p120ctn、Kaiso及matrilysin在非小细胞肺癌中的表达及其意义

目的探讨p120catenin(p120ctn)、Kaiso和matrilysin在非小细胞肺癌(non-small cell lung cancer,NSCLC)发生发展中的作用。方法采用免疫组织化学法检测98例NSCLC(52例肺鳞状细胞癌和46例肺腺癌)以及16例支气管扩张症组织中p120ctn、Kaiso和matrilysin的表达情况,分析NSCLC中p120ctn、Kaiso和matrilysin与临床病理特征的关系及三者表达的相关性。结果在NSCLC组织和支气管扩张症组织中,p120ctn蛋白异常表达率分别为74.5%、6.3%,两组之间表达率比较有统计学差异(P<0.001);在肺癌细胞、杯状细胞、纤毛细胞、基底细胞中Kaiso蛋白异位表达率分别为63.3%、6.3%、6.3%、37.5%,肺癌细胞与杯状细胞、纤毛细胞表达率比较有统计学差异(P<0.001)。p120ctn蛋白异常表达、matrilysin蛋白细胞质表达均与NSCLC组织学类型密切相关(P<0.05),Kaiso蛋白异位表达与NSCLC分化程度密切相关(P<0.05)。在NSCLC组织中,Kaiso细胞核表达与matrilysin细胞质表达存在显著负相关(r=-0.23,P=0.02)。结论 p120ctn可能通过与Kaiso在胞质内异位表达,解除了Kaiso对靶基因的抑制作用,促进matrilysin的细胞质表达,而参与肺癌的发生发展。     

Abnormal expression and clinicopathologic significance of p120-catenin in lung cancer

The aim of this study was to investigate the relationship between the expression of p120ctn in human lung squamous cell carcinoma, adenocarcinoma and its clinicopathologic significance. The expression of p120ctn in tumors and adjacent normal lung tissues from 143 patients was examined by immunohistochemistry and Western blot. Expression of p120ctn occurs mainly in the cell membrane of normal bronchial mucosa. Abnormal expression of p120ctn, including cytoplasmic and reduced membranous expression, was found in 114 of 143 specimens (79.7%) and was significantly associated with poor differentiation, high TNM stage, and lymph node metastasis (P<0.05 for each) but not with histologic subtype. The Kaplan-Meier survival test revealed that abnormal expression of p120ctn was related to poor survival (P<0.001). A Cox regression analysis revealed that abnormal p120ctn expression was an independent factor in predicting patient survival (P=0.024). Compared with that in normal lung tissues, membranous protein level was lower in tumors (P=0.003). Abnormal expression of p120ctn is associated with tumor progression and poor prognosis in lung squamous cell carcinoma and adenocarcinoma. Reduced expression or even the absence of p120ctn isoform 1 and 3 in tumor cell membranes may be responsible for the abnormal expression of p120ctn that has been found in lung cancer.     

Acadesine Kills Chronic Myelogenous Leukemia (CML) Cells through PKC-Dependent Induction of Autophagic Cell Death

CML is an hematopoietic stem cell disease characterized by the t(9;22) (q34;q11) translocation encoding the oncoprotein p210BCR-ABL. The effect of acadesine (AICAR, 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) a compound with known antileukemic effect on B cell chronic lymphoblastic leukemia (B-CLL) was investigated in different CML cell lines. Acadesine triggered loss of cell metabolism in K562, LAMA-84 and JURL-MK1 and was also effective in killing imatinib-resistant K562 cells and Ba/F3 cells carrying the T315I-BCR-ABL mutation. The anti-leukemic effect of acadesine did not involve apoptosis but required rather induction of autophagic cell death. AMPK knock-down by Sh-RNA failed to prevent the effect of acadesine, indicating an AMPK-independent mechanism. The effect of acadesine was abrogated by GF109203X and Ro-32-0432, both inhibitor of classical and new PKCs and accordingly, acadesine triggered relocation and activation of several PKC isoforms in K562 cells. In addition, this compound exhibited a potent anti-leukemic effect in clonogenic assays of CML cells in methyl cellulose and in a xenograft model of K562 cells in nude mice. In conclusion, our work identifies an original and unexpected mechanism by which acadesine triggers autophagic cell death through PKC activation. Therefore, in addition to its promising effects in B-CLL, acadesine might also be beneficial for Imatinib-resistant CML patients.     

Down-regulation of human<Emphasis Type="Italic">NDR</Emphasis> gene in megakaryocytic differentiation of erythroleukemia K562 cells

To study the control of hematopoietic cell differentiation, a human negative differentiation regulator (NDR) gene was identified by the comparative analysis of differentially expressed genes in hemato-lymphoid tissues.NDR is expressed preferentially in the adult bone marrow, fetal liver and testis. Immunocytochemistry with anti-NDR antiserum showed the presence of NDR in human erythroleukemia K562 cell line and CD34⁺ cells sorted from the umbilical cord blood. When fused to the green fluorescent protein (GFP), NDR was directed to the nucleus of mouse 3T3 and K562 cells. Fusion protein with a deletion from residues 7 to 87 was detected in the cytoplasm. NDR appeared not to affect the proliferation of K562 cells when overly expressed. However, its expression was down-regulated during megakaryocytic differentiation of K562 cells induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Down-regulation of NDR correlated well with up-regulation of megakaryocytic markers, CD41 and CD61. Overexpression of the nuclear NDR-GFP in K562 cells inhibited the expression of CD41 and CD61 in megakaryocytic differentiation. Treatment of K562 cells with GF-109203X (GFX), an antagonist of the protein kinase C (PKC), blocked NDR down-regulation, up-regulated expression of CD41/CD61 and TPA-induced megakaryocytic differentiation. These results suggest a novel function of nuclear NDR protein in regulating hematopoietic cell development.     

下调RALA表达抑制人白血病K562细胞迁移和侵袭

目的 研究小干扰RNA（siRNA）抑制v-ral 白血病致病因子RALA基因表达对人白血病K562细胞迁移和侵袭的影响.方法 利用LipofectamineTM 2000将化学合成的RALA siRNA转染体外培养的K562细胞,Real-time PCR检测细胞内RALA mRNA的表达水平;Western印迹检测细胞内RALA蛋白的表达水平;Boyden趋化小室实验检测细胞体外迁移和侵袭能力.结果与随机对照组相比,转染48 h后,RALA siRNA显著下调K562细胞内RALA mRNA和蛋白的表达（P＜0.05）.与随机对照组相比,转染RALA siRNA的K562细胞迁移和侵袭能力显著降低（P＜0.05）.结论 癌基因RALA在人白血病K562细胞迁移和侵袭过程中发挥重要作用,通过siRNA下调RALA的表达可抑制K562细胞迁移和侵袭能力.     

Spred2 Modulates the Erythroid Differentiation Induced by Imatinib in Chronic Myeloid Leukemia Cells

Differentiation induction is currently considered as an alternative strategy for treating chronic myelogenous leukemia (CML). Our previous work has demonstrated that Sprouty-related EVH1 domainprotein2 (Spred2) was involved in imatinib mediated cytotoxicity in CML cells. However, its roles in growth and lineage differentiation of CML cells remain unknown. In this study, we found that CML CD34⁺ cells expressed lower level of Spred2 compared with normal hematopoietic progenitor cells, and adenovirus mediated restoration of Spred2 promoted the erythroid differentiation of CML cells. Imatinib could induce Spred2 expression and enhance erythroid differentiation in K562 cells. However, the imatinib induced erythroid differentiation could be blocked by Spred2 silence using lentiviral vector PLKO.1-shSpred2. Spred2 interference activated phosphorylated-ERK (p-ERK) and inhibited erythroid differentiation, while ERK inhibitor, PD98059, could restore the erythroid differentiation, suggesting Spred2 regulated the erythroid differentiation partly through ERK signaling. Furthermore, Spred2 interference partly restored p-ERK level leading to inhibition of erythroid differentiation in imatinib treated K562 cells. In conclusion, Spred2 was involved in erythroid differentiation of CML cells and participated in imatinib induced erythroid differentiation partly through ERK signaling.     

N-terminal 1-54 amino acid sequence and Armadillo repeat domain are indispensable for P120-catenin isoform 1A in regulating E-cadherin

P120-catenin (p120ctn) exerts important roles in regulating E-cadherin and invasiveness in cancer cells. However, the mechanisms by which p120ctn isoforms 1 and 3 modulate E-cadherin expression are poorly understood. In the current study, HBE, H460, SPC and LTE cell lines were used to examine the effects of p120ctn isoforms 1A and 3A on E-cadherin expression and cell invasiveness. E-cadherin was localized on the cell membrane of HBE and H460 cells, while it was confined to the cytoplasm in SPC and LTE cells. Depletion of endogenous p120ctn resulted in reduced E-cadherin expression; however, p120ctn ablation showed opposite effects on invasiveness in the cell lines by decreasing invasiveness in SPC and LTE cells and increasing it in HBE and H460 cells. Restitution of 120ctn isoform 1A restored E-cadherin on the cell membrane and blocked cell invasiveness in H460 and HBE cells, while it restored cytoplasmic E-cadherin and enhanced cell invasiveness in SPC and LTE cells. P120ctn isoform 3A increased the invasiveness in all four cell lines despite the lack of effect on E-cadherin expression, suggesting a regulatory pathway independent of E-cadherin. Moreover, five p120ctn isoform 1A deletion mutants were constructed and expressed in H460 and SPC cells. The results showed that only the M4 mutant, which contains N-terminal 1-54 amino acids and the Armadillo repeat domain, was functional in regulating E-cadherin and cell invasiveness, as observed in p120ctn isoform 1A. In conclusion, the N-terminal 1-54 amino acid sequence and Armadillo repeat domain of p120ctn isoform 1A are indispensable for regulating E-cadherin protein. P120ctn isoform 1A exerts opposing effects on cell invasiveness, corresponding to the subcellular localization of E-cadherin.     

p120(ctn) acts as an inhibitory regulator of cadherin function in colon carcinoma cells

p120(ctn) binds to the cytoplasmic domain of cadherins but its role is poorly understood. Colo 205 cells grow as dispersed cells despite their normal expression of E-cadherin and catenins. However, in these cells we can induce typical E-cadherin-dependent aggregation by treatment with staurosporine or trypsin. These treatments concomitantly induce an electrophoretic mobility shift of p120(ctn) to a faster position. To investigate whether p120(ctn) plays a role in this cadherin reactivation process, we transfected Colo 205 cells with a series of p120(ctn) deletion constructs. Notably, expression of NH2-terminally deleted p120(ctn) induced aggregation. Similar effects were observed when these constructs were introduced into HT-29 cells. When a mutant N-cadherin lacking the p120(ctn)-binding site was introduced into Colo 205 cells, this molecule also induced cell aggregation, indicating that cadherins can function normally if they do not bind to p120(ctn). These findings suggest that in Colo 205 cells, a signaling mechanism exists to modify a biochemical state of p120(ctn) and the modified p120(ctn) blocks the cadherin system. The NH2 terminus-deleted p120(ctn) appears to compete with the endogenous p120(ctn) to abolish the adhesion-blocking action.