Expression and activity of matrix metalloproteases in human malignant mesothelioma cell lines

The extracellular matrix metalloproteases (MMPs) secreted by various human tumor cells play a crucial role in tumor cell invasion and metastasis, but their expression in malignant mesothelioma (MM) cells has not been examined. In this study, we have investigated the spectrum of MMPs and tissue inhibitors of metalloproteases (TIMPs) produced by 8 MM cell lines. Using RT-PCR, we found that all investigated MM cell lines expressed genes encoding mRNA for MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin-1), MMP-9 (gelatinase B) and TIMPs 1, 2 and 3. We also found that 6/8 MM cell lines expressed MMP-7 (matrilysin) and 3/8 MM cell lines expressed MMP-10 (stromelysin-2). MMP-11 (stromelysin-3) was not detected in any of the MM cell lines. Production of MMP-2 and MMP-9 was confirmed using gelatin zymography. In addition, all MM cell lines secreted a 66 kDa metalloprotease, while 3/8 MM cell lines secreted 46, 48, 51 and 63 kDa metalloproteases which specifically degraded the extracellular matrix components fibronectin, vitronectin and laminin. The 66 kDa protease was identified as MMP-3 by Western blot. Our results reveal a broad spectrum of MMPs and TIMPs produced by MM cells and indicate that different substrate specificities of MMPs may play a role in MM cell invasion.     

Retinoic acid inhibits fibronectin and laminin synthesis and cell migration of human pleural mesothelioma in vitro.

The effects of retinoic acid (RA) on cell replication, fibronectin and laminin synthesis, integrin expression and haptotactic migration of three mesothelioma cell cultures of different histotype, one epithelioid, one fibromatous and one biphasic, were evaluated. Cell growth was not affected by RA, while RA treatment decreased the synthesis of fibronectin and laminin and inhibited the migration of all three mesotheliomas on substrates of fibronectin and laminin; on the contrary, the expression of some integrins was not significantly modified by RA. These data indicate that RA may lead to a decrease of mesothelioma cell local invasion; this can correlate with a modification induced by RA on mesothelioma tumor progression in vivo.     

Calmodulin modulates Akt activity in human breast cancer cell lines

Growth factor-induced activation of Akt occurs in the majority of human breast cancer cell lines resulting in a variety of cellular outcomes, including suppression of apoptosis and enhanced survival. We demonstrate that epidermal growth factor (EGF)-initiated activation of Akt is mediated by the ubiquitous calcium sensing molecule, calmodulin, in the majority of human breast cancer cell lines. Specifically, in estrogen receptor (ER)-negative, but not ER-positive, breast cancer cells, Akt activation is abolished by treatment with the calmodulin antagonist, W-7. Suppression of calmodulin expression by siRNAs against all three calmodulin genes in c-Myc-overexpressing mouse mammary carcinoma cells results in significant inhibition of EGF-induced Akt activation. Additionally, transient expression of constitutively active Akt (Myr-Akt) can overcome W-7-mediated suppression of Akt activation. These results confirm the involvement of calmodulin in the Akt pathway. The calmodulin independence of EGF-initiated Akt signaling in some cells was not explained by calmodulin expression level. Additionally, it was not explained by ER status or activation, since removal of estrogen and ablation of the ER did not convert the ER-positive, W-7 insensitive, MCF-7 cell line to calmodulin dependent signaling. However, forced overexpression of either epidermal growth factor receptor (EGFR) or ErbB2 did partially restore calmodulin dependent EGF-stimulated Akt activation. This is consistent with observation that W-7 sensitive cells tend to be estrogen independent and express high levels of EGFR family members. In an attempt to address how calmodulin is regulating Akt activity, we looked at localization of fluorescently tagged Akt and calmodulin in MCF-7 and SK-BR-3 cells. We found that both Akt and calmodulin translocate to the membrane after EGF-stimulation, and this translocation to the same sub-cellular compartment is inhibited by the calmodulin inhibitor W-7. Thus, calmodulin may be regulating Akt activity by modulating its sub-cellular location and is a novel target in the poor prognosis, ER-negative subset of breast cancers.     

Mutational analysis of 9 different tumour-associated genes in human malignant mesothelioma cell lines

Seven tumour suppressor genes (Chk1, Chk2, Apaf1, Rb1, p53, p16(INK4a) and p14(ARF)) and two oncogenes (N-ras and BRAF) were screened in nine human malignant melanoma (HMM) cell lines for point mutations or small deletions/insertions by DGGE, TGGE and SCCP analysis. For the first time in human mesothelioma, Chk1 gene mutations were detected in two of the nine investigated HMM cell lines. P53 gene mutations were found in three cell lines and p16(INK4a) mutations in 5. Mutation of the Chk1 gene implies a novel disruption mechanism of the p53 pathway in HMM, without affecting p53 itself. According to our knowledge, this is the first mutation screening of Chk1, Chk2, Apaf1 and Rb1 in human malignant mesothelioma.     

Doxorubicin retention and chemoresistance in human mesothelioma cell lines

Eight cell lines were established from the pleural effusion of 4 patients with malignant mesothelioma. The most sensitive (FCCMES-4) and the most resistant (FCCMES-2) mesothelioma cell lines had IC₅₀ of 0.66 and 1.85 μM for doxorubicin in clonogenic assays, respectively. In comparison with murine leukemic P388 cells, mesothelioma cell lines were 7.5- to 21 -fold more resistant to doxorubicin. Co-incubation with verapamil significantly increased doxorubicin retention in one of the cell lines (FCCMES-2) expressing P-glycoprotein in 16.8% of the cells. These results indicate that doxorubicin resistance may be intrinsic in refractory mesothelioma patients and P-glycoprotein-mediated drug efflux may be involved in resistance of some of the mesotheliomas. © 1994 Wiley-Liss, Inc.     

纤维连接蛋白在不同类型肺癌细胞侵袭过程中的作用及机制

目的 观察和探讨纤维连接蛋白(FN)对不同类型肺癌细胞侵袭能力的影响及机制。方法 在FN包被的培养板和FN滤膜的Boyden浸润小室等体外侵袭模型中，比较小细胞肿癌细胞系054A和肺腺癌细胞系A549粘附及迁移能力的差别，并检测FN对肿瘤细胞增殖活性的影响。分别给予细胞抗α3整合素、抗α5整合素、抗β1整合素单抗预处理后，观察侵袭性的变化。结果 FN增强A549细胞粘附、迁移及增殖活性的作用明显大于054A细胞。这种作用在A549细胞能被抗α3、抗α5和抗β1单抗抑制，而在054A细胞能被抗α3和抗β1单抗抑制。结论 FN对不同类型肺癌细胞侵袭能力的影响不同，可能与细胞膜整合素受体的差异有关。     

p53 and Kirsten-ras mutations in human mesothelioma cell lines.

Twenty cell lines from 17 individuals with malignant mesothelioma have been examined for p53 alterations by direct sequencing of genomic DNA, by evaluation of mRNA expression levels, and by immunocytochemical analysis of p53 protein expression in comparison with normal human pleural mesothelial cells. The results of this study show p53 abnormalities in cell lines from 3 individuals. These include 2 point mutations and one null cell line. Interestingly, while both cell lines with point mutations exhibit high levels of p53 protein, normal mesothelial cells as well as 12 of the mesotheliomas evaluated express low but significant levels. In addition, sequencing of K-ras at codons 12, 13, and 61 reveals wild-type sequence in all 20 mesothelioma cell lines. The capacity to induce tumors in athymic nude mice did not correlate with the presence of a p53 mutation or elevated p53 protein levels. These data suggest that neither p53 alteration nor K-ras activation constitutes a critical step in the development of human mesothelioma.     

Malignant mesothelioma: the antiproliferative effect of cytokine combinations on three human mesothelioma cell lines

Tumour necrosis factor (TNF) combined with gamma-interferon (IFN gamma) is known to have antiproliferative effects on many tumour cells, both in vitro and in vivo. We investigated whether human mesothelioma cells would respond in a similar way. Mesothelioma cell lines established from primary tumours did not respond significantly in vitro to either TNF or IFN gamma alone but were inhibited by combinations of TNF and IFN gamma at concentrations as low as 5 ng/ml. In contrast, a mesothelioma cell line established from a metastatic tumour was sensitive to IFN gamma both alone and in combination with TNF but not to TNF alone. We also looked at the responses of one primary tumour cell line and the metastatic tumour cell line to alpha-interferon (IFN alpha) both alone and in combination with TNF. Both cell lines were sensitive to IFN alpha but were more sensitive to the THF/IFN alpha combination. We conclude that these low dose combinations of cytokines are worth further investigation in the development of mesothelioma therapy.     

A novel folate transport activity in human mesothelioma cell lines with high affinity and specificity for the new-generation antifolate,pemetrexed

Pemetrexed is a novel antifolate effective in the treatment of mesothelioma. Studies were undertaken to characterize the transport of this antifolate in this tumor. We report the presence of a novel, concentrative high-affinity transport activity in three human mesothelioma cell lines, characterized in detail in the NCI-H28 line, with a pemetrexed influx K(t) of 30 nM and V(max) of 10 nmol/g protein/min. This route is highly specific for pemetrexed, with a substrate specificity pattern quite different from that of the reduced folate carrier and folate receptors. In particular, there is an apparent relatively low affinity for other antifolate inhibitors of dihydrofolate-reductase (MTX, aminopterin, PT523) and thymidylate synthase (ZD1694, ZD9331). Besides its impact on the transport of pemetrexed, this high-affinity route may represent another pathway by which physiological folates are transported into human cells.     

FAK在不同迁移特性骨肉瘤亚克隆细胞系中的表达及其意义

目的 探讨不同迁移特性骨肉瘤亚克隆细胞系粘着斑激酶（FAK）的表达及其对骨肉瘤细胞增殖和迁移的影响。方法 通过有限稀释法获得不同迁移能力的骨肉瘤细胞143B亚克隆细胞系A1、A2、A3、A4、A5,运用Western blottting技术检测不同亚克隆细胞系FAK的表达。小干扰RNA（siRNA）序列研究FAK对骨肉瘤细胞增殖及迁移能力的作用,免疫荧光染色法比较形成板状伪足细胞数的比例。结果 骨肉瘤细胞143B亚克隆细胞系A2、A3的迁移能力较A1、A4和A5强（P＜0.05）,其细胞中FAK表达也明显增高。外源转入FAK siRNA后,骨肉瘤亚克隆细胞系A3的迁移能力明显下降（P＜0.05）,形成板状伪足细胞数的比例由33.3％降至12.8％（P＜0.05）,但增殖能力无明显变化。结论 迁移能力较强的骨肉瘤细胞143B亚克隆细胞系中FAK呈高表达,FAK可能通过影响板状伪足的形成从而增强骨肉瘤细胞的迁移能力,但是对骨肉瘤细胞的增殖无明显作用。     

in vitro effects of recombinant human interferon gamma on human mesothelioma cell lines

Malignant mesothelioma is a tumor arising from serous surfaces and often related to asbestos exposure. Malignant mesothelioma is resistant to various forms of therapy. Radiotherapy, surgery or chemotherapy only slightly improve prognosis. IFN-γ produces complete or partial responses in stage-l patients with malignant mesothelioma. The in vitro biological effect of IFN-γ on malignant mesothelioma cells remains poorly elucidated. In the present study, 32 well-characterized human mesothelioma cell lines (HMCL) were treated with r-hu IFN--γ at 4 doses and cell growth was determined by a colorimetric method (MTT assay). Among the 32 HMCLs tested, 11 exhibited significant cell-growth inhibition; 16 were insensitive to r-hu IFN-γ, and 5 were slightly inhibited. The sensitive cell lines were strongly inhibited by r-hu IFN-γ. Our results show that HMCL exhibit a large range of responses to r-hu IFN-γ, some of which can be compared with those obtained in vivo in humans.     

Telomerase activity of cultured human pancreatic carcinoma cell lines correlates with their potential for migration and invasion

BACKGROUND: Despite the recent clinical finding that high telomerase activity is an unfavorable prognostic marker for various human malignant tumors, there has been no experimental evidence supporting the link between telomerase and tumor aggressiveness. In the current investigation, the authors examined the relation between telomerase activity and potential for biologic aggressiveness in human pancreatic carcinoma cells. METHODS: Telomerase activity was measured in a poorly metastatic cell line HPC-3 and its highly metastatic variant HPC-3H4, as well as in many pancreatic carcinoma cell lines. Aggressive behavior of cancer cells was assessed by in vitro migration and invasion assay. RESULTS: Compared with parental HPC-3, HPC-3H4 displayed higher telomerase activity, which was associated with a scattered phenotype and enhanced migration activity. Furthermore, the authors found that relative telomerase levels correlated well with both motility (P = 0.0041) and invasion (P = 0.0114) in 13 pancreatic carcinoma cell lines. There was, however, no significant association between telomerase activity and cell proliferation. When telomerase activity of KP-1N cells was inhibited by transfection with antisense oligonucleotides, their motility and invasion rates were significantly decreased. CONCLUSIONS: The authors concluded that the magnitude of telomerase activation may reflect the potential for aggressive behavior within cancer cells. These findings support the clinical utility of telomerase activity as a prognostic indicator. Their results also suggest a therapeutic potential for telomerase inhibitors to prevent tumor invasion and possibly metastasis.     

Over-expression of 14-3-3sigma in budding colorectal cancer cells modulates cell migration in the presence of tenascin-C

Epigenetic silencing of the 14-3-3sigma gene by CpG hypermethylation has been reported in many kinds of cancers, but has been considered inapplicable in the colorectal variety. The expression of 14-3-3sigma in colorectal cancer is located primarily in the invasive area. The interaction between tumor cells and the extracellular matrix (ECM) is involved in tumor invasion. In the current study, we investigated the correlation between 14-3-3sigma expression and the ECM, focusing especially on the presence of tenascin-C (TNC) at the invasive area of colorectal cancers. Correlations between the immunohistochemical expression of 14-3-3sigma and TNC, as well as other clinicopathological factors, were evaluated in 123 colorectal carcinoma tissues. 14-3-3sigma expression was frequently observed in budding tumor cells in the invasive area and expression was significantly correlated with budding formation (p=0.001), pTNM classification (p=0.001) and stromal TNC expression (p=0.004). Using colorectal cancer cell lines and ECMs, the up-regulation of 14-3-3sigma mRNA levels was investigated by semi-quantitative RT-PCR. TNC surrounding the tumor cells increased 14-3-3sigma mRNA expression 1.8- to 2.2-fold in HCT116 cells. The effect of 14-3-3sigma over-expression on tumor cell migration was investigated using an agarose-cell droplet migration assay. Over-expression of 14-3-3sigma up-regulated HCT116 cell migration on TNC (p<0.001). We concluded that the expression of 14-3-3sigma in the invasive area modulates tumor cell migration in certain types of colorectal cancer and thus facilitates tumor progression.     

Signal transduction in adherent and non-adherent human cell lines after fibronectin stimulation

It is shown that adherent and non-adherent human ovarian carcinoma cells (OVP 10) secrete MMPs and their production was stimulated by fibronectin as documented by gelatinise zymography. These cells also presented an increase of ERK phosphorylating activity following fibronectin stimulation, regardless of their adhesion. Contrary to OVP 10 cells, the human urothelial cells (HCV-29) are more anchorage-dependent. They only secreted the MMPs under adherent conditions and they revealed a lower level of basal and fibronectin stimulated ERK phosphorylation activity. In addition, non-adhering HCV-29 cells showed post translational down-regulation of focal adhesion kinase.     

Diamine oxidase activity in human melanoma cell lines with different tumorigenicity in nude mice.

The activity of diamine oxidase (DO, EC 1.4.3.6.) which converts putrescine into gamma-aminobutyraldehyde in the degradative pathway of polyamine, was studied in 4 human melanoma cell lines, 2 of which produce tumours in greater than 80% of nude mice (M3Dau, M4Beu), whereas the other 2 induce tumours in less than 25% (M1Dor, M2GeB). The activity of DO in these cells varies with the growth rate: 24 h after seeding there is an initial increase in DO activity, followed by a steep decline during exponential growth. At 96 h, when cells reach saturation density, the activity of DO is significantly greater in the highly tumorigenic cell lines than in the poorly tumorigenic cell lines. Kinetic studies show that for the highly tumorigenic lines apparent Km values are 10.6 X 10(-6)M +/- 0.2 (M3Dau) and 14.2 X 10(-6) M +/- 0.6 (M4Beu), whereas for the poorly tumorigenic lines the values are 4.5 X 10(-6) M +/- 0.3. After transplantation into nude mice, the M1Dor cell line, which exhibits a low Km (app.) for DO of which had high Km (app.) value. Km (app.) determination of DO could be an approach for characterizing human melanoma cells differing in their tumorigenic potential in nude mice.     

Production of fibronectin by human tumor cells and interaction with exogenous fibronectin: comparison of cell lines obtained from colon adenocarcinomas and squamous carcinomas of the upper aerodigestive tract

Cell lines derived from 13 different human colon adenocarcinomas were examined for production of fibronectin by ELISA and for cell-surface expression of fibronectin by indirect immunofluorescence. Two squamous epithelial cell lines obtained from tumors of the upper aerodigestive tract were used as controls. None of the 13 colon carcinoma lines produced detectable amounts of fibronectin or showed detectable cell-surface staining with anti-fibronectin. The 2 squamous epithelial cell lines, in contrast, produced large amounts of fibronectin which could be detected in the culture medium and bound to the substratum. The squamous carcinoma cells also stained brightly when examined in the viable state by immunofluorescence with anti-fibronectin. In addition to being studied for fibronectin production, each cell line was also examined for the ability to interact with exogenous fibronectin in an adhesion assay. None of the colon carcinoma cells were adherent to fibronectin-coated culture dishes while the 2 squamous carcinoma cells rapidly attached and spread on this substratum. These data suggest that cell lines derived from adenocarcinomas of the colon are deficient in production of fibronectin and in their ability to interact with exogenous fibronectin. In their degree of deficiency, the colon carcinoma cells are significantly different from several different types of human tumor cell. The failure of the colon carcinoma cells to synthesize detectable amounts of fibronectin endogenously or to interact with exogenous fibronectin may explain, in part, the low degree of adhesive interaction which these cells have for their substratum. This, in turn, may influence the in vitro and in vivo properties of colon carcinoma cells.     

Unique expression of integrin fibronectin receptors in human neuroblastoma cell lines.

Cultured human neuroblastoma cells can be classified morphologically into 3 types: neuroblastic (N), intermediate (I) and substrate adherent (S). Neuroblastoma cells of all types were found to attach and display distinct morphological characteristics on fibronectin, with S-type cells attaching better than N-type cells. Studies of the expression of integrin fibronectin receptors (alpha 3 beta 1, alpha 4 beta 1, alpha 5 beta 1 and alpha V beta 1) were carried out using a total of 26 morphologically distinct cell lines and their subpopulations. Fluorescence-activated cell sorting (FACS) analysis and immunoprecipitation revealed that all S-type cells expressed abundant alpha 5 beta 1, while N-type cells barely expressed this molecule. Although alpha 3 beta 1 expression of S-type cells was also higher than that of N-type cells, some N-type cells had significantly increased levels of this molecule. alpha 4 beta 1 was found to be randomly expressed. All cell lines tested expressed alpha V beta 1. Human neuroblastoma cells, the majority of which are N-type cells with very low alpha 5 beta 1 expression, are also contrasted with other childhood cancer cells (rhabdomyosarcoma, Ewing's sarcoma, and glioma), all of which expressed high levels of alpha 5 beta 1. The characteristic expression of integrin fibronectin receptors may account for the clinically unique tumor behavior, and the immunohistochemical staining for integrins may become a useful alternative to conventional histology in differential diagnosis and a marker for prognosis in neuroblastoma.     

Interference of tenascin-C with syndecan-4 binding to fibronectin blocks cell adhesion and stimulates tumor cell proliferation.

Tenascin-C is an adhesion-modulatory extracellular matrix molecule that is highly expressed in tumors. To investigate the effect of tenascin-C on tumor cells, we analyzed its antiadhesive nature and effect on tumor cell proliferation in a fibronectin context. Glioblastoma and breast carcinoma cell adhesion was compromised by a mixed fibronectin/tenascin-C substratum, which concomitantly caused increased tumor-cell proliferation. We identified the antiadhesive mechanism as a specific interference of tenascin-C with cell binding to the HepII/syndecan-4 site in fibronectin through direct binding of tenascin-C to the 13th fibronectin type III repeat (FNIII13). Cell adhesion and proliferation levels were restored by the addition of FNIII13. Overexpression of syndecan-4, but not syndecan-1 or -2, reverted the cell adhesion defect of tenascin-C. We characterized FNIII13 as the binding site for syndecan-4. Thus we describe a novel mechanism by which tenascin-C impairs the adhesive function of fibronectin through binding to FNIII13, thereby inhibiting the coreceptor function of syndecan-4 in fibronectin-induced integrin signaling.     

Cadherin-7 interacts with melanoma inhibitory activity protein and negatively modulates melanoma cell migration

Melanoma inhibitory activity (MIA) has been identified as a small protein secreted from malignant melanoma cells, which strongly enhances melanoma cell migration and invasion. Detailed analyses performed by our group showed interaction of MIA with extracellular matrix proteins and integrin α4β1 and α5β1 leading to cellular detachment. In this study, we identified cadherin-7 as a new MIA-binding protein using surface-enhanced laser desorption/ionization-mass spectrometry technology and co-immunoprecipitation. Cadherin-7 is a classical cell–cell adhesion molecule which was shown to be upregulated in malignant melanoma. We demonstrated enhanced expression of cadherin-7 in primary tumor cells compared to metastatic cells. Upregulation of cadherin-7 expression in metastatic cell lines but also downregulation of expression in cells derived from primary melanomas resulted in reduced cell migration. In addition, we speculate that MIA/cadherin-7 interaction may regulate cell–cell adhesion of malignant melanoma cells influencing the migration of the cells. Interestingly, overexpression of cadherin-7 resulted in a decreased MIA mRNA expression. In addition, MIA effects on cell migration were abrogated in cell clones overexpressing cadherin-7. In conclusion, these findings suggest that cadherin-7 regulates the expression and activity of MIA and the migration of melanoma cells playing a role in tumor development of malignant melanoma. ( Cancer Sci 2009; 100: 261–268)     

The relation between deoxycytidine kinase activity and the radiosensitising effect of gemcitabine in eight different human tumour cell lines

Background  

Gemcitabine (dFdC) is an active antitumour agent with radiosensitising properties, shown both in preclinical and clinical studies. In the present study, the relation between deoxycytidine kinase (dCK) activity and the radiosensitising effect of gemcitabine was investigated in eight different human tumour cell lines.