Beta 1 integrin expression on human small cell lung cancer cells.

The integrins are a supergene family of cell surface glycoproteins that promote cellular adhesion. Each member of the family is an alpha/beta heterodimer composed of a distinct alpha subunit noncovalently linked to one of at least six common beta subunits. These include the six beta 1 integrins (alpha 1-6/beta 1) which represent receptors for extracellular matrix proteins and the three beta 2 integrins (alpha L, alpha M, alpha X/beta 2) that are expressed by leukocytes and which bind to C3bi and/or endothelial ligands. Recently, it was reported that certain human tumor cells express the beta 1 integrins and that small cell lung cancer (SCLC) cell lines express the beta 2 integrin Mo1 (alpha M/beta 2). To extend these initial observations, we examined SCLC cell lines for integrin expression at the glycoprotein and mRNA levels and assessed the potential function of these integrins in promoting SCLC adhesion. An indirect immunofluorescence analysis of five SCLC cell lines (NCI-H187, H345, H146, H209, and N417) using alpha and beta subunit-specific monoclonal antibodies demonstrated the uniform expression of beta 1 (beta 1 much greater than beta 2 greater than or equal to beta 3 congruent to beta 4). Among the beta 1-associated alpha subunits, alpha 3 was uniformly expressed at high surface density by all five cell lines (as confirmed in H345 cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of anti-beta 1 and anti-alpha 3 immunoprecipitates), while alpha 5 was not detected. The leukocyte (beta 2-associated) alpha M and alpha L subunits were also variably expressed by the five lines. Consistent with the surface expression of beta 1 integrin gene products, beta 1 (but not beta 2) mRNA was detected in SCLC cells by Northern blot analysis. That beta 1 integrin expression was involved in SCLC adhesion was suggested by the adherence of H345 cells to laminin, a known ligand for the alpha 3 beta 1 integrin. Moreover, an antibody specific for the beta 1 subunit inhibited this adhesion, indicating that the beta 1 subunit promotes adhesion to laminin. We conclude that beta 1 integrin molecules are expressed by human SCLC cells (with uniform expression of alpha 3/beta 1) and promote their adhesion to laminin.     

Transcriptional gene expression profiling of small cell lung cancer cells

A global gene expression analysis using oligonucleotide microarrays was performed on many human small cell lung cancer (SCLC) cell lines in cell culture and/or as xenografts. The expression was compared with the expression profiles of 18 normal tissues. In a hierarchical cluster analysis the cell lines clustered distinctly from normal tissues and grouped into four clusters. One cluster consisted of two related cell lines and was markedly different from the other SCLC cell lines, whereas the rest of the clusters grouped together. Two subclusters contained the classical SCLC types and one subcluster the variant SCLC type, thus identifying many genes with differential expression between the two variants of SCLC. All of the xenografts clustered closest to the cell lines from which they originated and had the same expression levels as the cells grown in culture for the majority of genes. The analysis confirmed the high expression of many genes identified previously as highly expressed in SCLC cells including neuroendocrine markers, oncogenes, and genes involved in cell proliferation and division. The analysis furthermore identified a number of molecules not identified previously as expressed in SCLC. Several of these are expressed in low or undetectable amounts in the majority of normal tissues and, therefore, are potential targets for new therapeutic approaches. By including the published array profiles of six ressected SCLC tumors from Bhattacharjee et al. (A. Bhattacharjee et al., Proc. Natl. Acad. Sci. USA, 98: 13790-13795, 2001.), the analysis revealed that most of the novel potential targets expressed by SCLC cell lines and xenografts were also expressed in the tumors. This analysis demonstrates the value of using cell lines and xenografts for expression profiling, when a limited quantity of tumor material is available.     

Increased expression of Id family proteins in small cell lung cancer and its prognostic significance.

PURPOSE: To study the molecular pathology of human small cell lung cancer (SCLC), molecular biology approaches were used to identify genes involved in malignant progression of the cancer cells. EXPERIMENTAL DESIGN: Microquantity differential display was used initially to identify genes expressed differentially between normal and malignant cell lines. The differences were verified by Western blot. Immunohistochemical analysis was done on paired normal and malignant lung tissues and on tissues taken by biopsy to assess the expression status of candidate genes and their prognostic significance. RESULTS: Inhibitor of DNA/differentiation (Id)1 gene was up-regulated in SCLC cells. Levels of Id1 in 8 of 10 cell lines were increased by 1.7- to 21.4-fold when compared with the benign cells. A similar increase was also found in levels of Id2 and Id3. On 26 pairs of lung tissues, all four Id proteins were significantly (Wilcoxon Signed Rank Test, P < 0.001-0.005) overexpressed in cytoplasm of the malignant cells. In nuclei of SCLC cells, Id1 expression was significantly reduced, whereas the levels of Id2, Id3, and Id4 were significantly (Wilcoxon Signed Rank Test, P < 0.001) increased. Immunohistochemical staining on biopsy specimens showed that the increased expression of Id2 in cytoplasm of cancer cells, not the other three proteins, was significantly associated with the increased survival of SCLC patients. CONCLUSION: Changed expression profiles of Id proteins may play important roles in malignant progression of SCLC, and the increased Id2 in cytoplasm is a novel prognostic factor to predict the patient outcomes.     

CEA、VEGF标志物与小细胞肺癌预后相关性

目的:探讨不同评价标志物与小细胞肺癌预后相关性。方法:64例小细胞肺癌患者以及同期36例行肺叶切除术的正常患者,分为观察组与对照组。免疫组化SP法测定LRP,酶联免疫吸附法(ELISA)对血液中血清癌胚抗原（CEA）、血清血管内皮生长因子(VEGF)进行检测,同时测定观察组在化疗前后LRP阳性率、以及CEA与VEGF的水平变化情况。结果:小细胞肺癌患者与肺部良性病变的VEGF检测值分别为:(290.7±48.52)pg/ml、(123.5±31.48)pg/ml,CEA检测值分别为:(9.78±2.58)ng/L、(4.06±0.84)ng/L,LRP阳性率分别为:53.1%、5.6%,两组比较有显著性差异（P<0.05)。观察组化疗前后的VEGF检测值分别为:(286.7±47.52)pg/ml、(167.5±34.48)pg/ml,CEA检测值分别为:(9.68±2.48)ng/L、(5.16±1.25)ng/L,LRP阳性率分别为:51.6%、15.6%,两组比较有显著性差异（P<0.05)。结论:LRP、VEGF以及CEA等标记物在肺癌组织的表达水平显著高于正常组织,可用于对肺癌疾病的诊断以及对小细胞肺癌预后预测。     

PDGFR-beta expression in small cell lung cancer patients

BACKGROUND: Platelet derived growth factor (PDGF) and PDGFR-beta are expressed and have been found to have prognostic value in several human cancers. Data in non-small-cell cancer cell lines have suggested that PDGFR is a therapeutic target for drug development. In the current study PDGFR-beta expression and prognostic value in small cell lung cancer (SCLC) was investigated. METHODS AND MATERIALS: Paraffin-embedded tissue blocks from 53 patients with limited and extensive stage SCLC were obtained for immunohistochemical staining. Tumors from each patient were sampled 3 times and stained with PDGFR-beta specific antibody. Patients were divided into low and high staining groups based on intensity. RESULTS: There was high intensity PDGFR-beta staining in 20 patients with SCLC. Another 29 expressed low intensity PDGFR-beta staining, with only 4 patients showing no PDGFR-beta staining. There was no statistically significant difference in 5 year overall survival between patients with low levels of PDGFR-beta staining vs. those with high level staining SCLC tumors (p = 0.538). CONCLUSIONS: The present study found that the majority of SCLC patients express, at least, a low level of PDGF-beta. However, the level of PDGFR-beta expression was not a statistically significant predictor of 5 year overall survival in SCLC.     

神经内分泌分化与p53对非小细胞肺癌预后的影响

目的探讨神经内分泌(neuroendocrine,NE)分化标志物嗜铬蛋白A(CgA)、突触素(Syn)、神经内分泌颗粒和p53对非小细胞肺癌(non-small cell lung carcinoma,NSCLC)预后的影响。方法62例NSCLC进行含铂类方案新辅助化疗1~2个周期,然后手术治疗,以免疫组化法检测术后标本中NE分化标志物CgA、Syn及p53,以电镜观察标本中神经内分泌颗粒,随访3年,结果采用SPSS11.0统计软件,进行生存率分析。结果p53及NE分化标志物联合p53对非小细胞肺癌患者的无病生存期在统计学上均有显著性差异(P<0.05),NE( )p53(-)患者生存率较高。说明p53和NE分化联合p53与非小细胞肺癌患者的预后有关。结论神经内分泌分化和p53两者联合,用于非小细胞肺癌患者无进展生存期评价具有一定的临床价值。     

The effect of adenovirus-mediated gene expression of FHIT in small cell lung cancer cells

The candidate tumor suppressor fragile histidine traid (FHIT) is frequently inactivated in small cell lung cancer (SCLC). Mutations in the p53 gene also occur in the majority of SCLC leading to the accumulation of the mutant protein. Here we evaluated the effect of FHIT gene therapy alone or in combination with the mutant p53-reactivating molecule, PRIMA-1(Met)/APR-246, in SCLC. Overexpression of FHIT by recombinant adenoviral vector (Ad-FHIT)-mediated gene transfer in SCLC cells inhibited their growth by inducing apoptosis and when combined with PRIMA-1(Met)/APR-246, a synergistic cell growth inhibition was achieved.     

小细胞肺癌神经内分泌起源标志物的研究进展

[摘要] 小细胞肺癌（small cell lung cancer,SCLC）具有生长分数高、倍增时间短、侵袭性极高的特点,往往在诊断该病的时候就已发生远处转移,若不对其病情进行及时有效的治疗,患者将在2~3 个月内死亡。如果SCLC能够在局限期得到诊断,那么将有20%～25%的患者在接受联合化疗与放疗后,可以获得一个较长生存期。因此,寻找特异性和敏感性高的血液肿瘤标志物,为SCLC早期诊断提供可靠、简便、安全、耐受性好等依据对治疗SCLC极其重要。本文就近年来SCLC神经内分泌起源标志物嗜铬粒蛋白A、神经特异性烯醇化酶、促胃泌素释放肽、脑型肌酸激酶同工酶BB、神经细胞黏附分子、突触素等的研究进展进行综述。     

Neuronal differentiation by indomethacin and IBMX inhibits proliferation of small cell lung cancer cells in vitro

Background

Small cell lung cancer (SCLC) is one of the most aggressive malignancies implying a very poor prognosis for patients even under therapy. Since it is known that SCLC cells exhibit neurone-like characteristics, we investigated whether a neuronal induction medium (NID) consisting of indomethacin (200 μM), 3-isobutyl-1-methylxanthine (IBMX, 500 μM) and insulin (5 μg/ml) induces neuronal differentiation and by this reduces malignancy of SCLC in vitro.

Methods

Anti-proliferative effects were tested by incubating five SCLC cell lines (OH1, OH3, SW2, H69 and H82) with NID for 72 h (XTT-assay). Afterwards, anti-proliferative as well as cytotoxic effects (lactate dehydrogenase [LDH] assay, electron microscopy) of a range of drug concentrations (indomethacin 6.25-800 μM, IBMX 15.625-2000 μM and combinations of both) regarding H82 and SW2 were analysed. We further investigated the presence of cyclooxygenase- (COX-) 1 and 2 (IHC, Western blot) as well as levels of COX-2 before and after treatment. Neuronal differentiation was evaluated by morphological analyses (electron microscopy), detection of CD 56 and CD 171 (FACS) and recording Na⁺ and K⁺ currents (patch clamp).

Results

Proliferation of all cell lines was inhibited significantly in a dose dependent manner (linear regression), whereas SW2 and H82 were most sensitive. Treatment with insulin alone had no effect at all. Cytotoxic effects were only observed after incubation with high concentrations of indomethacin (H82) and combined treatment (SW2). COX-1 and 2 were detectable in H82 and SW2, whereas the level of COX-2 remained unaffected under treatment. By electron microscopy, we could not observe distinct neurone-like morphological changes after 72 h of treatment. However, the majority of H82 and SW2 cells expressed both CD 56 (NCAM) and CD 171 (L1), showing an increase of NCAM and L1 intensity at the cell surface after 7 and 14 days of treatment. We further demonstrated an up-regulation of neurone-specific Na⁺ currents as well as a significant down-regulation of herg K⁺ currents after NID treatment.

Conclusion

Our findings demonstrate significant anti-proliferative, non-toxic effects of indomethacin and IBMX on SCLC cells in vitro. Treated SCLC cells further possess increased neuronal characteristics in vitro, possibly leading to a reduced malignant potential.     

Cross-linked envelope-related markers for squamous differentiation in human lung cancer cell lines

Lung carcinoma cell lines were analyzed in culture and in nude mouse xenograft for both morphological appearance and expression of specific proteins that participate in cross-linked envelope formation during normal squamous cell terminal differentiation. Cross-linked envelope formation, induced by artificial influx of millimolar Ca2+ into the cultured cells, was an exclusive trait of squamous, adenosquamous, and mucoepidermoid carcinomas. Small cell lung carcinoma and non-squamous non-small cell lung carcinoma lines, such as adenocarcinoma and large cell carcinoma, were uniformly negative for cross-linked envelope formation. Involucrin, which is incorporated into the cross-linked envelope by the enzyme transglutaminase, was expressed at highest levels in squamous tumors, but several of the non-squamous non-small cell lung carcinoma lines also expressed comparable amounts. On the other hand, transglutaminase activity was consistently higher in squamous as opposed to non-squamous lines, so that in cell culture, a clear contrast between the groups could be observed. A Mr 195,000 protein that is incorporated into cultured human epidermal cell cross-linked envelopes was also observed in some but not all of the squamous lines. Two forms of transglutaminase are expressed in cultured keratinocytes. One of them, tissue transglutaminase, was expressed in the majority of squamous cell lines even though it is not a normal product of squamous differentiation in vivo. Keratinocyte transglutaminase, which is distinct from the tissue form and is normally expressed during terminal differentiation in squamous epithelia. was measurably present in only one of the six squamous cell lines tested. In nude mouse xenografts, keratinocyte transglutaminase, localized immunohistochemically with a biotinylated mouse monoclonal antibody, was again present only in a minority of the squamous lines whereas involucrin was expressed in all. In contrast to involucrin, keratinocyte transglutaminase is not an obligatory component of squamous differentiation in the pulmonary carcinoma cell lines tested. Its expression may be of value in further refining their classification.     

小细胞肺癌干细胞信号通路的研究进展

小细胞肺癌是具有高度侵袭性的肺肿瘤,其主要临床特征是化疗有效率高但易在短时间内复发转移,这一特点可能与肿瘤干细胞的存在有关。肿瘤干细胞被认为是恶性肿瘤发生发展、耐药、复发及转移的根源。目前多认为肿瘤干细胞与正常干细胞有着相同的信号通路,如Hedgehog、Notch、Wnt等通路。本文就这几条信号通路在小细胞肺癌干细胞中所起的作用以及针对这几条信号通路治疗药物的研究进展和可能的信号通路交互作用等方面进行综述。     

Prognostic value of KIT expression in small cell lung cancer

BACKGROUND: Small cell lung cancer (SCLC) is a very aggressive disease, with poor survival rates despite standard treatment with combination chemotherapy with or without radiotherapy. Further insights into the molecular biology of this malignant tumour are needed to improve the therapeutic approaches and outcome. KIT protein is expressed in SCLC, and its kinase activity has been implicated in the pathophysiology of many tumours, including SCLC. The purpose of this study was to evaluate the prevalence of KIT expression in patients with SCLC and its prognostic value. METHODS: We performed an inmunohistochemical analysis of 204 SCLC samples to determine KIT protein expression. The relationship between KIT expression and clinicopathological parameters was evaluated. Univariate and multivariate analyses were performed to define its prognostic significance. RESULTS: KIT expression was observed in 149 of 204 tumour tissues (73%). KIT expression was associated with advanced disease and with decreased incidence of bone metastases. No significant differences were observed for time to disease progression (TTP) (9.1% versus 6.2% at 3 years, p=0.6) or overall survival (OS) (10.7% versus 6.9% at 3 years, p=0.37) among patients with KIT positive versus negative tumours, respectively. Multivariate analysis showed that sex, tumour stage, albumin levels and response to therapy were the only independent predictors for survival. CONCLUSION: KIT protein is expressed in a high percentage of SCLC tumours. In our study population, however, the expression of KIT had no significant impact on survival.     

Changes of protein expression during tumorosphere formation of small cell lung cancer circulating tumor cells

Small cell lung cancer (SCLC) is frequently disseminated and has a dismal prognosis with survival times ofapproximately two years. This cancer responds well to initial chemotherapy but recurs within a short time as aglobally chemoresistant tumor. Circulating tumor cells (CTCs) are held responsible for metastasis, the extremely highnumbers of these cells in advanced SCLC allowed us to establish several permanent CTC cell lines. These CTCs aredistinguished by the spontaneous formation of large spheroids, termed tumorospheres, in regular tissue culture. Thesecontain quiescent and hypoxic cells in their interior and are associated with high chemoresistance compared to singlecell cultures. Nine CTC lines were compared for their expression of 84 proteins associated with cancer either as singlecells or in the form of tumorospheres in Western blot arrays. With the exception of the UHGc5 line, all other CTClines express EpCAM and lack a complete EpCAM-negative, vimentin-positive epithelial-mesenchymal transition(EMT) phenotype. Upon formation of tumorospheres the expression of EpCAM, that mediates cell-cell adhesion ismarkedly upregulated. Proteins such as E-Cadherin, p27 KIP1, Progranulin, BXclx, Galectin-3, and Survivin showedvariable changes for the distinct CTC cell lines. In conclusion, EpCAM presents the most critical marker forindividual SCLC CTCs and the assembly of highly chemoresistant tumorospheres.