Organization of F-actin filaments in human glioma cell lines cultured on extracellular matrix proteins

Extracellular matrix (ECM) constituents likely play an important role in cell proliferation and the invasion of malignant human gliomas. We examined the formation of stress fibers and the growth of the human glioblastoma cell lines A172 and T98G cultured on collagen types I, IV, and V laminin (LN), and fibronectin (FN). A172 cells cultured on LN and FN formed complete F-actin filaments after 24 h of culture and grew logarithmically after 48 h. In contrast, T98G cells on LN and FN reorganized only short F-actin filaments after 24 h of culture and grew rapidly after 72 h. However, on the collagen preparations, neither cell line formed definite stress fibers and both showed lower rates of cellular proliferation. Significantly positive correlation was observed between the relative intensity of F-actin filaments and the cell proliferation. The results indicate that the ability of ECM components to modulate the growth and differentiation of malignant glioma cells may be mediated, in part, by the assembly and disassembly of F-actin filaments.     

Cytogenetic analysis of eight human papillomavirus immortalized human keratinocyte cell lines

Cytogenetic analysis was performed on human papillomavirus (HPV)-immortalized cell lines. Lines were established by co-transfection of primary human keratinocyte cells with HPV type 16 or 18 DNA and pSV2neo. The resulting clonal lines contained integrated HPV DNA and exhibited extended life spans in culture but were non-tumorigenic in nude mice. Two HPV16-immortalized lines (FEPE1L8 and FEPE1L9) and 4 HPV18-immortalized lines (FEA, FEH18L, FEP18-5 and FEP18-11) were established. Two additional lines were derived by subsequent treatment of the FEA line with TPA and by further transfection with HSVII DNA. Cytogenetic analysis revealed that all lines were abnormal, containing a variety of numerical and structural aberrations. Six of the 8 lines were hyper-triploid and 2 were near-diploid. Examination of lines FEA, FEH18L and FEP18-11 at multiple passages in culture revealed that the lines were clonal and chromosomally stable over extended passage in culture. Structural rearrangements were most common in chromosomes 1 and 3 but also occurred in chromosomes 5, 7, 8, 12, 16 and 22. Marker chromosomes were present in all cell lines. A small metacentric marker, possibly an isochromosome for the short arm of chromosome 5, was consistently present in the FEA line and its derivatives (FEAB10 and FEAT) as well as the FEH18L line. A loss or reduction in copy number of chromosome 13 was seen in 5 of the 8 cell lines.     

Relationship between extracellular matrix interactions and degree of differentiation in human colon carcinoma cell lines

Human colon carcinoma cell lines that vary in their degree of differentiation were examined for their ability to interact with extracellular matrix components. For this purpose, established cell lines were classified on the basis of several criteria that relate to degree of differentiation. These criteria include histology of the original tumor, histology of xenografts, in vitro morphology, and carcinoembryonic antigen expression. On this basis, the cell lines used were either moderately well or poorly differentiated. The poorly differentiated cell lines adhered to surfaces coated with laminin or reconstituted basement membrane extract (Matrigel) to a significantly greater extent than the moderately well differentiated lines with the exception of one moderately well differentiated line that was derived from a highly aggressive signet ring cell carcinoma. In addition, the poorly differentiated cell lines exhibited considerable spreading on laminin and Matrigel after adherence that was not evident for the moderately well differentiated lines. The adherence of these cell lines on fibronectin-coated surfaces did not correlate as well with differentiation although, in general, poorly differentiated cell lines adhered better than moderately well differentiated lines. None of the cells that adhered to fibronectin exhibited the extensive spreading seen on laminin. The specificity of tumor cell interactions with extracellular matrix glycoproteins was examined using synthetic peptides which correspond to sequences within these proteins that are recognized by cell surface receptors. The pentapeptide YIGSR-NH2 significantly inhibited the adherence and spreading of the tumor cell lines on laminin, but not on fibronectin. The peptide RGDS, however, did not inhibit tumor cell interactions with laminin although it did inhibit their interactions with fibronectin. Thus, the interactions of colon carcinoma cells with laminin and fibronectin are probably mediated by separate receptors. Taken together, the data demonstrate that cells derived from colon carcinomas exhibit considerable variation in their ability to interact with extracellular matrix components, and that this variability is related to the degree of differentiation of original tumor.     

Plasminogen activator mediated degradation of subendothelial extracellular matrix by human squamous carcinoma cell lines

Extracellular matrix (ECM) produced by bovine corneal endothelial cells was used to investigate the role of the plasminogen activator/plasmin system in the degradation of ECM by human squamous cell carcinoma (SqCCs) and human foreskin epidermal cells (HFEC). SqCCs caused an 8- to 34-fold greater solubilization of 3H-glucosamine-labeled ECM than HFEC. This action in SqCCs was dependent upon the presence of acid-treated serum, indicating that tumor-associated proteinases were sensitive to the inhibitory action of acid-labile proteinase inhibitors present in the serum. SqCC mediated digestion of radiolabeled ECM was decreased by 14- to 55-fold in plasminogen depleted serum, and the addition of 100 micrograms/mL of purified human plasminogen resulted in up to a 30-fold increase in the degradation of the ECM. Inhibitors of this proteinase system and murine monoclonal antibodies (MAb) specific for human urokinase plasminogen activator (uPA) decreased the SqCC mediated digestion of radiolabeled ECM in a concentration dependent manner. SqCCs exhibited 10- to 30-fold higher extracellular uPA levels than HFEC, as assayed by substrate hydrolysis, zymography, micro-ELISA, western analysis, and northern analysis. These findings reflect the differential ability of these cell types to degrade the ECM. In addition, immuno-cross-reactive plasminogen activator inhibitor type I (PAI type 1) and type II (PAI type 2) were identified in cell-free conditioned medium produced by both tumor cells and normal epidermal cells, using a micro-ELISA assay. Indirect immunofluorescence flow cytometry, employing MAbs directed against uPA, detected the presence and localization of uPA on the SqCC cell surface. These findings were specific for uPA, since cell surface associated tissue plasminogen activator was not detected in these cell types under analogous conditions. In addition, partially purified SqCC plasma membrane preparations exhibited 2- to 10-fold higher uPA-like activity than HFEC, as determined by zymography. The findings support the concept that the plasminogen activator system is important in the breakdown of ECM by SqCCs and suggest that regulatory mechanisms involved in this proteolytic system may be important targets for chemotherapeutic intervention to limit tumor cell invasion and metastasis.     

Regulation of extracellular matrix metalloproteinase inducer and matrix metalloproteinase expression by amphiregulin in transformed human breast epithelial cells

Amphiregulin (AR) and epidermal growth factor effects on expression and activity of the extracellular matrix metalloproteinase inducer (EMMPRIN) were examined in NS2T2A1 breast tumor cells. Both growth factors induced mRNA and protein expression of EMMPRIN, and matrix metalloproteinase (MMP) -2 and -9 enzymatic activity. The induction of EMMPRIN by AR was mediated by epidermal growth factor receptor (EGFR) tyrosine kinase activation and inhibited by ZD1839. AR and EGFR antisense (AS) cDNAs inhibited EMMPRIN expression and MMP activity. Coculture of NS2T2A1V expressing AR- or EGFR-AS with fibroblasts and endothelial cells showed a decreased MMP activity. In parallel, nude mice tumors derived from AR and EGFR-AS cells revealed reduced level of EMMPRIN and MMP activity. AR and epidermal growth factor, therefore, regulate EMMPRIN and its MMP-mediated expression, identifying EGFR signaling as critical to this regulation.     

Extracellular matrix proteins characterize human tumor cell lines

Extracellular matrix proteins synthesized and secreted by adherent human tumor cell lines were analyzed using metabolic labelling with glycine and proline in the presence of ascorbate, polypeptide analysis and polyacrylamide gel electrophoresis, affinity chromatography, collagenase digestion, and immunofluorescence staining. The results showed a characteristic pattern of matrix proteins for each tumor cell type. Tumor cell lines of mesenchymal origin produced mostly interstitial types (I and II) of collagen and fibronectin. Carcinoma cell lines secreted only basement membrane proteins, type IV collagen, laminin and fibronectin, but not interstitial collagen. A melanoma and a rhabdomyosarcoma cell line produced type V of procollagen that has not previously been described in cell culture. Neuroblastoma cells were shown to be phenotypically heterogeneous also with respect to matrix protein production. We propose that the analysis of extracellular matrix proteins may serve as an adjunct in the classification of human tumors.     

胞外环境Ca^2＋变化诱导人肿瘤细胞凋亡

目的：观察胞外环境Ca^2 变化对人肿瘤细胞的直接诱导凋亡作用。方法：通过EGTA螯合胞外Ca^2 或添加CaCl2改变胞外环境Ca^2 。用PI和Hoechst33342荧光双染观察细胞核形态，用流式细胞仪检测凋亡百分率。分别借助荧光探针Fluo-3和Rh123检测胞内Ca^2 和线粒体膜电位（△ψm)变化。用环孢菌素A（CsA)阻断PT孔，研究其在凋亡中的作用。结果：胞外环均Ca^2 升高或降低均诱导人胃癌MGC-803和喉癌Hep细胞凋亡，恢复胞外Ca^2 阻断凋亡。随胞外Ca^2 变化胞内Ca^2 相应升高或降低，但线粒体△ψm均表现为急剧下降。CsA对MGC-803细胞凋亡无明显抑制作用，轻微促进Hep细胞凋亡。结论：胞外环境Ca^2 变化引起胸内Ca^2 相应变化和线粒体△ψm下降并通过PT孔非依赖性途径诱导肿瘤细胞凋亡。     

胞外环境Ca~(2 )变化诱导人肿瘤细胞凋亡

目的 :观察胞外环境Ca2 变化对人肿瘤细胞的直接诱导凋亡作用。方法 :通过EGTA螯合胞外Ca2 或添加CaCl2 改变胞外环境Ca2 。用PI和Hoechst3334 2荧光双染观察细胞核形态 ,用流式细胞仪检测凋亡百分率。分别借助荧光探针Fluo 3和Rh12 3检测胞内Ca2 和线粒体膜电位 (ΔΨm)变化。用环孢菌素A(CsA)阻断PT孔 ,研究其在凋亡中的作用。结果 :胞外环境Ca2 升高或降低均诱导人胃癌MGC 80 3和喉癌Hep细胞凋亡 ,恢复胞外Ca2 阻断凋亡。随胞外Ca2 变化胞内Ca2 相应升高或降低 ,但线粒体ΔΨm均表现为急剧下降。CsA对MGC 80 3细胞凋亡无明显抑制作用 ,轻微促进Hep细胞凋亡。结论 :胞外环境Ca2 变化引起胞内Ca2 相应变化和线粒体ΔΨm下降并通过PT孔非依赖性途径诱导肿瘤细胞凋亡。     

Rearrangement of the same chromosome regions in different SV40 transformed human skin keratinocyte lines is associated with tumourigenicity

Twelve different human keratinocyte strains were transformed with recombinant plasmid pSV6-1 which contained an origin defective SV40 genome. When injected into athymic nude mice lines produced either squamous cell carcinomas (SCC) in all animals, SCC in some animals and epidermal cysts in others, or epidermal cysts only in all the animals. The tumourigenic capacity of the lines could be correlated with the chromosomal changes present initially in the transformed cells. Lines which produced SCC in all the animals within a short period of time all showed simultaneous loss of part of chromosomes 3p, 8p and 11p in one homologue. Lines which were not tumourigenic did not show these simultaneously appearing rearrangements. These specific rearrangements are acquired in vitro and the time taken for a recognisable tumour to appear is related to the proportion of such cells in the line. The rearrangement of the same chromosome regions in different tumourigenic cell lines suggests that genes in these regions are important in the development of squamous cell carcinoma, possibly by loss of heterozygosity, at particular loci.     

Collagen biosynthesis in human neuroblastoma cell lines: evidence for expression of glial cell properties

An analysis was done on the synthesis of collagen, an extracellular matrix protein synthesized by Schwann cells, by several human neuroblastoma lines. Cultured cells were incubated in the presence of L-[2,3-3H]proline, and the collagens synthesized and secreted into the culture medium were analyzed by electrophoresis on acrylamide gels, ion exchange chromatography, and immunoprecipitation. The amount of collagen secreted by 4 cell lines tested represented less than 3% of the total protein synthesis, indicating a low degree of collagen biosynthesis by this tumor type. Analysis of collagen types secreted by all cell lines revealed the presence of a high-molecular-weight collagen precursor (Mr = 165,000) identified as type IV procollagen. In addition, several cell lines synthesized stromal type I and type III collagens. These studies show that neuroblastoma cells produce collagenous proteins including basement membrane collagen (type IV) and stromal collagens (types I and III), indicating that these cells express properties of glial cells such as Schwann cells.     

Identity of tumorigenic human urothelial cell lines and 'spontaneously' transformed sublines.

Restriction fragment length polymorphism (RFLP) analysis, comparative marker chromosome analysis, and polymorphic enzyme analysis was carried out on a total of eight human urothelial cell lines and sublines selected according to our knowledge of their HLA-A,B phenotype. RFLP analysis and cytogenetic analysis showed that the cell lines Hu1703He, Hu1922, and T24 are genuine cell lines of different origin. The identity of Hu1703He could not be confirmed by its isozyme phenotype which was identical to the T24 phenotype. RFLP analysis and isozyme analysis revealed that three cell lines, Hu456, Hu549, and Hu961a, and two transformed sublines, HCV-29Tmv and Hu609Tmv, are sublines of T24. A common origin of Hu456, Hu549, Hu961a, HCV-29Tmv, and Hu609Tmv was confirmed by marker chromosome analysis. However, the T24 origin of these cytogenetically related cell lines was not supported by chromosome analysis of T24. RFLP analysis and HLA phenotyping of two tumorigenic and invasive sublines isolated from a culture of non-tumorigenic Hu609 cells showed that non-tumorigenic Hu609 cells can transform ''spontaneously'' in vitro into tumorigenic Hu609T cells. The results emphasise the need for careful monitoring and screening of cell lines for their identity using more than one identification parameter.     

Anti-tumor surveillance of non-contact-inhibited transformed cell lines

In order to determine if the correlated expression of transformation and tumorigenicity is affected by the agents used to induce transformants or by the immune status of the host used to test the tumorigenicity of transformants, we derived a series of cloned cell lines from foci of transformed cells induced by treatment of the contact-inhibited mouse cell line B/C-N7.ICI with the DNA demethylating agent 5-azacytidine (5-AZC) or the DNA demethylating and mutating agent benzo(a)pyrene dihydrodiol epoxide (BPDE). The transformed cell lines were injected into syngeneic nude and normal mice to determine their tumorigenicity. The results of this analysis showed that 93% of the transformants induced by 5-AZC treatment grew as tumors when injected into nude mice. Of those lines capable of growing as tumors in nude mice, 86% were also tumorigenic when injected into normal mice. In contrast, only 64% of BPDE-induced transformants grew as tumors in nude mice, and of those, only 44% were also tumorigenic in normal mice. The existence of non-contact-inhibited transformants that are tumorigenic only in nude mice indicates that host anti-tumor immune surveillance mechanisms are operative in normal mice. Further, the difference in both the percentage of transformed cell lines that are tumorigenic and the percentage of tumorigenic transformants that are susceptible to immune surveillance when transformants are induced by BPDE as compared to 5-AZC indicates that the transforming agent can affect both the correlation between the expression of transformation and tumorigenicity, and the interaction between the immune system and tumorigenic transformants.     

Immunohistochemical pattern of cytokeratins and MMPs in human keratinocyte cell lines of different biological behaviour

We investigated three clonally related human keratinocyte cell lines of different biological behaviour, HaCaT (non-tumorigenic), A5 (benign, tumorigenic) and II-4RT (malignant, tumorigenic), with regard to the cell-associated localization of the major MMPs -1, -2, -3, -9, -10 and -11. These observations were correlated with the pattern of cytokeratins (CK) 10, 13 and 14 which served as markers for cellular. In all three cell lines, we detected immunohistochemically various MMPs within the cytoplasm of the tumor cells, however, with differences between the MMPs and the various cell lines. MMP-1, -10 and -11 were strongly and equally present in all cell lines tested. MMP-2 was seen only faintly in all cells. In contrast, MMP-3 was only faintly seen in the cytoplasm of the non-tumorigenic, more extensive in the benign A5 cells, but strongly and extensive in the malignant II-4RT cells. MMP-9 was also seen in increasing intensity corresponding to the tumorigenicity of the three cells lines, however, with less intense staining than MMP-3. A semiquantitative immunoreactive score confirmed these observations. In parallel, the CK pattern indicated advanced cellular maturation of HaCaT (as seen by expression of basal CK 14 and suprabasal CKs 10 and 13). In the A5 cells a reduced expression of suprabasal CKs-10 and -13 indicated lesser maturation. The malignant II-4RT cells revealed even fewer cells with suprabasal keratinocyte differentiation. Our study clearly confirms differences in the concentration of cell-associated MMPs. These analyses parallel and supplement our previous biochemical studies. In addition, we provide circumstantial evidence that the expression of several major MMPs is associated with changes in the maturation pattern as evidenced by expression of CKs.     

Expression pattern of matrix metalloproteinases in human gynecological cancer cell lines

Background  

Matrix metalloproteinases (MMPs) are involved in the degradation of protein components of the extracellular matrix and thus play an important role in tumor invasion and metastasis. Their expression is related to the progression of gynecological cancers (e.g. endometrial, cervical or ovarian carcinoma). In this study we investigated the expression pattern of the 23 MMPs, currently known in humans, in different gynecological cancer cell lines.     

Expression of matrix metalloproteinase genes in transformed rat cell lines of high and low metastatic potential.

The enzymes that comprise the family of matrix metalloproteinases (MMPs) share the capacity to degrade extracellular matrix components. A large body of evidence indicates that certain members of this metalloproteinase gene family play critical roles in determining the malignant phenotype of solid tumors. We previously have derived transformed cell lines with vastly different metastatic potentials by transfecting different combinations of oncogenes into primary rat embryo cells. Conditioned medium from those cell lines was assayed by Western blot analysis for the production of four separate matrix metalloproteinases to see whether a correlation could be found between protease expression and the metastatic phenotype. The transformed rat embryo cell lines with high metastatic potential were found to produce high levels of the stromelysin 1 (MMP-3) and stromelysin 2 (MMP-10) proteases, while the nonmetastatic lines produced low or undetectable levels of these two enzymes. No correlation was seen between the metastatic phenotype of the cell lines and the level of expression of two other matrix metalloproteinases, the M(r) 72,000 type IV collagenase (MMP-2) and the M(r) 92,000 gelatinase (MMP-9). These data suggest that the differential regulation of the stromelysin proteases may contribute to the difference seen in the metastatic potential of these cell lines.     

Regulation and function of the extracellular matrix protein tenascin-C in ovarian cancer cell lines.

The extracellular matrix glycoprotein tenascin-C (TN) is overexpressed in the stroma of malignant ovarian tumours particularly at the interface between epithelia and stroma leading to suggestions that it may be involved in the process of invasion (Wilson et al (1996) Br J Cancer 74: 999-1004). To define regulation of TN further and investigate its function in ovarian cancer, a range of cell line models were studied. Concentrations of secreted TN in media from cultures of ovarian fibroblast cell lines were at least 100-fold greater than from carcinoma cell lines. Evidence for paracrine regulation of TN secretion was obtained by co-culture of carcinoma cells with fibroblast cells wherein secretion into the media was greater than from fibroblasts alone. Transforming growth factor (TGF)-beta1, insulin-like growth factor (IGF)-II and progesterone all stimulated TN secretion while human choriogonadotropin (hCG), follicle-stimulating hormone (FSH) and gamma-interferon inhibited secretion. TGF-beta1 produced the greatest stimulation of TN in cultured fibroblasts and its co-expression with TN was examined in primary ovarian tumours. There was a significant association between the presence of moderate-strong expression of TN and TGF-beta1. Evidence for TN having a functional role in ovarian carcinoma was obtained from adhesion and migration assays. The PE01, PE04, SKOV-3 and 59M cell lines all demonstrated marked adhesion to plastic coated with TN relative to the control protein bovine serum albumin (BSA) and expressed alpha2beta1 and alpha3beta1 integrins. The SKOV-3 cell line migrated more rapidly through TN than through BSA indicating that TN can facilitate migration of ovarian carcinoma cells.     

Nuclear matrix protein composition of human lung carcinoma cell lines

The nuclear matrix is the RNA-protein skeleton within the nucleus that contributes to the structural and functional organization of DNA. Differences in the nuclear matrix protein composition between cancer and normal cells have been reported in various cell lines and tissues, suggesting altered gene expression. This study examined the nuclear matrix protein composition of various human lung cell lines. Using high resolution two-dimensional electrophoresis, at least ten common proteins, as well as specific differences, were identified in each category of lung cell lines. These protein differences may be responsible, at least in part, for the different phenotypes of human lung cancer.     

人膀胱癌组织中细胞外基质金属蛋白酶诱导因子的表达

背景与目的:基质金属蛋白酶(matrix metalloproteinases,MMPs)在恶性肿瘤的发生、浸润和转移中发挥关键作用,肿瘤细胞表达的细胞外基质金属蛋白酶诱导因子(extracellular matrix metalloproteinase inducer,EMMPRIN)司以诱导间质成纤维细胞合成MMPs,从而促进肿瘤的浸润和转移。本研究拟通过检测EMMPRIN在人膀胱癌组织和正常膀胱粘膜中的表达,探讨EMMPRIN在膀胱癌发生、浸润和转移中的作用。方法:应用免疫组化SP方法检测EMMPRIN在45例膀胱癌组织和9例正常膀胱粘膜中的表达,并与临床病理指标结合起来分析。结果;EMMPRIN蛋白定位于癌细胞的胞膜和胞浆,阳性率为73.3％,正常膀胱粘膜和肿瘤间质均为阴性表达。EMMPRIN在Ⅱ、Ⅲ级和术后复发组的阳性表达率分别为84.0％、84.6％和100％,高于Ⅰ级和无复发组(分别为14.3％和55.6％)(P<0.05);在淋巴结转移组的强阳性表达率(85.7％)高于无淋巴结转移组(23.7％)(P<0.05)。结论:EMMPRIN在人膀胱癌组织中过度表达,与膀胱癌的恶性程度有关,可作为判断膀胱 癌是否具备高侵袭转移潜能的指标。     

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.