p63基因在肺癌组织中的表达

目的研究肺鳞状细胞癌、腺癌、大细胞肺癌、小细胞肺癌以及淋巴结或肺内转移性肿瘤标本组织中p63基因的mRNA转录因子和蛋白表达水平,探讨p63基因表达与其定位在3q 27-q29区域改变的关系.方法采用cDNA微阵列技术同时检测72例不同病理组织学类型的原发性肺癌(包括鳞状细胞癌、腺癌、大细胞癌、小细胞癌)和肺癌肺内转移及淋巴结转移灶内的p63基因mRNA水平.另外,利用组织芯片技术构建150例原发性肺癌石蜡包埋标本组织芯片,采用免疫组织化学LSAB法检测p63基因蛋白表达情况.同时应用比较基因组杂交(CGH)技术对70例原发性肺癌标本进行了3号染色体长臂改变的分析.结果p63mRNA转录因子在肺鳞状细胞癌标本表达明显增强,与腺癌、大细胞癌、小细胞癌相比增多10倍以上.肺癌转移灶内p63基因mRNA表达水平明显高于其相对应的原发性灶,差异有统计学意义(P＜0.001).免疫组织化学染色结果显示肺鳞状细胞癌阳性表达率为94.64%;腺癌是1.79%;4例大细胞肺癌中2例为阳性染色;但所有小细胞肺癌标本免疫组织化学染色均为阴性.pT1分期肺癌的p63基因蛋白表达阳性率与pT2分期肺癌相比有统计学差异(P＜0.05).比较基因组杂交结果发现肺鳞状细胞癌3号染色体长臂27-29区域有显著的扩增,腺癌表现为缺失.鳞状细胞癌p63基因的表达增强与3q27-q29区域的显著扩增有明显正相关性(P＜0.000 1),说明定位于3号染色体长臂27-29区域的p63基因的拷贝有明显的扩增.结论p63mRNA转录因子和蛋白在肺鳞状细胞癌较其他肿瘤表达显著增高,转移灶高于原发灶;肺鳞癌p63表达增强与3号染色体长臂3q27-q29区域的扩增显著相关.     

c-erbB-2、p53、bcl-2和nm23-H1在肺癌中的表达

目的：探讨ｃｅｒｂＢ２、ｐ５３、ｂｃｌ２和ｎｍ２３Ｈ１基因在肺癌发生发展过程中的作用。方法：用免疫组化ＡＢＣ法对原发性肺癌组织中４种基因的表达和突变进行检测。结果：５８例肺癌中,３１例（５３４５％）ｐ５３过度表达,１８例（３１０３％）ｂｃｌ２过度表达。ｃｅｒｂＢ２与ｎｍ２３Ｈ１在１０例小细胞肺癌（ＳＣＬＣ）中未见表达。而在４８例非小细胞肺癌（ＮＳＣＬＣｓ）中两者过度表达率均为５０％。ｃｅｒｂＢ２与ｎｍ２３Ｈ１表达呈正相关（Ｐ＜００５）。腺癌ｎｍ２３Ｈ１的表达明显高于鳞癌（Ｐ＜００５）。ｐ５３、ｂｃｌ２蛋白表达在肺癌分化程度中呈负相关（Ｐ＜００５）。ｎｍ２３Ｈ１、ｐ５３和ｂｃｌ２的表达与患者的生存率有关（Ｐ＜００５）。结论：ｃｅｒｂＢ２、ｐ５３、ｂｃｌ２和ｎｍ２３Ｈ１基因蛋白产物的检测对肺癌患者的诊治和预后评估有积极意义。     

Expression and localization of thrombomodulin in preneoplastic bronchial lesions and in lung cancer

Thrombomodulin (TM) is an endothelial surface glycoprotein that acts as a natural anticoagulant. It inhibits thrombin and accelerates the activation of the anticoagulant protein C. TM has been detected in dermal keratinocytes, where it is associated with terminal differentiation. It can also be detected in various types of squamous malignant neoplasms and in malignancies of endothelial and mesothelial origin, such as Kaposi's sarcoma or malignant mesothelioma, but is absent in pulmonary adenocarcinomas (AC). Seventy-two lung tumour specimens [33 squamous cell carcinomas (SQCC), 23 AC, 1 large cell carcinoma, 8 small cell lung cancers (SCLC) and 7 multidifferentiated tumours (MT)] were analysed immunohistochemically by staining with an anti-TM antibody in order to assess TM expression. All of the SQCC stained positively for TM. In contrast, only 9 AC and 4 MT and none of the SCLC showed positive anti-TM staining. Seven hyperplastic bronchial epithelial specimens and eight preneoplastic bronchial lesions (five cases of moderate dysplasia, two cases of severe dysplasia and one case of carcinoma in situ) were used as controls.Normal or hyperplastic areas of bronchial epithelium revealed no positive reaction. However, a distinct positive anti-TM staining pattern related to the degree of keratiniziation of dysplastic lesions was seen. The present results suggest that anti-TM immunostaining is a useful marker for squamous cell carcinoma in the differential diagnosis of pulmonary carcinoma, also indicating keratinocyte differentiation in dysplastic bronchial epithelium.     

Expression profiling of integrins in lung cancer cells

Integrins are heterodimeric transmembrane receptors consisting of 18 α and 8 β subunits. Heterodimer composition of α and β subunits has a potential for determining tumor subtypes of human lung cancer. The purpose of this study was to investigate the expression profile of integrins in lung cancer cells. Expression profiling of integrins in a panel of lung cancer cell line, including A549 (adenocarcinoma, ADC), Calu-1 (squamous carcinoma, SCC), H1650 (bronchioloalveolar carcinoma, BAC) and DMS-53 (small cell lung cancer, SCLC), was analyzed by cDNA microarrays, restriction analysis of gene expression (RAGE) and flow cytometry. Seventy-nine lung cancer specimens were used to further validate the data from cell lines using immunohistochemistry. Integrins are obviously expressed in a cell type-specific manner, such as α3 in A549, Calu-1 and H1650 except in DMS53, α4 in H1650, α5 and β1 in all cell lines. The integrins detected with cDNA microarrays were all unequivocally detected with RAGE and by flow cytometry at the protein level. In all lung cancer specimens, α3 integrin was strongly expressed in ADC, SCC and BAC, but was infrequent in SCLC. α4 integrin was solely expressed in BAC. α5 and β1 integrins were expressed in all four histological types of lung cancer specimens. Integrin α3 and α4 may be useful as diagnostic markers for adenocarcinoma, squamous cell carcinoma and bronchioloalveolar carcinoma. RAGE is a promising technique for studying the expression profiles of genes, such as integrins in cancer cells.     

人肺癌细胞凋亡与p53、bcl-2、Bax的表达

目的：探讨人肺癌组织细胞自发凋亡的发生,ｐ５３基因及凋亡相关基因ｂｃｌ－２、Ｂａｘ在肺癌组织中的表达及与肺癌细胞凋亡和生物学行为的关系。方法：用甲基绿－派诺宁染色检测５０例肺癌组织切片中凋亡细胞,ＬＳＡＢ免疫组化法检测ｐ５３和Ｂａｘ的蛋白表达,原位杂交方法检测ｂｃｌ－２ ｍＲＮＡ的表达。结果：１００％小细胞肺癌凋亡发生数１０个／ＨＰＦ,２１％的肺鳞癌和１５％的肺腺癌凋亡发生数〉１０个／ＨＰＦ。ｐ５     

Decreased Beclin-1 expression is correlated with the growth of the primary tumor in patients with squamous cell carcinoma and adenocarcinoma of the lung

Beclin-1 is a Bcl-2-interacting protein, and it may delay cell cycle progression and induce autophagy. The function and expression of Beclin-1 and Bcl-2 in squamous cell carcinoma and adenocarcinoma of the lung remain largely unknown. Herein, we investigated the expression of Beclin-1 and Bcl-2 in squamous cell carcinoma and adenocarcinoma of the lung. Tissue samples from 262 cases were used in this study. Immunohistochemical staining for Beclin-1 and Bcl-2 were conducted using a tissue microarray. In squamous cell carcinoma, Beclin-1 expression was strongly positive in 48 (28.6%) of 168 samples, it was moderately positive in 42 (25.0%) of 168 samples, and it was negative or weakly positive in 78 (46.4%) of 168 samples. In adenocarcinoma, Beclin-1 expression was strongly positive in 26 (27.7%) of 94 samples, it was moderately positive in 27 (28.7%) of 94 samples, and it was negative or weakly positive in 41 (43.6%) of 94 samples. Beclin-1 expression was inversely correlated with the tumor size and the primary tumor stage (pT) in both types of tumor. Especially, the TNM stage of adenocarcinoma was inversely correlated with Beclin-1 expression. Our results suggest that a progressively reduced Beclin-1 expression is correlated with the primary tumor growth of squamous cell carcinoma and adenocarcinoma of the lung.     

Bcl-2 protein expression in lung cancer and close correlation with neuroendocrine differentiation.

For determination of the cellular distribution of bcl-2 expression in lung cancer and clarification of its correlation with cell neuroendocrine differentiation, Bcl-2 immunostaining was carried out on a large series of formalin-fixed, paraffin-embedded lung cancer samples, and four general neuroendocrine marker and seven peptide hormone stainings were carried out on all Bcl-2-positive squamous cell carcinomas and adenocarcinomas of the lung as well as on 8 pulmonary neuroendocrine carcinomas histologically diagnosed. In addition, 3 small cell lung cancer cell lines were studied by Western blotting. Neuroendocrine differentiation in Bcl-2-negative squamous cell carcinomas and adenocarcinomas was examined with chromogranin A and alpha-subunit of Go protein stainings. Bcl-2 protein was detected in 104/111 small cell carcinomas, 8/8 neuroendocrine carcinomas, 0/6 typical (well differentiated) carcinoids, 23/64 squamous cell carcinomas, 4/65 adenocarcinomas, and all 3 small cell lung cancer cell lines. All 8 neuroendocrine carcinomas, 11 of the Bcl-2-positive squamous cell carcinomas, and all 4 Bcl-2 positive adenocarcinomas expressed multiple neuroendocrine markers. The distributions of Bcl-2 and neuroendocrine marker immunoreactivity closely paralleled each other on consecutive sections. In squamous cell carcinomas, Bcl-2-positive cells could be roughly subdivided into those with neuroendocrine differentiation features, usually demonstrating intense Bcl-2 staining, with basaloid tumor cells usually expressing weak to moderate Bcl-2 staining. The present study clearly shows Bcl-2 protein expression to be remarkably differentially regulated according to histological types of lung cancers and to appear to quite likely be closely associated with neuroendocrine differentiation of tumor cells, indicating that bcl-2 is importantly involved in cell development and differentiation, in addition to protecting cells from apoptosis. Bcl-2 might be usable as a neuroendocrine marker in lung cancers and possibly also in neural-crest-derived tumors.     

钾离子通道蛋白Kir2.1/KCNJ2及多药耐药蛋白MRP1/ABCC1在小细胞肺癌中的表达及相关性

 目的：探讨内向整流钾通道2.1（Kir2.1/KCNJ2）及多药耐药相关蛋白1（MRP1/ABCC1）在小细胞肺癌(SCLC) 中的表达情况及两者的相关性。方法：采用实时定量PCR和蛋白印迹法检测小细胞肺癌多药耐药细胞株H69AR及敏感株H69细胞中Kir2.1/KCNJ2及MRP1/ABCC1 mRNA和蛋白的表达水平,采用免疫组化EnVision两步法检测44 例SCLC组织中Kir2.1/KCNJ2和MRP1蛋白的表达水平;采用蛋白免疫共沉淀实验检测Kir2.1/KCNJ2与MRP1/ABCC1之间的相互作用。
结果：H69AR细胞株中Kir2.1/KCNJ2及MRP1/ABCC1 mRNA和蛋白的表达均显著高于H69细胞株,二者均有显著差异（P<005）;免疫组化结果表明SCLC组织中Kir2.1/KCNJ2和MRP1/ABCC1阳性表达率分别为47.7%(21/44) 和52.3%(23/44),两者表达在SCLC 中呈正相关(r=0853,P<0.01); Kir2.1/KCNJ2与MRP/ABCC1在H69AR细胞内可形成复合物。结论：Kir2.1/KCNJ2与小细胞癌多药耐药有关。Kir2.1/KCNJ2和MRP1/ABCC1之间可能存在共同作用通路。Kir2.1/KCNJ2可能通过调控MRP1/ABCC1的表达促进SCLC多药耐药的发生。     

Expression of epithelial and neural antigens in small cell and non small cell lung carcinoma

Seventy-one lung carcinomas from 66 different patients were stained with a panel of monoclonal antibodies. Twenty-nine were small cell lung carcinoma (SCLC), 15 adenocarcinomas, 17 squamous carcinomas and 10 large cell carcinomas. Three of the monoclonal antibodies recognize different cytokeratins, three recognize other epithelial antigens and one recognizes a neural antigen. Both formalin-fixed and cryopreserved tumours were studied using an indirect immunoperoxidase method. 23/29 SCLC reacted with all but one of the antibodies which recognize epithelial antigens. This staining was similar to that seen in non small cell lung carcinomas (NSCLC) and provides further evidence that SCLC are true epithelial tumours. All but one of the SCLC stained with the antibody recognizing a neural antigen. This antibody did not stain squamous or adenocarcinomas. However, four of the large cell carcinomas stained well with this antibody, suggesting that SCLC and some large cell carcinomas share a common pathway of differentiation. There were variations of staining seen both within and between tumours. This has obvious implications if immunotargetting with monoclonal antibodies is to be used diagnostically or therapeutically.     

Non-receptor tyrosine kinase Etk is involved in the apoptosis of small cell lung cancer cells

Epithelial and endothelial tyrosine kinase (Etk), also known as Bmx (bone marrow X kinase) plays an important role in apoptosis of epithelial cells. The goal of this study was to investigate whether Etk is involved in apoptosis of small cell lung cancer (SCLC) cells and correlated with the expression levels of apoptosis-associated proteins such as Bcl-2, Bcl-X_L and p53. One hundred and seventy-one cases of lung cancer specimens including seventy-one SCLCs and one hundred NSCLCs were immunostained for Etk, Bcl-2, Bcl-X_L and p53. Parental SCLC H446 cell line, and its subline (H446-Etk) that overexpresses Etk, were used to study the role of Etk in apoptosis induced by doxorubicin. It was found that high expression of Etk occurs in 74.6% of SCLC cases, but only in 40% of NSCLC cases, and there is marked difference in the expression levels of Bcl-2, Bcl-X_L and p53 between Etk-positive and Etk-negative SCLC cases. Furthermore, the levels of Bcl-2 and Bcl-X_L significantly increased in H446-Etk cells than that in H446 cells after doxorubicin treatment, and were positively associated with Etk expression. However, p53 did not correspond with Etk expression although its expression decreased greatly with apoptosis both in H446-Etk and H446 cells. After doxorubicin treatment, the cell viability was significantly higher in H446-Etk cells than in parental H446 cells. Downregulation of Etk by Etk siRNA sensitized H446 cells to doxorubicin. Our results indicate that upregulation of tyrosine kinase Etk may be a new mechanism involved in protection of SCLC cells from apoptosis. Bcl-2 and Bcl-X_L but not p53 may contribute to doxorubicin-induced apoptosis through Etk pathway.     

Dihydroartemisinin inhibits cell proliferation via AKT/GSK3β/cyclinD1 pathway and induces apoptosis in A549 lung cancer cells

Lung cancer is the most common cause of cancer-related death in the world. The main types of lung cancer are small cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC); non small cell lung carcinoma (NSCLC) includes squamous cell carcinoma (SCC), adenocarcinoma and large cell carcinoma, Non small cell lung carcinoma accounts for about 80% of the total lung cancer cases. Dihydroartemisinin (DHA) inhibits cell proliferation and induces apoptosis in several cancer cell lines. The effects of DHA on cell growth and proliferation in lung cancer cells remain to be elucidated. Here, we demonstrate that DHA inhibited cell proliferation in the A549 lung cancer cell line through suppression of the AKT/Gsk-3β/cyclin D1 signaling pathway. DHA significantly inhibited cell proliferation of A549 cells in a concentration and time dependent manner as determined by MTS assay. Flow cytometry analysis demonstrated that DHA treatment of A549 cells resulted in cell cycle arrest at the G1 phase, which correlated with apparent downregulation of both mRNA and protein levels of both PCNA and cyclin D1. These results suggest that DHA is a potential natural product for the treatment of lung cancer.     

Expression of Bcl-2 and Amplification of c-myc Are Frequent in Basaloid Squamous Cell Carcinomas of the Esophagus

Basaloid squamous cell carcinoma (BSCC) of the esophagus is a rare, poorly differentiated variant of typical esophageal squamous cell carcinoma (SCC) characterized by high proliferative activity and frequent spontaneous apoptoses. In the present study, we investigated the expression of the apoptosis-suppressing protein Bcl-2 in 23 BSCC of the esophagus and 23 stage-matched typical esophageal SCC by means of immunohistochemistry. In addition, amplification of the apoptosis- and proliferation-inducing gene c-myc was determined by means of differential polymerase chain reaction. Bcl-2 expression was found significantly more often in BSCC than in SCC (86.9% vs. 17.4%, P < 0.0001). Amplification of c-myc was nearly twice as common in BSCC as in SCC (47.8% vs. 26.1%, not significant). Bcl-2 protein expression together with c-myc amplification was detected in 43.5% of the BSCC but in none of the typical SCC (P < 0.0001). Taken together, our findings indicate that the molecular pathogenesis of esophageal BSCC differs from that of typical SCC and frequently involves coactivation of c-myc and Bcl-2.     

HIF-2α expression is suppressed in SCLC cells, which survive in moderate and severe hypoxia when HIF-1α is repressed

Small cell lung carcinoma (SCLC) is extremely aggressive and frequently metastasizes widely in its early stage. Because tumor hypoxia is related to aggressive tumor behavior and the hypoxic adaptation of SCLC is poorly documented, we stained SCLC tumors arranged in a tissue microarray for hypoxia-inducible factor (HIF)-1α and HIF-2α proteins. We found an overall lack of HIF-2α protein expression, which was confirmed in large tumor sections. HIF-1α protein was strongly expressed in most tumors, frequently adjacent to necrotic regions. In concordance, cultured SCLC but not non-small cell lung carcinoma cells showed no or extremely low levels of HIF-2α mRNA and no HIF-2α protein at hypoxia. HIF-1α was stabilized after 4 hours at hypoxia, and its accumulation increased up to 96 hours. SCLC cells survived well and showed net proliferation and low cell death in modest (1% oxygen) and severe (0.1% oxygen) hypoxia. HIF-1α repression virtually did not influence cell death or viability despite reduced levels of hypoxia-inducible genes, such as BNIP3 and BNIP3L. At 1% oxygen no increased autophagy (LC3B-II activation) or NF-κB signaling were detected, whereas the unfolded protein response was activated at severe hypoxia. Our data indicate that HIFs are not exclusively required for SCLC cell survival at modest or severe hypoxia and that additional, yet uncharacterized, hypoxia-driven adaptation pathways may become activated.     

p53 expression in oat and non-oat small cell lung carcinomas: correlations with proliferating cell nuclear antigen.

AIMS--To investigate the immunohistochemical expression of p53 protein in small cell lung carcinoma (SCLC), comparing it with that in non-small cell carcinoma (NSCLC); and to evaluate the correlation between p53 and proliferating cell nuclear antigen (PCNA) expression as well as between p53 and PCNA expression and survival. METHODS--Paraffin wax embedded tissues from 61 cases of primary lung carcinoma were stained for p53 protein and PCNA using the monoclonal antibodies 1801 and PC-10, respectively, in a standard avidin-biotin immunoperoxidase method. RESULTS--Of the 61 lung carcinomas 35 (57%) were positive for p53 (range 1% to 90%). Ninety percent of the non-oat SCLC contained positive cells and the labelling index (LI) was significantly higher than that of the oat SCLC (p < 0.001). SLCC also displayed a higher p53 LI than NSCLC (p < 0.01); no difference was found between squamous carcinoma, adenocarcinoma, and oat cell carcinoma. A p53 LI of greater than 1% tended to be associated with nodal metastases (p < 0.05), and p53 expression in node positive tumours as well as in oat cell carcinomas was indicative of poorer survival (p < 0.01 and p < 0.1, respectively). A p53 Li of greater than 60% was a negative prognostic factor in non-oat SCLC (p < 0.05). PCNA LI was highest in non-oat SCLC and lowest in NSCLC; oat cell carcinomas had a mean LI intermediate between NSCLC and non-oat SCLC (NSCLC v oat cell carcinoma p < 0.05 and oat cell v non-oat cell carcinomas p < 0.01). A PCNA LI was not correlated with nodal metastases or survival, but there was a significant positive correlation between PCNA LI and p53 LI (rs = 0.484, p < 0.001). CONCLUSIONS--p53 and PCNA expression differ substantially among the various types of lung carcinomas. Substantial differences were also found between oat and non-oat types of SCLC, indicating that SCLC is heterogeneous as far as proliferation rate and altered p53 expression is concerned. p53 seems to be of some prognostic value. The relation between PCNA and p53 expression indicates that the PCNA gene is slightly upregulated by p53.     

Increased activity of nm23-H1 gene in squamous cell carcinoma of the head and neck is associated with advanced disease and poor prognosis

The present study was undertaken to determine whether the nm23-H1 gene is expressed in squamous cell carcinoma of the head and neck (SCCHN) and whether the level of nm23-H1 protein or mRNA in cells vary as they progress to a more malignant phenotype. Of the 120 SCCHN studied 54 (45%) stained positively for nm23-H1 protein. Protein expression was significantly higher in more advanced stages of disease. Expression of nm23-H1 was significantly higher in cancer tissues than in normal, adjacent tissue, dysplasia, or carcinoma in situ. The nm23-H1 rate increased with progression of synchronous lesions from dysplasia to carcinoma in situ and finally to carcinoma (P<0.05). Northern blot analyses of tissues with various clinicopathological characteristics also revealed differences in nm23-H1 mRNA expression. When levels of nm23-H1 mRNA were compared to tumor stage, intensity of expression was found to be higher in stages 3 and 4 than stages 1 and 2 (P<0.01). Malignant tumors had a higher level of mRNA nm23-H1 expression than normal or premalignant tissues. The nm23-H1 negative patients survived significantly longer than nm23-H1 positive ones (P<0.05). To study the possible relationship between nm23-H1 gene expression and cell growth rate in tumor cells, the mRNA level in each tumor was compared to proliferative activity. The nm23-H1 gene expression levels were directly related to the [3H]thymidine labeling index in tumor cells (R=0.6681). Our results strongly indicate that the nm23-H1 gene is involved in progression of SCCHN. Together with results obtained on lung cancer, our observations suggest that increased expression of nm23-H1 in cancers of the upper aerodigestive tract may have different implications than elsewhere in the body.     

PTEN/MMAC1/TEP1在肺癌中的丢失和失活

目的 了解抑制癌基因ＰＴＥＮ／ＭＭＡＣ１／ＴＥＰ１（以下简称ＰＴＥＮ）在肺癌中的缺失和失活。方法 选用２４例具有正常对照的肺癌新鲜标本,１８例小细胞肺癌石蜡切片,用聚合酶链反应及杂合性丢失分析法检测ＰＴＥＮ的杂合笥丢失;并采用原位杂交法、免疫组化化学染色法、Ｗｅｓｔｅｍ ｂｌｏｔ法观察肺癌标本中ＰＴＥＮ ｍＲＮＡ和蛋白水平的表达。对１株肺腺癌和３株小细胞肺癌系进行ＰＴＥＮ的Ｓｏｕｔｈｅｒｎ、Ｎｏｒ