Expression of Pirh2, a p27(Kip1) ubiquitin ligase, in hepatocellular carcinoma: correlation with p27(Kip1) and cell proliferation

p53-Induced ring-H2 protein (Pirh2), a recently identified ubiquitin-protein ligase, interacts with p27(Kip1) to promote ubiquitination of p27(Kip1) independently of p53. High Pirh2 and low p27(Kip1) immunoreactivity are associated with a poor prognosis in several cancers, including resistant phenotypes. In the present study, we investigated the role of Pirh2 and p27(Kip1) in human hepatocellular carcinoma (HCC) progression. Immunohistochemical analysis was performed on formalin-fixed paraffin sections of 87 specimens. Statistical analysis showed that expression of Pirh2 was negatively related to p27(Kip1) expression (r = 0.787; P < .05), and Pirh2 expression correlated significantly with histologic grade (P < .001), venous invasion (P = .004), tumor size (P = .024), and the presence of multiple tumor-bearing lymph nodes (P = .017), whereas p27(Kip1) expression correlated significantly with histologic grade (P < .001), venous invasion (P = .048), and cirrhosis (P = .028). By Kaplan-Meier analysis, the survival curves of low versus high expressers of Pirh2 and p27(Kip1) showed significant separation (P < .01). Molecular interaction could be demonstrated between Pirh2 and p27(Kip1) in three HCC cell lines. In vitro, following release of two HCC cell lines from serum starvation, the expression of Pirh2 was upregulated, whereas p27(Kip1) was downregulated. Our results suggest that Pirh2 mediates the degradation of p27(Kip1) and participates in cell proliferation in human HCC. These findings provide a rational framework for further development of Pirh2 inhibitors as a novel class of anti-tumor agents.     

HMGB1 enhances smooth muscle cell proliferation and migration in pulmonary artery remodeling

HMGB1 is a necessary and critical mediator of acute lung injury and can act as a chemoattractant and anti-apoptosis factor in injury or repair in diseases. In this study we sought to determine whether HMGB1 is involved in the remodeling of pulmonary artery and investigate the mechanism. A rat model of pulmonary artery remodeling was successful induced with LPS infusion and the increasing of pulmonary arteries media was obviously inhibited in rats treated with thrice inject of HMGB1 neutralizing antibody. The percent of areas of tunica media to total artery wall was (0.53±0.15), (0.81±0.10) and (0.59±0.11) in control, LPS and antibody group respectively (p<0.05). Meanwhile, treatment with HMGB1 neutralizing antibody not only decreased the level of HMGB1 mRNA and protein significantly, but inhibited the expression of PCAN and Bcl-2 as well. On the contrary, Bax, a gen which represented the apoptosis, revealed an absolutely reversed trend to Bcl-2 in pulmonary arteries. Experiments in vitro showed that HMGB1 could stimulate the proliferation of hPASMC in MTT test and increase the number of migrated cells in a concentration-dependent manner in chemotaxis assay using modified Boyden chambers. In conclusion, data from this study support the concept that HMGB1 is involved in the remodeling of pulmonary artery by enhancing proliferation and migration of smooth muscle cell. Inhibiting HMGB1 may be a new target to deal with the remodeling of pulmonary artery.     

NHE-1与大鼠肺动脉平滑肌细胞增殖和凋亡

目的:探讨Na⁺/H⁺交换器-1(NHE-1)、细胞内pH对肺动脉平滑肌细胞增殖和凋亡的作用。方法:实验分对照组和低氧3周组,大鼠常压低氧3周后,分离肺动脉平滑肌层,测定细胞内pH,并应用RT-PCR技术,检测NHE-1mRNA表达的变化。体外培养肺动脉平滑肌细胞,诱导细胞酸化后,原位细胞凋亡检测不同浓度及时间NHE-1特异性抑制剂作用后,细胞凋亡率的改变。结果:低氧组肺动脉平滑肌细胞内pH及NHE-1mRNA表达均明显高于正常对照组。随药物浓度增加和作用时间延长,细胞凋亡率明显增高。结论:NHE-1参与调节细胞内pH,在肺动脉平滑肌细胞增殖和凋亡中起重要的作用。     

Bottom-up cell proliferation with cyclin A and p27Kip1 expression in ulcerative colitis-associated dysplasia

To analyze the cell kinetics of ulcerative colitis (UC)-associated dysplasia, cyclin A, cyclin D1, cyclin E, cdk2, cdk4, p21(Waf1), and p27(Kip1) were immunohistochemically examined, in comparison with sporadic tubular adenomas. Immunohistochemical labeling indices for each marker in formalin-fixed paraffin-embedded tissue sections were assessed in a total of 23 low-grade dysplasias, 27 high-grade dysplasias, and 14 invasive adenocarcinomas associated with UC. For comparison, 21 sporadic tubular adenomas with low-grade dysplasia, 33 with high-grade dysplasia, and 21 invasive adenocarcinomas were also examined. In UC-associated dysplasias, cyclin A and p27(Kip1) were located in the lower parts of the crypts and p21(Waf1) in the upper regions. In tubular adenomas, cyclin A, cdk4, p27(Kip1), and p21(Waf1) were all expressed in the upper parts of the crypts. The expression levels of cyclin D1, cyclin E, and cdk2 were low. The cell proliferation zone in UC-associated dysplasia is located towards the bases of the crypts with the strong expression of cyclin A and p27(Kip1), in contrast to tubular adenomas, which have their cell proliferation zone in the upper parts of neoplastic crypts. It is considered that tumorigenesis with UC-associated dysplasia is of the bottom-up type, related to altered expression of cyclin A and p27(Kip1).     

Expression of p27(Kip1) protein in transitional cell carcinoma of the upper urinary tract.

The expression of p27(Kip1), a negative regulator of the cell cycle, has been reported to correlate with the biological behavior and prognosis of several tumors. However, its prognostic importance in transitional cell carcinoma of the upper urinary tract (TCC-UUT) has not previously been investigated. We investigated p27(Kip1) protein expression using immunohistochemistry in 132 cases of TCC-UUT and also its relation to proliferating cell nuclear antigen (PCNA) immunoreactivity, p53 oncoprotein immunoreactivity, clinicopathologic parameters, and clinical outcome. A positive expression of p27(Kip1) protein was recognized in 94.7% of the samples and was apparent within tumor nuclei. In the normal urothelium, its expression was identified in all cell layers. A positive expression of p53 oncoprotein was recognized in 27.2% of the patients. The PCNA index was 7.4 to 93.1% (mean, 66.4%). Examination of the relationships between the expression of p27(Kip1) protein and clinicopathologic findings, PCNA index, and the expression of p53 oncoprotein revealed that the expression of p27(Kip1) protein decreased significantly with stage and grade. In a univariate analysis of disease-free and overall survival rates, no correlation was found between the expression of p27(Kip1) protein and prognosis. The expression of p27(Kip1) protein appears to be of little or no value in informing the prognosis in TCC-UUT.     

Reduced expression of p27/Kip1 is associated with the development of pulmonary adenocarcinoma

p27/Kip1 (p27), a negative regulator of cell proliferation, is a powerful prognostic marker in non-small cell lung carcinoma. To clarify the significance of p27 aberrations in the tumourigenesis of lung adenocarcinoma, p27 expression was investigated by immunohistochemistry in lung adenocarcinoma and its precursor lesion, atypical adenomatous hyperplasia (AAH), and correlated with the expression of Ki-67, cyclin D1, and cyclin E. The p27 labelling index decreased in parallel with tumour progression (24.0% to 4.5%) and was found to be lower in neoplastic lesions than in normal bronchiolar epithelial cells (48.8%). There was a negative correlation between p27 and Ki-67 expression (rho=-0.384, p<0.001). Cyclin E-positive lesions (with labelling index >/=5%) were found only in overt adenocarcinomas. The Ki-67 labelling index of cyclin E-positive, high (>/=10%) p27 expressers was lower than that of cyclin E-positive, low (<10%) p27 expressers (16.8% vs. 42.6%; p=0. 046) and was similar to that of cyclin E-negative adenocarcinomas (15.0%). These results indicate that reduced p27 expression is associated with and may play a role in progression during the development of pulmonary adenocarcinoma.     

SIRT1上调大鼠血管平滑肌细胞P27Kip1的表达

目的 研究III类去乙酰化酶SIRT1在血管平滑肌中对p27Kip1表达的影响及其可能的机制。方法 用H2O2和oxLDL刺激大鼠平滑肌细胞A7r5,Western blot检测平滑肌细胞内源性SIRT1的表达变化;在动物血管损伤模型中,Western blot检测血管损伤处平滑肌的SIRT1表达水平。用SIRT1重组腺病毒感染A7r5细胞和原代大鼠平滑肌细胞,Western blot观察SIRT1过表达引起的p27表达的改变;用SIRT1抑制剂NAM处理平滑肌细胞并观察p27的表达改变;用免疫共沉淀技术探寻SIRT1上调p27基因表达可能的分子机制。 结果 在H2O2和oxLDL氧化应激刺激的A7r5细胞中,内源性SIRT1的表达随作用时间逐渐增加;动物血管损伤模型中,内源性SIRT1的表达明显上调;过表达SIRT1能够在10%血清刺激的早期上调p27表达,而SIRT1抑制剂NAM则能够减少p27表达;在原代平滑肌细胞中,腺病毒介导的SIRT1过表达同样能够显著上调p27的表达。在血管平滑肌细胞中检测到SIRT1能够与FOXO3a相互作用。结论 SIRT1在血管平滑肌细胞中上调p27的表达。     

Reduced expression of cyclin-dependent kinase inhibitor p27(Kip1) in oral malignant tumors.

p27(Kip1), a cyclin-dependent kinase (CDK) inhibitor, blocks progression from the G(1) to S phase by binding cyclin E-CDK2 and inhibiting their activities. We studied the expression of p27 in oral tumors by immunohistochemistry to determine whether lack of p27 plays a role in the development and progression of oral cancer. Reduced expression of p27 was detected in 86% of the squamous cell carcinomas and 95% of the mucoepidermoid carcinomas, respectively, while p27 expression was well preserved in the pleomorphic adenomas. The expression of p27 showed an inverse correlation with the expression of cyclin E in the squamous cell carcinomas and mucoepidermoid carcinomas. However, there was no relationship between clinicopathological parameters and p27 expression. These results suggest that the reduction of p27 protein may confer the development of oral squamous cell carcinoma and mucoepidermoid carcinoma partly through the increased expression of cyclin E.     

Regulation of p27Kip1 during gentamicin mediated hair cell death

The INK4 and Kip/Cip families of Cyclin Dependent Kinase inhibitors (CKIs) are regulators of the cell cycle. In addition, CKIS including p27(Kip1) can protect cells from apoptosis in vitro. However, little is known about protective effect of p27(Kip1) in vivo. We used systemic treatment with aminoglycosides to induce hair-cell death in the basilar papilla (BP), the auditory organ of the avian inner ear, and characterised the expression of p27(Kip1) with confocal and immunofluorescence microscopy. In contrast to the adult mammalian cochlea where p27(Kip1) is expressed only in supporting cells, p27(Kip1) is found in the nuclei of both hair cells and supporting cells in the BP of the normal, mature bird. Forty-eight hours after gentamicin treatment, hair cells with TUNEL positive nuclei and hair cells with pyknotic nuclei were both detected, suggesting many hair cells die by apoptosis. When the BP was double labelled for p27(Kip1) and myosin VIIa, a hair-cell specific protein, all dying hair cells that had been ejected from the epithelium were found to be myosin VIIa positive but negative for p27(Kip1) even though nuclear remnants were still visible. In the transition zone where partial hair-cell loss occurs, freshly ejected hair cells lying immediately above the surface of the BP no longer expressed p27(Kip1). Damaged hair cells within the epithelium in the transition zone contained p27(Kip1) in their cytoplasm but not in their nuclei. These data support recent in vitro findings suggesting that p27(Kip1) protects cells from apoptosis and that its downregulation may be a general feature of programmed cell death.     

低氧诱导因子1在低氧致肺动脉平滑肌细胞增殖中的作用

目的: 探讨低氧对肺动脉平滑肌细胞(PASMC)增殖和凋亡的影响,以及HIF-1α、P-ERK1/2、iNOS蛋白表达变化在其中的作用与意义。方法: 体外培养大鼠PASMC，设计常氧组、低氧组及ADM、L-NAME、PD98059干预组，用MTT比色法和PCNA的免疫组化法测定细胞增殖反应，用流式细胞仪检测细胞凋亡，用Western blotting法检测HIF-1α、P-ERK1/2、iNOS的蛋白表达。结果: （1）低氧24 h组的A值明显高于常氧组(P<0.01)，而PD98059及ADM干预组明显低于低氧组(P<0.01)， L-NAME干预组明显高于低氧组和常氧组(P<0.01)。（2）免疫组化表明，低氧24 h组呈阳性表达(P<0.01)。PD98059、ADM抑制了PCNA的表达(P<0.01)， L-NAME促进了PCNA的表达(P<0.01)。（3）各组在低氧培养24 h后，凋亡指数差异无显著(均P＞0.05)。（4）Western blotting表明常氧组少量HIF-1α、iNOS、 P-ERK1/2表达，低氧4 h后均表达增高(P<0.01),8 h仍维持在高峰(P<0.01)，而HIF-1α、P-ERK1/2在低氧24 h后表达下调。L-NAME促进了HIF-1α表达(P<0.01),PD98059部分抑制了HIF-1α、iNOS及P-ERK1/2表达(P<0.01)；ADM部分抑制了HIF-1α表达，促进iNOS表达(P<0.01)。结论: 低氧能促进肺动脉平滑肌细胞增殖，对细胞的凋亡无影响；HIF-1在低氧诱导肺动脉平滑肌细胞增殖中起重要作用。     

p27 Kip1 protein expression in Hashimoto's thyroiditis

AIMS: Hashimoto's thyroiditis (HT) is an autoimmune disease in which both proliferation and apoptosis are enhanced. p27(Kip1) protein protects tissues from disease mechanisms that involve excessive cell proliferation and apoptosis. This study investigated whether there is loss of p27(Kip1) expression in HT and whether p27(Kip1) immunoreactivity has any relation to the proliferative indicator Ki-67. Because p27(Kip1) is regulated through either degradation, mediated by the S phase kinase associated protein 2 (Skp2), or sequestration, via D3 cyclin, the expression of these proteins was also investigated. METHODS: Immunohistochemistry was used to assess p27(Kip1), Ki-67, Skp2, and cyclin D3 expression in 19 cases of HT and in 10 normal thyroids. The results were evaluated by image analysis and reported as labelling indices (LIs) in both groups. RESULTS: The p27(Kip1) LI was lower in HT than in normal thyroid (28% v 75%; p < 0.001), whereas Ki-67 (1.13% v 0.13%), Skp2 (0.74% v 0.15%), and cyclin D3 (1.56% v 0.00%) LIs were higher in HT than in normal thyroids (p < 0.001). There was no correlation between p27(Kip1) and the expression of Ki-67, Skp2, and cyclin D3. CONCLUSIONS: p27(Kip1) downregulation is not exclusive to tumours but occurs also in HT, independently of the proliferative status and of changes in Skp2 and cyclin D3 expression. Further investigation is required to understand the mechanisms leading to p27 deregulation because these observations suggest that the regulation of p27(Kip1) expression in epithelial thyroid cells may play a role in HT pathogenesis.     

Expression of cell cycle inhibitor p27Kip1 and its inactivator Jab1 in melanocytic lesions.

Decreased expression of p27 (a cyclin-dependent kinase inhibitor) is an adverse prognostic marker in a diverse array of human cancers. The purpose of this study was to investigate the expression of p27 and Jab1 (a protein involved in p27 degradation) in melanocytic lesions, and to identify their possible participation in melanoma progression. A tissue microarray was constructed using formalin-fixed, paraffin-embedded archival tissue blocks of 94 melanocytic lesions including 19 benign nevi, 21 dysplastic nevi, 23 melanomas, and 31 metastatic melanomas. The expression of p27 and Jab1 was evaluated by immunohistochemistry. The association between p27, Jab1, and clinicopathological parameters was analyzed using chi2 and Fisher's exact tests. Nonparametric Pearson's rank correlation was applied to evaluate the relationship between p27 and Jab1 expression. p27 was expressed in 15 (88%) nevi, 18 (95%) dysplastic nevi, 11 (50%) melanomas, and only in four (13%) of the metastatic melanomas (P<0.001). Jab1 was expressed in 14 (82%) standard nevi, 18 (95%) dysplastic nevi, 17 (77%) melanomas, and 16 (53%) of the metastatic melanomas (P<0.01). In metastatic melanomas, there was a negative correlation between p27 and Jab1 expression (r=-0.166). The low levels of p27 in primary and metastatic melanoma cases may explain the high proliferation rate of such lesions. Also, the relative high expression of Jab1 in metastatic melanoma, associated with low levels of p27, suggests that Jab1 may be involved in survival and proliferation of metastatic melanoma cells.     

p27Kip1 modulates cell migration through the regulation of RhoA activation

The tumor suppressor p27(Kip1) is an inhibitor of cyclin/cyclin-dependent kinase (CDK) complexes and plays a crucial role in cell cycle regulation. However, p27(Kip1) also has cell cycle-independent functions. Indeed, we find that p27(Kip1) regulates cell migration, as p27(Kip1)-null fibroblasts exhibit a dramatic decrease in motility compared with wild-type cells. The regulation of motility by p27(Kip1) is independent of its cell-cycle regulatory functions, as re-expression of both wild-type p27(Kip1) and a mutant p27(Kip1) (p27CK(-)) that cannot bind to cyclins and CDKs rescues migration of p27(-/-) cells. p27(-/-) cells have increased numbers of actin stress fibers and focal adhesions. This is reminiscent of cells in which the Rho pathway is activated. Indeed, active RhoA levels were increased in cells lacking p27(Kip1). Moreover, inhibition of ROCK, a downstream effector of Rho, was able to rescue the migration defect of p27(-/-) cells in response to growth factors. Finally, we found that p27(Kip1) binds to RhoA, thereby inhibiting RhoA activation by interfering with the interaction between RhoA and its activators, the guanine-nucleotide exchange factors (GEFs). Together, the data suggest a novel role for p27(Kip1) in regulating cell migration via modulation of the Rho pathway.     

Expression of cyclin kinase inhibitor p27(Kip1) in skin tumours of dogs

Skin tumours (n=148) of epidermal or hair follicle origin were examined immunohistochemically to determine the expression of p27(Kip1)(p27), a cyclin-dependent kinase inhibitor (CDKI), and of Ki-67. In normal skin, a large number of basal cells of the epidermis and hair follicles were positive for Ki-67 and many suprabasal epithelial cells were positive for p27. Most of the hair matrix cells were positive for Ki-67 but negative for p27. Hair papillae were strongly positive for p27. Squamous cell carcinomas had a p27 positive index (PI) significantly lower than that of trichoepitheliomas (P<0.005), basal cell tumours (P<0.05) and intracutaneous cornifying epitheliomas (P<0.001). In contrast, Ki-67 PIs of squamous cell carcinomas and pilomatrixomas were significantly higher than those of trichoepitheliomas, basal cell tumours and intracutaneous cornifying epitheliomas (P<0.01 to P<0.001). No significant difference was observed between the Ki-67 PI values of squamous cell carcinomas and pilomatrixomas. The results suggested that p27 is capable of suppressing cell proliferation in the differentiation of normal canine skin. In spite of being a benign neoplasm, pilomatrixomas had a low p27 expression; this may be a reflection of the proliferative potential of the hair matrix. The expression of p27 may be a useful marker for the analysis of cell kinetics.     

Expression of Jun activation domain-binding protein 1 and p27 (Kip1) in thyroid medullary carcinoma

AIMS: p27 is a prominent regulator of cell proliferation by universally inhibiting the cell cycle, while Jun activation domain-binding protein 1 (Jab1), a multifunctional cell signaling protein, contributes to carcinoma progression by degrading p27. In this study, we investigated the expression of these proteins in medullary thyroid carcinoma. METHODS: We immunohistochemically examined Jab1 and p27 expression in 64 medullary thyroid carcinomas. RESULTS: Of the 64 cases examined, decreased p27 expression was observed in 38 cases (59.4%). The p27 expression level was inversely linked to tumour size as well as plasma calcitonin level. Jab1 expression level was generally high, and 46 cases (71.9%) were classified as overexpressing Jab1. The incidence was higher than those in papillary and follicular carcinomas, which were previously reported. Jab1 expression level was inversely linked to that of p27, and all five cases with only cytoplasmic but not nuclear staining of p27 overexpressed Jab1. CONCLUSIONS: These findings suggest that (1) decrease in p27 expression may contribute to local tumour growth; (2) Jab1 expression is related to the progression of medullary carcinoma by decreasing the amount of p27 in the cell and accelerating its degradation; and (3) Jab1 may play a more vital role in the pathogenesis of medullary carcinoma than papillary and follicular carcinomas.