proliferative activity. CONCLUSIONS: These data indicate that paclitaxel induces cell arrest at G2/M phase followed by apoptosis in human FLSs, which have high proliferative activity, and possible therapeutic effects of paclitaxel on RA.     

Genetic instability promotes the acquisition of chromosomal imbalances in T1b and T1c breast adenocarcinomas.

In order to evaluate biological and genetic properties of early breast carcinomas we analyzed microdissected tissue from 33 primary breast carcinomas stage T1b and T1c with respect to the nuclear DNA content, the expression pattern of Ki-67, cyclin A, p27KIP1, p53 and p21WAF1, and chromosomal gains and losses. The results show that T1b carcinomas (6-10 mm, n=17) were frequently near-diploid (53%) with low proliferative activity and staining patterns of p53 and p21WAF1 that suggest the presence of wild type protein. The majority (12/16) of the T1c tumors (11-20 mm), however, was aneuploid, and proliferative activity and p53 expression were increased. Larger tumor size correlated with an increasing number of chromosomal copy number changes and in particular with regional amplifications. High level copy number increases (amplifications), however, were found exclusively in the aneuploid tumors. Amplification events correlated with elevated cyclin A and reduced p27 expression, respectively. Our results suggest that the sequential acquisition of genomic imbalances during tumor progression is accelerated in aneuploid tumors, and may contribute to the increased malignancy potential.     

An immunohistochemical study of the expression of cell-cycle-regulated proteins p53, cyclin D1, RB, p27, Ki67 and MSH2 in gallbladder carcinoma and its precursor lesions

Gallbladder carcinomas are rare but highly lethal neoplasms. We examined the expression of five cell-cycle-related molecules (p53, RB, cyclin D1, p27, Ki-67), and MSH2, in 46 carcinomas, 14 adenomas, 15 low-grade dysplasias, 9 intestinal metaplasias and 20 normal gallbladder epithelia. The expression of these molecules was altered in gallbladder carcinomas and adenomas. In gallbladder carcinomas we observed increased expression of p53, cyclin D1, Ki-67, and MSH2 together with decreased expression of RB and p27 protein. Aberrant expression of cyclin D1 and reduced expression of RB were noted in adenomas, and expression of cyclin D1 was elevated in low-grade dysplasias. However, there was no change in the levels of these cell-cycle molecules in metaplasia. Expression of p53, p27, Ki-67, and MSH2 was correlated with clinical stage (P<0.05) and there was also a correlation between the expression of Ki-67 and MSH-2 and patient age (P<0.05). These results suggest that altered expression of cell-cycle molecules p53, cyclin D1, RB, p27, and of MSH-2 is involved in the progression of gallbladder carcinomas.     

p53 Protein expression and proliferative activity in ductal breast carcinomas

The immunocytochemical expression of p53 protein and Ki-67 labelling index in tumour cells of 100 ductal breast carcinomas of different histological grade and stage was evaluated in cytological material. In order to investigate p53 expression and Ki-67 expression an avidin-extravidin immunocytochemical technique was applied to imprints. Monoclonal antibody (MoAb) DO-p53 and proliferating cell monoclonal antibody were used as primary antibodies. A statistically significant difference was observed between p53 protein expression and grade of malignancy and clinical stage (P = 0.001, P < 0.001, respectively). A statistically significant difference was also observed between Ki-67 LI and histological grade and stage of the tumours (P < 0.001, P < 0.001 correspondingly). A correlation was observed between p53 protein expression and Ki-67 LI (P < 0.001). The immunocytochemical study of p53 protein and Ki-67 expression in cytological material represents a simple method which can be applied in routine cytological laboratories for the investigation of potential malignancy of ductal breast cancer.     

p53 protein expression and proliferative activity in imprints of superficial transitional cell carcinoma of the bladder

OBJECTIVE: To investigate p53 protein expression and proliferative activity in imprints of tumor biopsies from superficial transitional cell carcinoma of the bladder in relation to the histologic grade of malignancy and recurrence status. STUDY DESIGN: The study group consisted of 70 cases of superficial transitional cell carcinoma of the bladder. In order to investigate p53 protein expression and Ki-67 expression, an immunocytochemical avidin-extravidin complex technique was performed using monoclonal antibodies p53 D0-7 and proliferating cells correspondingly. RESULTS: Thirty-seven percent of superficial transitional cell carcinoma cases showed positive expression of p53 protein. No correlation was found between p53 protein expression and grade of malignancy (P = .45). p53 Protein expression was statistically correlated with a high Ki-67 labeling index (LI) (P < .001) and recurrence status (P < .001). Forty-seven percent of cases showed a Ki-67 LI > 25%. No correlation was found between a high Ki-67 LI and grade of malignancy (P = .703). A significant difference in high Ki-67 LI between recurrent and nonrecurrent tumors of the same grade (P < .001) and between recurrent and nonrecurrent tumors was found independently of grade (P < .001). CONCLUSION: These results on cytologic material could provide useful information on the biologic behavior of superficial transitional cell carcinoma of the bladder at the time of diagnosis.     

Expression of p53, bcl-2 and Ki-67 in cervical intraepithelial neoplasia and invasive squamous cell carcinoma of the uterine cervix

OBJECTIVE: To assess the expression of p53, bcl-2 and Ki-67 in the progression of cervical neoplasia. STUDY DESIGN: A total of 131 cervical specimens, consisting of normal cervical epithelium (n = 43), cervical intraepithelial neoplasia (CIN) lesions (n =40) and cervical squamous cell carcinomas (SCCs) (n = 48) were examined immunohistochemically in paraffin sections for expression of p53, bcl-2 and Ki-67. RESULTS: Immunoreactivity of p53 was found in 27% of SCC cases, but it had no significant relationship with SCC staging (p = 0.791). Immunoreactivity of bcl-2 was observed in 33% of CIN 3 cases. We found a significant relationship (chi2 test: p = 0.009) between the expression of bcl-2 and CIN grading. Ki-67 index was higher in high grade CIN (HGCIN: CIN 2 and 3) and SCC lesions compared to normal cervices. Ki-67 index showed a correlation with bcl-2 protein expression (p = 0.030), but not with p53 protein expression (p = 0.239). CONCLUSION: HGCIN is an early stage to demonstrate the alteration of bcl-2 and Ki-67 expressions. Progression of neoplasia in the uterine cervix is accompanied by an increase of antiapoptotic protein, bcl-2 as well as cellular proliferation.     

High expression of MDM2 protein and low rate of p21(WAF1/CIP1) expression in SCID mice Epstein Barr virus-induced lymphoproliferation.

To study the prevalence of p53 inactivation and MDM2/p21(WAFI/CIP1) expression in severe combined immunodeficient (SCID) mice Epstein-Barr virus (EBV)-induced lymphoproliferation, 19 samples obtained after ip injection of peripheral blood mononuclear cells (PBMCs) from EBV-seropositive donors or lymphoblastoid cell lines (LCL) were analyzed. In all samples tested, overexpression of Ki-67 antigen was shown by immunohistochemistry, indicating a high proliferative index of SCID mice EBV-induced lymphoproliferation. P53 mutations were screened by functional assay in yeast in 14 samples. With this test, a p53-inactivating mutation was found in only one case; the remaining cases exhibited a wild-type p53 pattern. However, an accumulation of p53 protein was detected by immunohistochemistry in six of 19 samples. P21 expression was found in seven of 19 samples but was not correlated with the rate of p53 protein in tumors. In contrast, high levels of nuclear accumulation of MDM2 were found in all samples by immunohistochemistry. These results suggest that a high Ki-67 proliferative index in SCID mice EBV-induced lymphoproliferation is not due to the inactivation of p53 by mutation, but could be associated with an overexpression of MDM2, which would act by a p53-independent mechanism.(J Histochem Cytochem 47:1315-1321, 1999)     

Immunohistochemical analyses of cell cycle-related proteins, apoptosis, and proliferation in pituitary adenomas.

To analyze the cell cycle regulatory mechanisms in the growth of pituitary adenomas, we investigated immunohistochemically the expression of the cell cycle-related proteins cyclin A and p27 in 48 pituitary adenomas. The frequency of apoptosis and the proliferative potential were also examined. The percentage of apoptotic cells was evaluated by immunohistochemical analysis using the anti-single-strand DNA antibody. The proliferative potential was assessed using the anti-Ki-67 antibody. The mean cyclin A labeling index (LI) for the non-recurrent group was 1.03% and for the recurrent group 2.31%. A positive linear correlation between cyclin A LI and Ki-67 LI was found. The mean p27 LI for the non-recurrent group was 67.4% and for the recurrent group 47.0%. There were significant differences in cyclin A LI and p27 LI between the non-recurrent group and the recurrent group. The mean apoptotic rate for the non-recurrent group was 0.87% and for the recurrent group 1.05%. There was no significant difference. Multivariate regression analysis revealed that high cyclin A LI and high Ki-67 LI were significant factors for shorter progression-free survival. The results suggest that the cyclin A LI is a useful prognostic factor in pituitary adenomas. (J Histochem Cytochem 49:1193-1194, 2001)     

Epithelial cell kinetics of the gastric mucosa during Helicobacter pylori infection

Helicobacter pylori is an important pathogen in major gastroduodenal diseases, including inflammation with ulceration and gastric malignancies. Alterations in H. pylori associated cell turnover in gastric epithelial cells are examined in relation to inflammatory activity, bacteria load and cytokines which may improve knowledge concerning the outcome of gastric diseases caused by H. pylori. Antral biopsies from 42 dyspeptic patients including 27 H. pylori-positive and 15 H. pylori-negative patients were tested for apoptotic activity by the TUNEL assay, and immuno-histochemically for p53 and the proliferative marker Ki-67. H. pylori infection, bacteria load and inflammatory activity were associated with increased cell turnover as judged by enhanced activities of TUNEL, p53 and Ki-67. Only p53 was significantly correlated to IFN-gamma, IL-8 and IL-10. The H. pylori-positive state was furthermore accompanied by varying degrees of altered distribution pattern of the markers studied, with occasional presence of apoptosis in the deeper pit zones, upward extension of Ki-67 and to a lesser degree of p53. Given a similar pattern of change in proliferation and apoptosis in some neoplastic lesions in other parts of the gastrointestinal tract, such studies in cell turnover may provide insights valuable in the investigations of potential precursors of gastric malignancies.