黏着斑激酶相关非激酶质粒转染对肝星状细胞凋亡的影响

目的：应用黏着斑激酶相关非激酶（FRNK）表达质粒瞬时转染肝星状细胞（HSCs），探讨FRNK选择性抑制黏着斑激酶(FAK)磷酸化对FN刺激的HSCs凋亡的影响。方法:在体外培养HSCs，以FN刺激HSCs增殖，采用脂质体介导的方法进行FRNK表达质粒转染。应用膜联蛋白（Annexin-V）/碘化丙啶（PI）双标记流式细胞术、DNA凝胶电泳技术和透射电镜技术检测细胞的凋亡，Western blotting及RT-PCR方法检测FRNK、FAK、p-FAK（Tyr397）、caspase-3蛋白及其mRNA表达。结果:FRNK表达质粒成功转染HSCs，在翻译后水平抑制FAK磷酸化；FRNK表达质粒转染HSCs 48 h后，细胞凋亡率增加，与空质粒组之间有显著差异［（25.37±1.92）% vs（9.28±1.05）%］，P<0.01，伴随caspase-3在翻译和转录水平的增高［（264.17±12.60 vs 185.82±9.69），P<0.01；（4.19±0.48 vs 1.07±0.27），P<0.01］。结论:在脂质体介导下瞬时转染FRNK表达质粒，可使外源性的FRNK在HSCs内大量表达，在翻译后水平抑制FAK磷酸化；可以诱导FN刺激的HSCs发生凋亡。     

膜型基质金属蛋白酶-1在黏着斑激酶相关非激酶调控肝星状细胞胶原代谢中的作用

目的： 探讨黏着斑激酶相关非激酶（FRNK）对纤维连接蛋白（FN）刺激的肝星状细胞（HSC）膜型基质金属蛋白酶-1（MT1-MMP）表达的影响并探讨MT1-MMP在FRNK调控HSC胶原代谢中的作用。方法：应用体外细胞培养技术,脂质体介导法进行FRNK质粒瞬时转染;采用Western blotting法测定FRNK蛋白在瞬时转染时相应的表达,鉴定转染效果;分别应用Western blotting和RT-PCR方法测定MT1-MMP在HSC中的相应表达。结果：FRNK质粒成功转染HSC,于转染48 h时,FRNK蛋白表达最强,P<0.05;FRNK转染后在基因水平上：MT1-MMP mRNA表达明显增加;翻译蛋白水平上：FRNK质粒转染HSC 48 h后,MT1-MMP蛋白表达量显著升高。结论：脂质体介导下FRNK质粒转染,可使外源性FRNK在HSC内大量表达,FRNK可能通过上调MT1-MMP值来抑制HSC胶原合成。     

粘着斑激酶在血管内皮细胞迁移中的作用

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase (PTK) that can localize indirectly to sites of clustering integrin family of heterodimeric receptors. As an important structure and signaling molecule in the adhesive complexes, which are large and stable referred as ‘focal adhesions‘ or relatively small and transient within filopodia and lamellipodia named ‘focal complexes‘, FAK is closely related with cell death, proliferation and migration. In this review, we discuss the function of FAK in the regulation of endothelial cell migration based on current data.     

Epithelial (E-) and placentae (P-) cadherin cell adhesion molecule expression in breast carcinoma

Two members of the cadherin family of intercellular adhesion molecules are found in normal breast tissue: E- (epithelial) cadherin is present in both luminal and myoepithelial cells, whereas P- (placental) cadherin is confined to myoepithelium. There is experimental evidence that loss of E-cadherin is associated with increased invasiveness of malignant cells in vitro, which stimulated us to examine the presence and distribution of E- and P-cadherin in breast carcinomas by means of immunohistochemical staining. E-Cadherin was present in all in situ and invasive ductal carcinomas examined, although it had a patchy distribution and the staining was of variable intensity. However, in 83 per cent of invasive lobular carcinomas and all lobular carcinomas in situ there was complete loss of E-cauherin expression. In the remaining 17 per cent of invasive lobular tumours, E-cadherin appeared to have an abnormal distribution within the cytoplasm with variable expression on the cell membrane. P-Cadherin, by contrast, was absent from all benign breast luminal epithelium and 25 carcinomas of ductal and lobular type. It was found in only one carcinoma of lobular type. We suggest that loss of cell-cell adhesion mediated by E-cadherin plays a part in the characteristic morphology of lobular carcinomas.     

Focal adhesion kinase (FAK) expression in normal and neoplastic lymphoid tissues

Focal adhesion kinase (FAK) is a protein tyrosine kinase essential for intracellular regulatory events, such as cell growth, differentiation, migration and tumor metastasis. The aim of this study was to analyze the expression of FAK protein in a series of normal and neoplastic lymphoid tissues.An anti-FAK antibody was used to study the protein expression in paraffin-embedded samples of normal and neoplastic, hematolymphoid and non-hematolymphoid tissues by immunohistochemistry. In normal hematolymphoid tissue, the strongest expression of FAK was detected in germinal center and marginal-zone B cells; positive staining was also found in mantle zone B cells. In human lymphomas, FAK was expressed mostly in B-cell lymphomas and was predominantly negative in T-cell lymphoma. In Hodgkin lymphomas, FAK was found only in the neoplastic cells of lymphocyte predominant type, whereas the tumor cells of the classical form were FAK-negative.We demonstrate for the first time the expression of FAK in paraffin-embedded hematolymphoid tissue samples. Its differential expression in lymphomas may be of relevance for some B-cell neoplasms by using it as an additional marker to distinguish B- from T-lymphoblastic leukemia/lymphoma to further differentiate lymphocyte predominant from classical Hodgkin lymphoma.     

Expression of E-selectin, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 in non-small-cell lung carcinoma

Vascular cell adhesion molecules (VCAM) play an important part in the regulation of inflammation and are considered to be important in the process of malignant tumour growth. The present study describes the immunohistochemical staining patterns of E-selectin, intercellular adhesion molecule (ICAM)-1 and VCAM-1 on endothelial cells of the vessels in tumour stroma and other cell types in non-small-cell lung carcinoma (NSCLC; n=43) in association with inflammatory cells. Expression of E-selectin was dominant on endothelial cells in the stromal areas of the tumour, especially at the borders, and was confined to endothelial cells. Moderate to strong staining for ICAM-1 was demonstrated on endothelial cells irrespective of size or localization of the vessels. Compared with ICAM-1, fewer vessels were positive for VCAM-1, and stained with lesser intensity. ICAM-1 expression was demonstrated on NSCLC cells, the basal cells of bronchial epithelium, type II pneumocytes, lymphocytes and fibroblasts. VCAM-1 was clearly expressed on NSCLC cells in 4 of the 43 cases and on lymphocytes and fibroblasts. The staining patterns observed on endothelial cells support the idea of an active status of NSCLC vessels. This phenotypic pattern looks similar to the vascular component of inflammation. The presence of ICAM-1 and VCAM-1 on NSCLC cells suggests a functional role in the process of chemotaxis for tumour cells.     

Expression of focal adhesion kinase in uveal melanoma and the effects of Hsp90 inhibition by 17-AAG

IntroductionFocal adhesion kinase (FAK) is implicated in tumor progression and metastatic cascade, and has been shown to be overexpressed in a variety of human cancers. However, the role of FAK in human uveal melanoma (UM) is not well defined. The purpose of this study was to evaluate the expression of FAK in UM tumors and normal eyes, and to determine the effect of Hsp90 inhibition on FAK expression in UM cells.MethodsFAK expression was assessed in 39 UM specimens, FAK[pY397] expression was assessed in 51 UM specimens, and both FAK and FAK[pY397] expression were assessed in 20 normal eyes. The expression of FAK and FAK[pY397] was detected by Western blot in five UM cell lines after treatment with 10 μmol/L of 17-AAG.ResultsFAK was positive in 87.2% and FAK[pY397] in 90% of UM specimens. Low FAK expression was detected in non-tumor structures and in normal eyes. The cell lines with the most proliferative, invasive phenotype (92.1, SP6.5 and MKT-BR) displayed high expression of FAK[pY397], and the levels of FAK and FAK[pY397] were decreased in the presence of 17-AAG starting with 24 h of exposure.ConclusionFAK and FAK[pY397] were overexpressed in human UM tumors compared to normal ocular tissue and high levels of FAK[pY397] were seen in the most aggressive UM cell lines. Hsp90 inhibition led to downregulation of FAK expression. We propose a role for FAK in the pathogenesis of UM. Future studies are needed to explore the use of Hsp90 inhibitors as a feasible approach for modulating FAK in UM.     

Role of focal adhesion kinase (FAK) in renal ischaemia and reperfusion

Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, plays important roles in cell migration, cell proliferation and cell survival. Because these processes participate in the restoration of tubular integrity in renal ischaemia and reperfusion, FAK expression and phosphorylation at Tyr-397, the latter indicative of its activity, were examined in the different kidney zones by Western blot analysis and immunohistochemistry. Expression and phosphorylation of FAK were also studied in Madin-Darby canine kidney (MDCK) and medullary thick ascending limb (mTAL) cells after ATP depletion and repletion. In control rat kidneys, FAK expression in outer and inner medulla exceeded that in cortex, and phosphorylation of FAK at Tyr-397 was most pronounced in the inner medulla. Although this expression pattern was not affected by 20 (40, 60)-min ischaemia and 20 (40, 60)-min ischaemia followed by 60-min or 24-h reperfusion, FAK phosphorylation was significantly reduced in all kidney zones immediately after ischaemia, but increased during reperfusion, exceeding control values in the outer and inner medulla. ATP depletion and repletion of MDCK and mTAL cells were associated with a decrease in FAK phosphorylation during ATP depletion, followed by an increase during repletion. Rephosphorylation of FAK after ATP repletion was enhanced by N-acetylcysteine, a reactive oxygen species scavenger. ATP depletion disrupted focal adhesions in MDCK cells. Their reformation after ATP repletion paralleled the increase in FAK phosphorylation. These findings suggest an essential role for FAK-signalling during renal ischaemia and early reperfusion.     

实验性高血压大鼠左心室肥大心肌细胞中黏着斑激酶的表达

目的研究黏着斑激酶（FAK）在高血压心室肥大发生发展机制中的作用。方法以特发性高血压心力衰竭（SHHF）大鼠为研究对象,通过免疫荧光标记、共聚焦显微镜及Western blot等方法,检测不同月龄SHHF大鼠左心室心肌细胞中FAK的表达及其分布。结果实验组SHHF大鼠与对照组WKY大鼠比较,FAK的表达在2月龄时无明显变化（50．5±6．9比49．8±5．0,n=6,P〉0．05）,而6、12、18月龄的SHHF大鼠心肌细胞中,FAK表达明显增加（130．6±3．0比47．3±1．3,144．7±5．4比46．4±3．1,141．4±9．8比48．5±2．2,各组n=6,P〈0．05）,并且聚集在心肌细胞两端的闰盘和细胞核等部位。结论在心肌肥大的发展过程中,FAK表达增加并有闰盘及核内的聚积,提示FAK与心肌肥大关系密切,可能参与了心肌肥大细胞信号转导的调控。     

Expression of the neural cell adhesion molecule (CD56) by human myeloma cells.

Recent studies in multiple myeloma indicate that molecules associated with different haematopoietic lineages may be expressed aberrantly by myeloma cells. In order to investigate this phenomenon further, we studied the immunophenotype of bone marrow cells from 21 patients with multiple myeloma using a panel of monoclonal antibodies against T,B, myelomonocytic, and natural killer (NK)-cell antigens. Leu-19/NKH1 (CD56), a molecule identical to N-CAM, which is normally expressed by neuroectodermal and NK cells, was found in 13 patients (62%). Dual-parameter flow cytometry was used to correlate N-CAM positivity with DNA aneuploidy or cytoplasmic immunoglobulin expression as markers of myeloma cells. When N-CAM was found positive, other haematopoietic antigens were expressed only in three out of 13 cases (23%). In contrast, myeloma cells not expressing N-CAM frequently exhibited pre-B cell markers, myeloid antigen, and HLA-DR, respectively (seven out of eight cases, 88%). Six out of eight N-CAM-negative myelomas were of the IgG lambda isotype, otherwise no clearcut association with basic clinical and laboratory parameters was noted. We conclude that N-CAM expression is a common finding in multiple myeloma. Whether its expression and the observed antigenic heterogeneity is just a manifestation of malignancy or N-CAM may play a role in the biology of multiple myeloma regarding tumour cell spread, remains to be explained.     

骨桥蛋白在食管鳞癌中的表达及其临床意义

目的观察骨桥蛋白（OPN）在食管鳞癌组织、癌旁组织和转移淋巴结的表达状况,探讨OPN在食管鳞癌中表达的临床意义。方法运用免疫组化S-P法检测44例食管鳞癌组织、癌旁组织和20例转移淋巴结免疫组化染色。结果OPN在食管癌组织、癌旁组织及转移淋巴结中的表达率分别为86．3％、0％和100％。癌组织中OPN主要表达于肿瘤细胞的细胞浆中。OPN的表达与肿瘤TNM分期、淋巴结转移状态有关,而与肿瘤位置、肿瘤直径、浸润深度及病理学分级无关。OPN在癌组织、癌旁组织及转移淋巴结表达强度亦存在显著性差异。结论食管鳞癌组织中OPN主要由肿瘤细胞产生。OPN与肿瘤的浸润、转移有关,反映了肿瘤的生物学特性。     

Expression of cell adhesion molecules alpha-2, alpha-5 and alpha-6 integrin,E-cadherin,N-CAM and CD-44 in renal cell carcinomas

Renal cell carcinoma is known to metastasize early independent of tumour grade. Invasion of the renal vein plays an important role in the prognosis. Cell adhesion molecules have been investigated, including the expression of alpha-2, alpha-5, and alpha-6 integrin, E-cadherin, neural-cell adhesion molecule and CD-44 in 34 renal cell carcinomas, using the alkaline phosphataseantialkaline phosphatase technique. Our results indicate a differential expression of these cell adhesion molecules (alpha-2, alpha-5 and E-cadherin) depending on histological type and tumour grade.     

Expression of apoptotic and cell proliferation regulatory proteins in keratoacanthomas and squamous cell carcinomas of the skin

The aim of this study was to investigate the expression of p53, caspase-3, bcl-2, MIB-1, and PCNA to validate more objective methods to differentiate squamous cell carcinoma and keratoacanthoma, as well as to understand their pathogenesis with accuracy. A total of 52 cases of histopathologically diagnosed keratoacanthoma in the proliferative stage and 56 cases of well-differentiated squamous cell carcinoma were selected in this study. The expression was evaluated by means of immunohistochemistry. Bcl-2 immunoreactivity was weak or absent in the majority of cases, either in squamous cell carcinoma or in keratoacanthoma. PCNA-positive cells did not show differences between two lesions evaluated. On the other hand, MIB-1 was statistically significant (p<0.05) between squamous cell carcinomas and keratoacanthomas. Moreover, p53 and caspase-3 were overexpressed in squamous cell carcinomas. Together, these results suggest that the biological behavior of the well-differentiated squamous cell carcinomas of the skin may be associated with cellular proliferation and/or deregulation of cell death, indicated by increased expression of the MIB-1 and apoptotic proteins p53 and caspase-3, respectively.     

高龄早期非小细胞肺癌单纯放射治疗临床观察

目的观察分析70岁以上早期（Ⅰ、Ⅱ期）非小细胞肺癌患者接受单纯放射治疗后的临床疗效及死亡原因。方法选择1992年至2003年48例70岁以上早期非小细胞肺癌患者，均为男性，年龄70～94岁，平均年龄78岁。Ⅰ期患者38例，Ⅱ期10例。既往有肺部阻塞性疾病26例，心血管疾病18例．糖尿病18例，脑血管疾病2例．共有15例（31．3％）患者同时合并两种或以上疾病。全部患者接受放射治疗，照射剂量60～66Gv（平均64Gy）／30～33次，常规分割照射2Gy／次。结果治疗后总有效率为58．3％。存活时间3～90个月（平均30．5个月），1、2、3年存活率分别为56．3％、45．8％、39．6％，平均疾病专项生存时间34．0个月，1、2、3年疾病专项生存率分别为62．6％、53．0％、45．8％。6例（12．5％）老年患者在放射治疗结束后3～6个月出现局灶性放射性肺炎，27例（56．3％）患者出现2级以下自限性的放射性食管反应。死亡的30例患者中，16例（53．3％）死于肺部感染、呼吸衰竭；9例（30．0％）死于肿瘤局部复发或广泛转移；3例（10．0％）死于心脑血管疾病；1例（3．3％）死于消化道大出血：1例（3．3％）死于甲状腺未分化癌。结论单纯放射治疗高龄早期非小细胞肺癌的临床疗效可靠且毒副反应较小．并且不会降低患者生活质量。对于高龄患者尤其是对于不能耐受或拒绝手术的高龄患者是很好的选择。从死亡原因分析．肺部感染仍是致死的重要因素。     

Expression of fibroblast growth factor receptor 1, fibroblast growth factor 2, phosphatidyl inositol 3 phosphate kinase and their clinical and prognostic significance in early and advanced stage of squamous cell carcinoma of the lung

Aim: Non-small cell lung carcinoma is the leading cause of cancer related to death in the world. Squamous cell lung carcinoma (SqCLC) is the second most frequent histological subtype of lung carcinomas. Recently, growth factors, growth factor receptors, and signal transduction system-related gene amplifications and mutations are extensively under investigation to estimate the prognosis and to develop individualized therapies in SqCLC. In this study, besides the signal transduction molecule phosphatidyl inositol-3-phosphate kinase (IP3K) p110α, we explored the expressions of fibroblast growth factor 2 (FGF2) and receptor-1 (FGFR1) in tumor tissue and also their clinical and prognostic significance in patients with early/advanced SqCLC. Materials and methods: From 2005 to 2013, 129 patients (23 early, 106 advanced disease) with a histopathological SqCLC diagnosis were selected from the hospital files of Cukurova University Medical Faculty for this study. Two independent pathologists evaluated FGFR1, FGF2, and PI3K (p110α) expressions in both tumor and stromal tissues from 99 of the patients with sufficient tissue samples, using immunohistochemistry. Considering survival analysis separately for patients with both early and advanced stage diseases, the relationship between the clinical features of the patients and expressions were evaluated by univariate and multivariate analyses. Results: FGFR1 expression was found to be low in 59 (60%) patients and high in 40 (40%) patients. For FGF2; 12 (12%) patients had high, 87 (88%) patients had low expression and for IP3K; 31 (32%) patients had high and 66 (68%) patients had low expressions. In univariate analysis, overall survival (OS) was significantly associated with stage of the disease and the performance status of the patient (P<0.0001 and P<0.001). There was no significant difference in OS of the patients with either low or high expressions of FGFR1, FGF2, and IP3K. When the patients with early or advanced stage disease were separately taken into consideration, the relationship did not differ, either. Any of FGFR1, FGF2 or IP3K expressions was not found predictive for the treatment of early or advanced staged patients. On the other hand, the expressions of both FGFR1 and FGF2 were significantly different with respect to smoking, scar of tuberculosis and scar of radiotherapy (P=0.002; P=0.06 and P=0.05, respectively). Discussion: There has not been identified an effective individualized treatment for SqCLC yet. Therefore, in order to be able to develop such a treatment in the future, it is essential to identify the genetic abnormalities that are responsible for the biological behaviors and carcinogenesis of SqCLC. Although we could not show the prognostic and predictive significance of FGFR1, FGF2 and IP3K expressions in SqCLC, we determined the expression rates of FGFR1, FGF2 and IP3K as a reference for Turkish patients. In conclusion, we want to put some emphasis on the fact that, pulmonary fibrosis which is a late complication of radiotherapy at stage III disease, and the scar of tuberculosis could be associated with FGFR1 and FGF2 expressions.     

Expression of cell adhesion molecules, CD44s and E-cadherin, and microvessel density in invasive micropapillary carcinoma of the breast

AIMS: Invasive micropapillary carcinoma (IMPC) is a rare variant of ductal carcinoma of the breast and is characterized by high metastatic potential and an aggressive clinical course. This tumour is hence ideal for studying the mechanism underlying tumour biological behaviour, especially metastasis. Cell adhesion molecules, such as CD44 and E-cadherin (Ecad), and angiogenesis are considered important in the invasion and metastasis of tumours. METHODS AND RESULTS: We immunohistochemically analysed 23 IMPCs for expression of a standard form of CD44 (CD44s), Ecad, and CD34 to measure microvessel density (MVD). Results are compared with the changes observed in 23 tubular carcinomas (TCs), another variant of ductal carcinoma that rarely metastasizes. Evaluation of haematoxylin and eosin (H&E) sections showed a higher prevalence of lymph-vascular invasion (19/23, 83%) and regional lymph node involvement (12/15, 80%) in IMPCs; whereas no lymph-vascular invasion or lymph node metastasis was identified in TCs. Loss or reduction of CD44s immunoreactivity was significantly frequent in IMPC (39%) compared with TC (4%) (P = 0.0098), and was associated with positive axillary lymph nodes and lymph-vascular invasion. All cases of IMPC and TC strongly expressed Ecad. MVD (in five 200x fields) was significantly higher in IMPC (88 +/- 37) than in TC (57 +/- 16) (P = 0.001). In the IMPC group, MDV was higher in cases with positive lymph node(s) (P = 0.048), and cases with loss or reduction of CD44s expression (P = 0.011). The same trend was also demonstrated in cases with lymph-vascular invasion (P = 0.077). Moreover, the vessels in IMPC had much smaller calibres with thinner walls than those in TC. CONCLUSIONS: Loss of the CD44 adhesion molecule and high MVD may play a significant role in the high incidence of lymph-vascular permeation and metastasis in IMPC.     

Expression of claudins 1, 4, 5, and 7 and occludin, and relationship with prognosis in squamous cell carcinoma of the tongue

Claudins and occludin are tight junctional proteins known to play a role in normal tissues and epithelial tumors. We analyzed the distribution patterns of claudins 1, 4, 5, and 7 and occludin in the superficial and invasive front of squamous cell carcinoma of the tongue of 97 patients and their relationship to cause-specific (squamous cell carcinoma of the tongue) patient survival (median follow-up period of 33.5 months; range, 1-234 months). Claudins 1 and 7 were strongly expressed, claudin 4 had moderate expression, whereas claudin 5 was least expressed. Occludin staining was mostly negative or very weak. Western blot analysis of tongue carcinoma (HSC-3) cells showed that all these proteins are also expressed in vitro. In cause-specific survival analysis, strong and low immunoreactivity of claudin 7 tended to be associated with decreased survival compared with medium immunoreactivity. We suggest that analyzing the immunohistologic staining levels of claudin 7 could be used for the prognostic purposes in patients with tongue squamous cell carcinoma.     

Expression of cell cycle and apoptosis regulatory proteins in keratoacanthoma and squamous cell carcinoma

Some authors view keratoacanthoma (KA) as a variant of squamous cell carcinoma (SCC), while others consider it a separate entity that must be distinguished from SCC. Involution displayed by KA is an important difference between these two entities. It has been suggested that apoptosis plays a role in the involution process of KA, although the exact trigger for it remains unclear. A hundred and fifty specimens were included in this study, 30 cases for each of the following groups: normal skin (NS), proliferative keratoacanthoma (pKA), regressing keratoacanthoma (rKA), well-differentiated squamous cell carcinoma (wdSCC), and poorly differentiated squamous cell carcinoma (pdSCC). They were immunohistochemically examined for the expression of p53, Ki-67, bak, and bcl-2. Significantly higher p53 and Ki-67 expressions were observed in all tumor lesions examined as compared with NS. There was higher bak expression in KAs compared to NS and a significant reduction of bak expression in pdSCC together with a significant reduction of bak expression in SCCs compared to pKA. Bcl-2 expression was similar in NS and SCCs, but was lower in rKA. We found a significant positive correlation between p53 and Ki-67, p53 and Bak in NS and examined skin tumors. Lower bcl-2 expression in conjunction with higher bak expression in rKA suggests a possible role of these apoptosis-regulating proteins in tumor regression. In contrast to this finding, a steady level of bcl-2 expression in pdSCC combined with lower bak expression levels and a high proliferation rate could contribute to progression and aggressiveness in these tumors. Bak and p53 expression is a sun-related and age-dependent process in NS and skin tumors.