Abnormal expression of the p53-binding protein MDM2 in Hodgkin's disease

The possible involvement of p53 tumor suppressor gene in the pathogenesis of Hodgkin's disease (HD) is suggested by the frequent finding of abnormal accumulation of p53 protein in the nuclei of Reed- Sternberg cells and their variants (H-RS) in a large proportion of cases. This finding, besides being consistent with the presence of p53 gene mutations, might represent a consequence of the inactivating interaction between p53 and p53-binding proteins such as the product of the MDM2 cellular oncogene. We have examined an unselected series of 77 HD cases of different histologic patterns for the expression of p53 and MDM2 proteins, using specific monoclonal antibodies and sensitive immunohistochemical techniques in single- and double-marker combination. In the large majority of cases (66/77), a variable proportion of H-RS cells expressed MDM2 that was strictly confined to the nuclei. Coexpression of both MDM2 and p53 was common in the same cells. The abnormal nuclear expression of p53 and MDM2 did not seem to correlate with the presence of Epstein-Barr virus infection, as shown by the results of LMP-1 antigen expression and EBER in situ hybridization analysis. Our data suggest that the abnormal accumulation of MDM2 and p53 proteins in HD might reflect a derangement of molecular mechanisms that could play a pathogenetic role in this disease.     

Overexpression of the MDM2 gene by childhood acute lymphoblastic leukemia cells expressing the wild-type p53 gene

The wild-type (wt) p53 tumor suppressor gene is commonly inactivated in human malignancies, either by mutations or by loss of expression. An additional proposed mechanism for inactivation of wt-p53 is amplification of the murine double minute 2 (MDM2) gene and overexpression of the MDM2 protein, which binds to p53 and eliminates its tumor suppressor function. To investigate a potential role for MDM2 in the inactivation of wt-p53 in pediatric acute lymphoblastic leukemia (ALL), we examined the expression of MDM2 and p53, as well as the occurrence of p53 mutations and possible amplification of the MDM2 gene, in 19 pediatric ALL cell lines and one pediatric acute myelogenous leukemia (AML) line. Although we did not find significant amplification of the MDM2 gene in any of the leukemic lines, we detected overexpression of MDM2 in all 10 lines that expressed wt-p53. Of the 10 lines without overexpression of the MDM2 gene, six (including the AML line) did not express p53, and four expressed mutant p53 with single point mutations in exons 7 and 8. To determine whether primary leukemic cells showed a similar correlation, we analyzed the original cryopreserved leukemic bone marrow cells from seven patients from whom cell lines were established. We obtained similar results from both the primary leukemic cells and the corresponding cell lines: overexpression of MDM2 was present in primary cells that expressed wt-p53 but not in cells that lacked expression of wt-p53. These findings suggest an important role for MDM2 in the pathogenesis of pediatric ALL in which leukemic cells express wt-p53.     

IGFBP-rP1 induces p21 expression through a p53-independent pathway, leading to cellular senescence of MCF-7 breast cancer cells

Objectives

Insulin-like growth factor-binding protein (IGFBP)-related protein 1 (IGFBP-rP1), a member of the IGFBP super family, was identified as a potent tumor suppressor in several carcinomas. IGFBP-rP1 was down-regulated in primary breast cancer tissues and several breast cancer cell lines but overexpressed in senescent human mammary epithelial cells (HMECs), suggesting that IGFBP-rP1 might be a tumor suppressor in breast cancer and the tumor suppressor role of IGFBP-rP1 might be associated with cellular senescence. The aim of the study was to observe the effect of IGFBP-rP1 on cellular senescence and the molecular events mediating this biological effect in MCF-7 breast cancer cells.

Methods

DNA fragment-encoding IGFBP-rP1 was cloned in-frame N-terminally to EGFP gene to generate IGFBP-rP1-EGFP fusion protein expression plasmid (pEGFP-IGFBP-rP1). The plasmid pEGFP-IGFBP-rP1 was then transfected into MCF-7 cells, and the proliferation, cell cycle distribution, cellular senescence, and cell cycle-related protein expression of MCF-7 cells were examined by trypan blue exclusion, flow cytometry, senescence-associated galactosidase (SA-β-gal) staining, and Western blot analysis, respectively. Two shRNA plasmid vectors against p21 or p53 gene were constructed and stably transfected into the MCF-7 cells to determine the involvement of p21 or p53 in cellular senescence induced by IGFBP-rP1.

Results

Transfection of IGFBP-rP1 or addition of condition medium (CM) from IGFBP-rP1-transfected cells in MCF-7 cells caused induction of a variety of senescent phenotypes, such as decrease in cell proliferation, increase in G0/G1 cell cycle arrest cells, change in cell morphology, and increase in senescence-associated galactosidase (SA-β-gal) activity. IGFBP-rP1-induced growth arrest is associated with enhanced expression of the cyclin-dependent kinase inhibitor p21 and dephosphorylation of the retinoblastoma protein (pRB). Cell proliferation block and cellular senescence induction in response to IGFBP-rP1 were partially reversed by p21 knockdown in MCF-7 cells. Knockdown of p53 in MCF-7 cells did not influence the growth inhibition, cellular senescence, and p21 expression of the cells in response to IGFBP-rP1 transfection.

Conclusions

Results from this study suggest that cellular senescence induced by IGFBP-rP1 is mediated at least in part by p21 enhanced expression, which regulated through the p53-independent pathway. IGFBP-rP1 might be one of the key molecules that trigger cellular senescence in breast cancer. Restoration of IGFBP-rP1 function might have therapeutic significance in breast cancer.     

Inhibition of PDE4 phosphodiesterase activity induces growth suppression, apoptosis, glucocorticoid sensitivity, p53, and p21(WAF1/CIP1) proteins in human acute lymphoblastic leukemia cells

Glucocorticoids are integral to successful treatment of childhood acute lymphoblastic leukemia (ALL) and other lymphoid malignancies. A large body of data indicates that in various model systems, elevation of cyclic adenosine monophosphate (cAMP) can potentiate glucocorticoid response, although this has not been well evaluated as a potential leukemia treatment. Although cAMP analogs have been studied, little data exist regarding the potential toxicity to leukemia cells of pharmacologic elevation of cAMP levels in leukemic blasts. Using MTT assays of cell proliferation on CEM ALL cells, we found that aminophylline and other nonspecific phosphodiesterase (PDE) inhibitors suppress cell growth. This effect is replicated by the PDE4-specific PDE inhibitor rolipram, but not by specific inhibitors of the PDE1 or PDE3 classes. We found that PDE inhibitors cause increased dexamethasone sensitivity and a synergistic effect with the adenylyl cyclase activator forskolin. We observed several important cellular characteristics associated with this treatment, including elevation of cAMP, induction of p53 and p21(WAF1/CIP1) proteins, G(1) and G(2)/M cell cycle arrest, and increased apoptosis. Sensitivity to forskolin and rolipram is shared by at least 2 pediatric ALL cell lines, CEM and Reh cells. Some cell lines derived from adult-type lymphoid malignancies also show sensitivity to this treatment. These findings suggest that PDE inhibitors have therapeutic potential in human ALL and characterize the molecular mechanisms that may be involved in this response.     

Apoptosis, bcl-2 expression and p53 accumulation in myelodysplastic syndrome, myelodysplastic-syndrome-derived acute myelogenous leukemia and de novo acute myelogenous leukemia

Apoptosis and its dysregulation have been implicated in dysplastic and ineffective hematopoiesis and the neoplastic transformation of bone marrow in myelodysplastic syndrome (MDS). To explore the role of apoptosis in hematological disorders, we examined the frequency of apoptotic cells by the in situ end labeling method in bone marrow specimens from 37 patients with MDS [refractory anemia (RA) 10 cases, RA with excess of blasts (RAEB) 27 cases including 12 cases with leukemic transformation], 12 patients with MDS-derived acute myelogenous leukemia (AML) and 13 patients with de novo AML. In addition, we investigated the relationship of apoptosis to the immunohistochemical expression of bcl-2 and p53 in these cases, and the association of apoptosis, bcl-2, and p53 with the leukemic evolution of MDS by examining sequential bone marrow samples of the same patient from the time of initial diagnosis to the time of overt leukemia. The percentage frequency of apoptotic cells was significantly greater in MDS (RA: 9.46 +/- 2.99%, m +/- SD; RAEB: 5. 60 +/- 3.09) as compared with those in MDS-derived AML (0.62 +/- 0. 37), de novo AML (0.28 +/- 0.11) and controls (1.00 +/- 0.59). On the other hand, the cases of RAEB with leukemic transformation exhibited a lower frequency of apoptotic cells and a higher frequency of bcl-2- and p53-positive cells than those without transformation. When the RAEB cases transformed to AML, the frequency of apoptotic cells was significantly reduced (2.96 +/- 1. 54 --> 0.62 +/- 0.37), while the frequencies of bcl-2-positive cells and p53-positive cells were greater (10.88 +/- 3.66 --> 30.54 +/- 7. 14, and 20.21 +/- 6.21 --> 32.34 +/- 14.71, respectively). In contrast to MDS-derived AML, over a half of de novo AML cases showed few p53-positive cells. These findings corroborate the earlier notion that apoptosis may play a substantial role in dysplastic and ineffective hematopoiesis in MDS. It is also suggested that the suppression of apoptosis associated with enhanced bcl-2 expression and p53 accumulation increases the probability of developing leukemia in MDS, and that oncogenetic development might be different between MDS-derived AML and de novo AML.     

Correlation of p53, MDM2 and p14ARF protein expression in human esophageal squamous cell carcinoma

Purpose  

To determine the interrelationships of p53, MDM2, and p14^ARF protein expression in primary esophageal squamous cell carcinoma (ESCC) and their prognostic value in ESCC.     

Mutant p53 protein, Bcl-2/Bax ratios and apoptosis in paediatric acute lymphoblastic leukaemia

PURPOSE: The Bcl-2 family of proteins regulates a late step in the apoptosis pathway. Bcl-2 protein is believed to be involved in imparting resistance to programmed cell death or apoptosis induced by chemotherapeutic agents and radiation. The anti-apoptotic function of the Bcl-2 protein appears to be modulated by its ability to heterodimerize with other members of the gene family, predominantly Bax, a protein favouring induction of apoptosis. Susceptibility to undergoing apoptosis may, therefore, be dependent on the ratio between Bcl-2 and Bax. Both Bax and Bcl-2 are regulated by the tumour-suppressor protein p53. The present study therefore aims to study the significance of the Bcl-2:Bax ratio, p53 expression and apoptosis in paediatric acute lymphoblastic leukaemia (ALL). METHODS: Expression of Bax, Bcl-2 and p53 was determined by immunocytochemistry, and apoptosis was evaluated by an enzymatic end-labelling technique using biotin-dUTP and further confirmed by annexin binding. The presence of mutant p53 was determined using a mutant-p53-specific enzyme-linked immunosorbent assay (ELISA). RESULTS: A total of 32 cases and 20 controls were evaluated. Bcl-2 was found to be expressed in 22/32 of the ALL cases. Pretreatment (spontaneous) apoptosis was observed in 23/32 cases. The mean pretreatment apoptotic index was 11.34 +/- 2.04% with a median value of 7.5%. CONCLUSIONS: There was a positive correlation between apoptosis and Bax expression (r = 0.5044; P = 0.0038). There was good correlation between the immunoreactivity of p53 and detection of mutant p53 by ELISA (r = 0.4605; P = 0.0079). The apoptosis index showed a negative borderline correlation to the expression of Bcl-2 protein (r = -0.3181; P = 0.076). There was an inverse correlation between extent of apoptosis and the presence of mutant p53 protein (r = -0.4732; P = 0.006). p53 protein expression also showed a correlation with both Bcl-2 (r = 0.4647; P = 0.007) and Bax (r = 0.4128; P = 0.018). The Bcl-2/Bax ratio, however, showed no significant correlation with apoptosis (r = -0.3131; P = 0.08) or with p53 expression. No significant association was evident between clinical and laboratory parameters with the Bcl-2/Bax protein expression except lymphadenopathy (r = 0.5774; P = 0.03). However, Bax expression showed a borderline correlation with the immediate tumour response to chemotherapy (r = -0.338; P = 0.0628). These patients are being followed-up to look for any association between clinical outcome, Bcl-2/Bax ratio and apoptosis.     

The novel histone deacetylase inhibitor, LBH589, induces expression of DNA damage response genes and apoptosis in Ph- acute lymphoblastic leukemia cells

We investigated the mechanism of action of LBH589, a novel broad-spectrum HDAC inhibitor belonging to the hydroxamate class, in Philadelphia chromosome-negative (Ph(-)) acute lymphoblastic leukemia (ALL). Two model human Ph(-) ALL cell lines (T-cell MOLT-4 and pre-B-cell Reh) were treated with LBH589 and evaluated for biologic and gene expression responses. Low nanomolar concentrations (IC(50): 5-20 nM) of LBH589 induced cell-cycle arrest, apoptosis, and histone (H3K9 and H4K8) hyperacetylation. LBH589 treatment increased mRNA levels of proapoptosis, growth arrest, and DNA damage repair genes including FANCG, FOXO3A, GADD45A, GADD45B, and GADD45G. The most dramatically expressed gene (up to 45-fold induction) observed after treatment with LBH589 is GADD45G. LBH589 treatment was associated with increased histone acetylation at the GADD45G promoter and phosphorylation of histone H2A.X. Furthermore, treatment with LBH589 was active against cultured primary Ph(-) ALL cells, including those from a relapsed patient, inducing loss of cell viability (up to 70%) and induction of GADD45G mRNA expression (up to 35-fold). Thus, LBH589 possesses potent growth inhibitory activity against including Ph(-) ALL cells associated with up-regulation of genes critical for DNA damage response and growth arrest. These findings provide a rationale for exploring the clinical activity of LBH589 in the treatment of patients with Ph(-) ALL.     

Nucleostemin knocking-down causes cell cycle arrest and apoptosis in human T-cell acute lymphoblastic leukemia MOLT-4 cells via p53 and p21Waf1/Cip1 up-regulation

Objectives

Nucleostemin (NS), a recently discovered nucleolar protein, is essential for maintaining self-renewal and proliferation of embryonic and adult stem cells as well as cancerous cells. The aim of this study was to determine biological function of NS in MOLT-4 cells as a human T-cell acute lymphocytic leukemia (T-ALL) model.

Methods

Efficacy of a specific small interference RNA on NS depletion was studied by quantitative polymerase chain reaction and western blotting. The growth rate and viability were analyzed by trypan blue exclusion test. Fluorescent microscopy was used for detecting apoptosis. Cell cycle and apoptosis were mechanistically studied by flow cytometry and western blotting.

Results

Knockdown of NS inhibited proliferation, arrested the cell cycle, and induced apoptosis through p53 and p21^Waf1/Cip1 pathways in MOLT-4 cells.

Discussion

These findings demonstrate critical roles of NS in MOLT-4 cells and may implicate on its therapeutic potential in this human T-ALL model.     

Frequent deletion of p16INK4a/MTS1 and p15INK4b/MTS2 in pediatric acute lymphoblastic leukemia

The tandemly linked p16INK4aMTS1 and p15INK4b/MTS2 genes on chromosome 9, band p21 encode proteins that function as specific inhibitors of the cyclin D-dependent kinases CDK4 and CDK6. This locus undergoes frequent bi-allelic deletion in human cancer cell lines, suggesting that the encoded proteins may function as tumor suppressors. However, more recent analysis of primary tumor samples has shown a much lower frequency of abnormalities affecting this region, raising doubt over the importance of these proteins in human malignancies. Hemizygous deletions and rearrangements of chromosome 9, band p21, are among the most frequent cytogenetic abnormalities detected in pediatric acute lymphoblastic leukemia (ALL), occurring in approximately 10% of cases. To determine if the p16INK4a/p15INK4b locus might be the target of these chromosomal lesions, we analyzed both genes in primary clinical samples from 43 pediatric ALL patients using interphase fluorescence in situ hybridization, Southern blot analysis, and the polymerase chain reaction. Deletions of p16INK4a/p15INK4b were identified in 18 of 20 cases with cytogenetically observed abnormalities of 9p and 5 of 23 with apparently normal chromosomes 9p, with the majority containing bi- allelic deletions (16 homozygous/7 hemizygous). Although most homozygous deletions involved both genes, Southern blot analysis showed an interstitial deletion in a single case that was confined to p16INK4a, suggesting that p15INK4b was not the critical target gene in this case. Sequence analysis of both p16INK4a and p15INK4b in all seven cases with hemizygous deletions failed to show mutations within the coding regions of the retained alleles. In this select group of patients, deletion of p16INK4a/p15INK4b was associated with T-cell phenotype, nonhyperdiploid karyotype (< 50 chromosomes), and poor event- free survival. These findings indicate that deletion of the p16INK4a/p15INK4b locus is one of the most common genetic abnormalities so far detected in pediatric ALL, and that loss of one or more of these cell cycle kinase inhibitors is important in leukemogenesis.     

Clinical significance of the ABCB1 and ABCG2 gene expression levels in acute lymphoblastic leukemia

Objectives: Acute lymphoblastic leukemia (ALL) is a clonal disease that accounts for 20% of acute leukemias in adults. A high percentage of adult patients (ranging from 70 to 80%) reach complete remission; however, the 5-year survival rate is only 20–40%. One of the main obstacles to treatment success is the drug resistance of leukemic cells. Therefore, our research group analyzed the ABCB1 and ABCG2 gene expression levels in 61 patients diagnosed with ALL and assessed whether the levels affected the clinical parameters and 40-month survival rate.

Methods: The ABCB1 and ABCG2 gene expression levels were analyzed using real-time polymerase chain reaction in 61 patients diagnosed with ALL and 99 healthy donors as controls. The association between ABCB1 and ABCG2 gene expression levels and clinical variables was determined using the Chi-square test and Fisher’s exact test. Overall survival (OS) was determined using the Kaplan–Meier method.

Results: The results showed high ABCB1 and ABCG2 gene levels, which were 4.5 and 2.3 times the levels of healthy donors, respectively. A total of 52% of the study patients expressed high ABCB1 levels and were significantly associated with the high-risk patient group and a decreased 40-month survival rate of 78%. Only 49% of the patients expressed high ABCG2 gene levels. No association was found between the clinical parameters and the ABCG2 gene expression levels.

Conclusions: Early detection of ABCB1 gene expression levels could be important for the diagnosis and monitoring of ALL patients.     

p27^kipl和p57^kip2与CyclinD1在急性白血病的表达及其临床意义

目的:探讨p27kipl和p57kip2与CyclinD1在急性白血病中的表达情况及其临床意义。方法:采用免疫组化S-P法检测39例急性白血病及10例正常对照者骨髓中p27kipl和p57kip2与CyclinD1蛋白的表达情况,并结合临床病理资料进行分析。结果:p27kipl和p57kip2与CyclinD1蛋白在急性白血病患者的阳性表达率分别为31%、33%、54%,在对照组表达率为70%、40%、0。p27kipl与cyclinD1在白血病与对照组中的表达差异有统计学意义。在37例接受化疗的急性白血病中,p27kipl和p57kip2阳性表达组化疗后的缓解率(66%、69%)明显高于p27kipl和p57kip2表达阴性组的缓解率(32%、29%),其差异有统计学意义(P<0.05)。p57kip2与CyclinD1在白血病中的表达具有正相关性。结论:p27kipl和p57kip2与CyclinD1在急性白血病患者中存在异常表达其蛋白的表达水平可能会影响化疗疗效。     

p27kip1和p57kip2与CyclinD1在急性白血病的表达及其临床意义

目的:探讨p27kip1和p57kip2与CyclinD1在急性白血病中的表达情况及其临床意义.方法:采用免疫组化S-P法检测39例急性白血病及10例正常对照者骨髓中p27kip1和p57kip2与CyclinD1蛋白的表达情况,并结合临床病理资料进行分析.结果:p27kip1和p57kip2与CyclinD1蛋白在急性白血病患者的阳性表达率分别为31%、33%、54%,在对照组表达率为70%、40%、0.p27kip1与cyclinD1在白血病与对照组中的表达差异有统计学意义.在37例接受化疗的急性白血病中,p27kip1和p57kip2阳性表达组化疗后的缓解率(66%、69%)明显高于p27kip1和p57kip2表达阴性组的缓解率(32%、29%),其差异有统计学意义(P＜0.05).p57kip2与CyclinD1在白血病中的表达具有正相关性.结论:p27kip1和p57kip2与CyclinD1在急性白血病患者中存在异常表达其蛋白的表达水平可能会影响化疗疗效.     

Frequency and prognostic significance of murine double minute protein-2 overexpression and p53 gene mutations in childhood acute lymphoblastic leukemia

Abstract

Aim: Determination of frequency and prognostic significance of murine double minute protein-2 (MDM-2) over expression and its association with p53 status in children with acute lymphoblastic leukemia (ALL).

Methods: MDM-2 expression by flow cytometry and p53 gene status by PCR were determined in peripheral blood or bone marrow of 46 ALL children (at initial diagnosis) and control group.

Results: MDM-2 was significantly overexpressed in 15 patients (32·6%). p53 mutation was detected in six out of 46 patients at initial diagnosis, three of them were out of 29 cases achieving complete remission (CR) and the other three cases were out of 17 of relapsed patients, which is significantly higher than CR group (P<0·05). Positive correlation was found between the MDM-2 overexpression and initial WBCs count, peripheral blast cell count and presence of CNS blasts (P<0·05, <0·05 and <0·05 respectively).

Conclusion: MDM-2 is overexpressed in a significant number of childhood ALL, and more often observed in the poor outcome group and its frequency is not related to p53 status. Measurement of MDM-2 as a bad prognostic marker even in cases with non-mutant p53 is very important. Moreover, MDM-2 may be a potential molecular target for production of new cancer therapy.     

Alterations in IRF1/IRF2 expression in acute myelogenous leukemia

The interferon response genes 1 and 2 have been shown to be involved in the regulation of differentiation and proliferation of cells of the myeloid series, with the former functioning as an anti-oncogene and the latter as an oncogene. In the study described here, the levels of expression of these two genes and the ratio of their expression were compared in AML and normal marrow. The ratio of gene expression was significantly less in AML marrow cells as compared to normal marrow cells [med ratio = 1.33 vs. 2.97, P = 0.003]. While the expression ratio was unaffected by the presence or absence of either ras or fms mutations, p53 mutations were associated with higher IRF1:IRF2 expression ratios that wt p53 genes [med = 1.701 vs. 1.135, P = 0.014]. Given the functional characteristics and the competitive nature of these two genes, it is possible that leukemic transformation is associated with a fall in IRF1:IRF2 ratios. Finally, the administration of IL4 can result in the normalization of the IRF1:IRF2 ratio in the marrow cells of some patients with AML.     

Asparagine synthetase expression is linked with L-asparaginase resistance in TEL-AML1-negative but not TEL-AML1-positive pediatric acute lymphoblastic leukemia

Resistance to L-asparaginase in leukemic cells may be caused by an elevated cellular expression of asparagine synthetase (AS). Previously, we reported that high AS expression did not correlate to L-asparaginase resistance in TEL-AML1-positive B-lineage acute lymphoblastic leukemia (ALL). In the present study we confirmed this finding in TEL-AML1-positive patients (n = 28) using microarrays. In contrast, 35 L-asparaginase-resistant TEL-AML1-negative B-lineage ALL patients had a significant 3.5-fold higher AS expression than 43 sensitive patients (P < .001). Using real-time quantitative polymerase chain reaction (RTQ-PCR), this finding was confirmed in an independent group of 39 TEL-AML1-negative B-lineage ALL patients (P = .03). High expression of AS was associated with poor prognosis (4-year probability of disease-free survival [pDFS] 58% +/- 11%) compared with low expression (4-year pDFS 83% +/- 7%; P = .009). We conclude that resistance to l-asparaginase and relapse risk are associated with high expression of AS in TEL-AML1-negative but not TEL-AML1-positive B-lineage ALL.     

High glucose induces plasminogen activator inhibitor-1 expression through Rho/Rho-kinase-mediated NF-kappaB activation in bovine aortic endothelial cells

Recently, it has become evident that elevated levels of plasminogen activator inhibitor-1 (PAI-1) are associated with myocardial infarction and stroke, especially in patients with diabetes. The molecular mechanisms involved in hyperglycemia-induced PAI-1 expression in bovine aortic endothelial cells (BAEC) were investigated. PAI-1 expression in BAEC was significantly increased in accordance with the concentration of glucose in media from 5.7 mM to 23 mM. Stimulation with high glucose (23 mM) significantly increased small GTPase Rho A activation. Pretreatment with a Rho-kinase inhibitor, Y-27632 (1-10 microM), significantly blocked high glucose-induced PAI-1 expression. NF-kappaB activity determined using the luciferase reporter gene assay was significantly enhanced by high glucose, and pretreatment with Y-27632 inhibited high glucose-induced PAI-1 expression at the basal level. An inhibitor of NF-kappaB action, namely parthenolide (0.1 microM), BAY 11-7082 (5 microM) and SN50 (1 microM), significantly blocked high glucose-mediated PAI-1 expression to a level with low glucose (5.7 mM). These data suggested that high glucose-induced PAI-1 expression in endothelial cells is mediated by NF-kappaB activation through the Rho/Rho-kinase pathway. Inhibition of Rho/Rho-kinase signaling might be a novel target for diabetes and metabolic syndrome.