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1.
《Cell communication & adhesion》2013,20(4):321-329
Interactions with the extracellular matrix constitute basic steps in cervix carcinoma cell invasion. In this study, we examined the adhesion and migration profiles of two human papillomavirus (HPV) DNA-transfected keratinocyte-derived cell lines, EIL8 and 18-11 S3, and of the cervix adenocarcinoma SiHa cell line, towards laminin-1, and the selective effect of a 24-72 h treatment of 1000 U/ml interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), a treatment that significantly decreases cervix carcinoma cell proliferation and progression in nude mice, on these parameters. Compared to normal cervix keratinocytes (CK) and two HPV DNA-transfected keratinocyte cell lines, in basal conditions, the SiHa cell line was characterized by increased attachment (SiHa, 48.74 ± 4.02 vs. normal keratinocytes, 4.32 ± 0.40, EIL8,17.80 ± 3.03 and 18-11S3,17.82 ± 1.48% of attached cells after 30min) and marked directed chemotactic migration towards laminin-1. Interestingly, treatment of the cells with the cytokines (1000U/ml IFN-γ and TNF-α) did not modulate the adhesion properties of the cells, but chemotactic migration of SiHa cells to laminin-1 was significantly decreased, while migration towards type I collagen was increased. Similar results were obtained with the Ca Ski cervix carcinoma cell line. Our results emphasize the altered pattern of interactions of cervix carcinoma cells with extracellular matrix components such as laminin-1, compared to normal and preneoplastic cells, and contributes to the understanding of the effects of cytokine treatment on cervix carcinoma cells. 相似文献
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3.
Extinction of the HPV18 upstream regulatory region in cervical carcinoma cells after fusion with non-tumorigenic human keratinocytes under non-selective conditions.
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F Rsl T Achtsttter T Bauknecht K J Hutter G Futterman H zur Hausen 《The EMBO journal》1991,10(6):1337-1345
'Universal fuser' clones of a human papillomavirus type 16 positive cervical carcinoma cell line (SiHa) were established to study the effect of a non-tumorigenic fusion partner on the regulation of a stably integrated chloramphenicol acetyltransferase (CAT) gene controlled by the HPV18 upstream regulatory region under non-selective conditions. The CAT expressing cells were fused with both non-tumorigenic, spontaneously immortalized human keratinocytes (HaCaT) and non-modified SiHa cells. The resulting hybrids were characterized by restriction enzyme fragment length polymorphism analysis and flow cytometry. While the non-selectable, HPV18-driven indicator gene is constitutively expressed in SiHa cells, the CAT activity is extinguished in SiHa x HaCaT cells, but still present in SiHa x SiHa hybrids. Examination of the cytokeratin expression pattern reveals that the keratinocyte phenotype seems not only to be dominant in terms of the extinction of the HPV18 regulatory region but also by the conservation of most of the differentiation markers of the non-tumorigenic fusion partner. Cycloheximide treatment and intracellular competition experiments using the transient COS7 fusion-amplification technique are accompanied by the reactivation of the marker gene in previously CAT- SiHa x HaCaT hybrids. These data strongly suggest that trans-acting negative regulatory factors derived from the non-malignant human keratinocytes are responsible for the extinction phenomenon. 相似文献
4.
During the evolution of epithelial cancers, cells often lose their characteristic features and acquire a mesenchymal phenotype, in a process known as epithelial-mesenchymal transition (EMT). In the present study we followed early stages of keratinocyte transformation by HPV16, and observed diverse cellular changes, associated with EMT. We compared primary keratinocytes with early and late passages of HF1 cells, a cell line of HPV16-transformed keratinocytes. We have previously shown that during the progression from the normal cells to early HF1 cells, immortalization is acquired, while in the progression to late HF1, cells become anchorage independent. We show here that during the transition from the normal state to late HF1 cells, there is a progressive reduction in cytokeratin expression, desmosome formation, adherens junctions and focal adhesions, ultimately leading to poorly adhesive phenotype, which is associated with anchorage-independence. Surprisingly, unlike "conventional EMT", these changes are associated with reduced Rac1-dependent cell migration. We monitored reduced Rac1-dependent migration also in the cervical cancer cell line SiHa. Therefore we can conclude that up to the stage of tumor formation migratory activity is eliminated. 相似文献
5.
Yongjun Wang Yanjie Weng Ying Shi Xi Xia Shixuan Wang Hua Duan 《The Journal of membrane biology》2014,247(7):591-599
Toll-like receptors are expressed in human immune cells and many tumors, but the role of toll-like receptor 4 (TLR4) in the development of tumors is controversial. We demonstrated the expression, distribution, and functional activity of TLR4 in tissues of normal cervix, cervical intraepithelial neoplasia (CIN), invasion cervical cancers (ICC), and different human papillomavirus (HPV)-infected cervical cancer cells. The results showed that TLR4 expression was in accordance with the histopathological grade: higher in ICC than in CIN, and low in normal cervical tissues and malignant cervical stroma. Expression was higher in SiHa (HPV16+) than in HeLa (HPV18+) cells, but was not observed in C33A (HPV?) cells. After treatment with its agonist, lipopolysaccharide (LPS), the expression levels of TLR4 was increased and apoptosis resistance was induced in SiHa cells, but not in HeLa or C33A cells. Meanwhile, LPS treatment did not alter the cell cycle distribution in SiHa cells. The mechanism of apoptosis resistance may be related to HPV16 infection and not correlated with the cell cycle distribution. Targeting TLR4 in combination with traditional drug treatment may serve as a novel strategy for more effectively killing cancer cells. 相似文献
6.
The effects of laminin-5 and its subunit gamma2 chain on cell adhesion and migration were studied, and a migration-related cis-acting element was identified in the gamma2 chain gene (LAMC2) using promoter-reporter gene constructs in transgenic mice. Intact laminin-5 molecules, but not recombinant gamma2 chain promoted cell adhesion of human keratinocytes and mouse squamous carcinoma cells, indicating that the gamma2 chain does not contain a cellular binding site. However, the gamma2 chain as such is probably involved in the process of cell locomotion, as antibodies against the short arm of the chain inhibited migration of carcinoma cells in an in vitro assay. Further evidence for the involvement of the gamma2 chain in cell migration was obtained by the identification of a cis-acting element in a promoter-lacZ reporter gene construct that was active in migratory epithelial cells of healing wounds in mice made transgenic by microinjection of the construct into fertilized oozytes. The migration active element was located in the sequence between -613 and +55. The same construct, and another one containing 5900 base pairs of the 5' flanking region, yielded very limited expression in cells of normal tissues. The limited expression was, however, only observed in epithelial cells of different tissues, i.e. cell types that normally express laminin-5 in vivo. The results show that the sequence between -613 and +55 contains elements that can drive expression during epithelial cell migration and that also partially confers more general epithelium expression. However, elements outside -5900 and +55 are needed for normal epithelium expression of the LAMC2 gene. 相似文献
7.
Both E6 and E7 oncoproteins of human papillomavirus 16 inhibit IL-18-induced IFN-gamma production in human peripheral blood mononuclear and NK cells. 总被引:16,自引:0,他引:16
S J Lee Y S Cho M C Cho J H Shim K A Lee K K Ko Y K Choe S N Park T Hoshino S Kim C A Dinarello D Y Yoon 《Journal of immunology (Baltimore, Md. : 1950)》2001,167(1):497-504
Cervical carcinoma is the predominant cancer among malignancies in women throughout the world, and human papillomavirus (HPV) 16 is the most common agent linked to human cervical carcinoma. The present study was performed to investigate the mechanisms of immune escape in HPV-induced cervical cancer cells. The presence of HPV oncoproteins E6 and E7 in the extracellular fluids of HPV-containing cervical cancer cell lines SiHa and CaSki was demonstrated by ELISA. The effect of HPV 16 oncoproteins E6 and E7 on the production of IFN-gamma by IL-18 was assessed. E6 and E7 proteins reduced IL-18-induced IFN-gamma production in both primary PBMCs and the NK0 cell line. FACS analysis revealed that the viral oncoproteins reduced the binding of IL-18 to its cellular surface receptors on NK0 cells, whereas there was no effect of oncoproteins on IL-1 binding to its surface IL-1 receptors on D10S, a subclone of the murine Th cell D10.G4.1. In vitro pull-down assays also revealed that the viral oncoproteins and IL-18 bound to IL-18R alpha-chain competitively. These results suggest that the extracellular HPV 16 E6 and E7 proteins may inhibit IL-18-induced IFN-gamma production locally in HPV lesions through inhibition of IL-18 binding to its alpha-chain receptor. Down-modulation of IL-18-induced immune responses by HPV oncoproteins may contribute to viral pathogenesis or carcinogenesis. 相似文献
8.
Hartwig B Borm B Schneider H Arin MJ Kirfel G Herzog V 《Experimental cell research》2007,313(8):1575-1587
Laminin-5 is a major adhesion protein of the skin basement membrane and crucially involved in integrin-mediated cell substrate attachment of keratinocytes, which is important for hemidesmosomal anchorage as well as for keratinocyte migration during epidermal wound healing. To investigate its role in keratinocyte migration, we analyzed laminin-5-deficient cells of patients with a lethal variant of junctional epidermolysis bullosa. Normal migrating keratinocytes adopted monopolar morphology with a distinct front lamella and employed a continuous mode of translocation. In contrast, laminin-5-deficient cells assumed a stretched bipolar shape with two lamella regions and migrated in a discontinuous, saltatory manner characterized by significantly decreased directional persistence and reduced migration velocity. The distinct morphology as well as the migratory phenotype apparently resulted from a defect in the formation of cell substrate adhesions that were completely missing in the cell body and less stable in the lamella regions. Accordingly in normal keratinocytes, a bipolar shape and a saltatory migration mode were inducible by blocking laminin-5-mediated substrate adhesion. Our findings clearly point to an essential role of laminin-5 in forming dynamic cell substrate adhesion during migration of epidermal keratinocytes and provide an explanation for the cellular mechanisms that underlie the lethal form of junctional epidermolysis bullosa. 相似文献
9.
Kalmykova NV Cherepanova OA Gorelik IuV Voronkina IV Blinova MI Pinaev GP 《Tsitologiia》2002,44(8):792-798
The cell-matrix interaction is one of the factors defining the cell behavior in normal and wounded tissues. To determine the function of laminin-2/4, one of components of the skin basement membrane in the process of reepithelization, we studied its interaction with human keratinocytes. The adhesive properties of laminin-2/4 and its effect on keratinocytes migration in vitro were analysed. For comparison with our present investigation, we used the earlier studied laminin-1 from EHS mouse sarcoma. Laminin-2/4 appeared to be a good substrate for human keratinocytes, and this correlates with a greater number of cell surface receptors compared with laminin-1. Laminin-2/4 alone does not stimulate keratinocyte migration, but, in contrast to laminin-1, supports EGF-mediated migration. The obtained results give an insight into the function of laminin-2/4 in normal skin and during wound healing. 相似文献
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11.
Cherepanova OA Kalmykova NV Koronkina IV Aré AF Gorelik IuV Pinaev GP 《Tsitologiia》2002,44(2):151-158
Laminins constitute a family of heterotrimeric glycoproteins of basement membranes. Laminins promote cell adhesion, migration, growth, and differentiation. So far, at least 12 different isoforms of laminin have been known. However, no sufficient knowledge is available on the nature of cell response on different laminins. The study was aimed to compare adhesive properties of two laminin isoforms, laminin-1 and laminin-2/4, with respect to normal (freshly isolated keratinocytes) and transformed (A-431) human skin cells. We have used the following assays: cell adhesion to the substrate covered with laminin isoformes, interaction of latex beads (D = 1 micron) coated with the same proteins with cells in suspension, and a comparative study of the cytoskeleton structure of cells spread on the immobilized laminins. It was demonstrated that laminin-2/4 is a more effective potent promotor of adhesion for both normal keratinocytes and transformed A-431 cells, compared with laminin-1. A comparison of many attached protein-covered beads allowed to estimate a relative quantity of cell surface receptors to laminin isoforms in different cell types. The relative number of receptors to laminin-2/4 on the keratinocyte surface is 7 times higher than that to laminin-1 after a 30 min incubation with cells, and is 6 times higher after 1 hour. As for A-431 cells, their attachment to laminin-2/4 beads is 5 times higher than that to laminin-1-beads after a 1 min incubation, but as early as after 5 min this distinction disappeared, owing to bead internalization. The presence of a specific receptor to laminin-2/4 but not to laminin-1 on the keratinocyte surface has been suggested. Keratin differences in cytoskeleton organization in normal and transformed skin cells spread on the substrates covered with laminin-1 and laminin-2/4 were demonstrated. 相似文献
12.
Monocyte chemotaxis and activating factor production by keratinocytes in response to IFN-gamma 总被引:7,自引:0,他引:7
J N Barker M L Jones C L Swenson V Sarma R S Mitra P A Ward K J Johnson J C Fantone V M Dixit B J Nickoloff 《Journal of immunology (Baltimore, Md. : 1950)》1991,146(4):1192-1197
Monocytes accumulate in the epidermis and along the dermo-epidermal junction in several different inflammatory skin diseases. To determine whether human epidermal keratinocytes elaborate a specific chemotaxin responsible for the accumulation of monocytes at these anatomic sites, monocyte chemotactic activity in conditioned 16-h cultured keratinocyte supernatants were assayed using human peripheral blood monocytes as the target cell. Dilutional analysis revealed directed monocyte migration in IFN-gamma-treated (100 U/ml) keratinocyte supernatants (80% maximal FMLP response) which was 10-fold more than IFN-gamma itself or untreated keratinocyte activity alone. Gel filtration chromatography revealed that this activity eluted just ahead of a 12.5-kDa molecular mass marker. Blocking studies demonstrated that a rabbit polyclonal antibody to monocyte chemotaxis and activating factor (MCAF) inhibited all monocyte chemotaxis by greater than 80%. Keratinocytes were metabolically labeled with 35S-cysteine/methionine, and after 16 h incubation the supernatants immunoprecipitated with the same anti-MCAF antibody. MCAF was detected as a protein doublet of 12 and 9 kDa only in IFN-gamma-treated (100 U/ml) keratinocyte supernatants. Incubation with IFN-gamma and TNF-alpha (250 U/ml) in combination resulted in increased production of MCAF protein. By Northern blot analysis, MCAF mRNA was constitutively expressed in keratinocytes and upregulated only in the presence of IFN-gamma. TNF-alpha, IL-1 beta, transforming growth factor-beta and phorbol esters had no positive or negative influence on MCAF mRNA. These studies demonstrate that biologically active MCAF is elaborated by human epidermal keratinocytes upon activation by IFN-gamma, a cytokine also required for the induction of adherence between monocytes and keratinocytes. Keratinocyte-derived MCAF is likely to be important in the regulation of cutaneous monocyte trafficking and may also be responsible for the recruitment of Langerhans cells and dermal dendrocytes, which share many phenotypic features with monocytes/macrophages, to their anatomic locations in skin. 相似文献
13.
In situ hybridization with digoxigenin-labeled DNA of human papillomaviruses (HPV 16/18) in HeLa and SiHa cells 总被引:9,自引:0,他引:9
To establish a nonradioactive method for demonstrating HPV DNA in routinely treated smears of the uterine cervix (alcohol fixation, staining according to Papanicolaou, preservation), in situ hybridizations were carried out in HeLa and SiHa cells grown on slides. After detailed investigations, the sensitivity and specificity of the biotin-avidin method (10) initially used proved to be inadequate for this purpose. Demonstration of HPV 16 DNA in SiHa cells (SiHa cells only contain 1-2 HPV genome copies) was possible only by use of digoxigenin-labeled HPV 16 gene probes, as well as an improved purification of the sample DNA from vector contaminations. Thus, for the first time a protocol for correlation of the results of an in situ hybridization with the cytological appraisal in the very same smear preparation has been developed for routine diagnostics. 相似文献
14.
Mohan K Ding Z Hanly J Issekutz TB 《Journal of immunology (Baltimore, Md. : 1950)》2002,168(12):6420-6428
Previous studies have shown that the CXC chemokine, IFN-gamma-inducible T cell alpha chemoattractant (I-TAC), was chemotactic for IL-2-activated human T lymphocytes, which express abundant CXCR3. However, because most memory T lymphocytes are also CXCR3(+), the ability of I-TAC to promote the migration of normal human blood T cells across HUVEC monolayers in Transwell chambers was examined. I-TAC induced a marked (4- to 6-fold) increase in transendothelial migration (TEM) of T cells across unstimulated HUVEC from 5.6 to 28% of input T cells and was substantially more active than IFN-gamma-inducible protein-10, another CXCR3 ligand. I-TAC significantly enhanced TEM of T cells across TNF-alpha, but not across IFN-gamma or IFN-gamma plus TNF-alpha-activated HUVEC. IFN-gamma or IFN-gamma plus TNF-alpha-activated HUVEC produced substantial amounts of I-TAC, in contrast to TNF-alpha-treated EC. Both CD4(+) and CD8(+) T cells migrated in response to I-TAC to a similar extent, while memory T cells migrated several fold better than naive T cells. Blockade of LFA-1 strongly inhibited I-TAC-induced T cell TEM across unstimulated HUVEC, and approximately 50-60% of the TEM across cytokine-activated HUVEC. However, blocking both LFA-1 and very late Ag-4 abolished I-TAC induced T cell TEM. In vivo significant levels of I-TAC were detected in arthritic synovial fluid. Thus, I-TAC is one of the most potent chemoattractants of normal human blood CD4 and CD8 T cell TEM and is likely a major mediator of blood memory T lymphocyte migration to inflammation. 相似文献
15.
Integrin beta4 regulates migratory behavior of keratinocytes by determining laminin-332 organization
Sehgal BU DeBiase PJ Matzno S Chew TL Claiborne JN Hopkinson SB Russell A Marinkovich MP Jones JC 《The Journal of biological chemistry》2006,281(46):35487-35498
Whether alpha6beta4 integrin regulates migration remains controversial. beta4 integrin-deficient (JEB) keratinocytes display aberrant migration in that they move in circles, a behavior that mirrors the circular arrays of laminin (LM)-332 in their matrix. In contrast, wild-type keratinocytes and JEB keratinocytes, induced to express beta4 integrin, assemble laminin-332 in linear tracks over which they migrate. Moreover, laminin-332-dependent migration of JEB keratinocytes along linear tracks is restored when cells are plated on wild-type keratinocyte matrix, whereas wild-type keratinocytes show rotation over circular arrays of laminn-332 in JEB keratinocyte matrix. The activities of Rac1 and the actin cytoskeleton-severing protein cofilin are low in JEB keratinocytes compared with wild-type cells but are rescued following expression of wild-type beta4 integrin in JEB cells. Additionally, in wild-type keratinocytes Rac1 is complexed with alpha6beta4 integrin. Moreover, Rac1 or cofilin inactivation induces wild-type keratinocytes to move in circles over rings of laminin-332 in their matrix. Together these data indicate that laminin-332 matrix organization is determined by the alpha6beta4 integrin/actin cytoskeleton via Rac1/cofilin signaling. Furthermore, our results imply that the organizational state of laminin-332 is a key determinant of the motility behavior of keratinocytes, an essential element of skin wound healing and the successful invasion of epidermal-derived tumor cells. 相似文献
16.
Variant isoforms of the cell surface glycoprotein CD44 (CD44v) are expressed during development, in selected adult tissues and in certain metastatic tumor cells. CD44v differ from the standard isoform (CD44s) by up to ten additional exon sequences included by alternative splicing. By cell fusion experiments, we have obtained evidence for the existence of cell-type specific trans-acting factors recruiting CD44 variant exon sequences. Stable cell hybrids of CD44s and CD44v expressing cells indicated a dominant mechanism for variant-exon inclusion. In transient interspecies heterokaryons of human keratinocytes and rat fibroblasts, the ability of the keratinocytes to include all variant exon sequences in CD44 was conferred completely on the rat fibroblast nucleus. Fusions of cells with complex CD44 splice patterns do not permit interpretation of splice control by the relative abundance of a single trans-acting factor, but rather by (a) positively acting factor(s) recruiting variant exon sequences in the 3' to 5' direction and additional factors selecting individual exons. Since the pancreatic carcinoma cell line BSp73ASML (in contrast to the cervix carcinoma cell lines SiHa and ME180) could not transfer its specific splice pattern in cell fusions, we conclude that in some tumors, splicing is also controlled by mutation of cis-acting recognition sites. 相似文献
17.
Higher expression and activity of metalloproteinases in human cervical carcinoma cell lines is associated with HPV presence. 总被引:6,自引:0,他引:6
Laura Beatriz da Silva Cardeal Carla Abdo Brohem Tatiana Caroline Silveira Corrêa Sheila Maria Brochado Winnischofer Fabio Nakano Enrique Boccardo Luisa Lina Villa Mari Cleide Sogayar Silvya Stuchi Maria-Engler 《Biochimie et biologie cellulaire》2006,84(5):713-719
Matrix metalloproteinases (MMPs) MMP-2, MMP-9, and MT1-MMP are required for basement membrane degradation in cervical carcinoma. We evaluated the expression and activity of MMPs and their inhibitors RECK and TIMP-2 in 3 human invasive cervical carcinoma cell lines. Two HPV16-positive cell lines (SiHa and CaSki) and an HPV-negative cell line (C33A) were cultured either onto a type-I collagen gel, Matrigel, or plastic, to recreate their three-dimensional growth environment and evaluate the expression of these genes using quantitative real-time PCR. We also analyzed the gelatinolytic activity of MMP-2 and MMP-9 by zymography. We found that HPV (human papillomavirus)-positive cell lines express higher levels of MMP-2, MT1-MMP, and TIMP-2 than the HPV negative cell line. In addition, MMP-9 was expressed at very low levels in both HPV-negative and HPV-positive cell lines. We also observed that the expression of the RECK gene is higher in CaSki cells, being associated with higher pro-MMP-2 activity. Furthermore, Matrigel substrate influences MMP-2 expression in both SiHa and CaSki cells. On the other hand, we found that type-I collagen gel, but not Matrigel, can enhance pro-MMP-2 activity in all cell lines. Our results suggest that the presence of HPV is related to increased expression of MMP-2, MT1-MMP, and TIMP-2, and that pro-MMP-2 activity is higher in HPV-positive than in HPV-negative cells. 相似文献
18.
Chen K Iribarren P Huang J Zhang L Gong W Cho EH Lockett S Dunlop NM Wang JM 《Journal of immunology (Baltimore, Md. : 1950)》2007,178(3):1759-1766
Human formyl peptide receptor (FPR)-like 1 (FPRL1) and its mouse homologue mFPR2 are functional receptors for a variety of exogenous and host-derived chemotactic peptides, including amyloid beta 1-42 (Abeta(42)), a pathogenic factor in Alzheimer's disease. Because mFPR2 in microglial cells is regulated by proinflammatory stimulants including TLR agonists, in this study we investigated the capacity of IFN-gamma and the CD40 ligand (CD40L) to affect the expression and function of mFPR2. We found that IFN-gamma, when used alone, induced mFPR2 mRNA expression in a mouse microglial cell line and primary microglial cells in association with increased cell migration in response to mFPR2 agonists, including Abeta(42). IFN-gamma also increased the endocytosis of Abeta(42) by microglial cells via mFPR2. The effect of IFN-gamma on mFPR2 expression in microglial cells was dependent on activation of MAPK and IkappaB-alpha. IFN-gamma additionally increased the expression of CD40 by microglial cells and soluble CD40L significantly promoted cell responses to IFN-gamma during a 6-h incubation period by enhancing the activation of MAPK and IkappaB-alpha signaling pathways. We additionally found that the effect of IFN-gamma and its synergy with CD40L on mFPR2 expression in microglia was mediated in part by TNF-alpha. Our results suggest that IFN-gamma and CD40L, two host-derived factors with increased concentrations in inflammatory central nervous system diseases, may profoundly affect microglial cell responses in the pathogenic process in which mFPR2 agonist peptides are elevated. 相似文献
19.
Relationship between cell kinetics and apoptotic effects in TNF-alpha and IFN-gamma-treated human tumour cell lines. 总被引:4,自引:0,他引:4
H Baisch 《European cytokine network》2001,12(4):604-613
TNF-alpha is a potent inducer of apoptosis and also affects the transit of cells through the phases of the cell cycle. It is thought that the proliferation signalling pathway is related to the apoptosis pathway, the details of this cross-talk are not yet fully understood. In this report, the effects of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on human tumour cell lines with respect to proliferation and apoptosis are examined. The TNF-alpha-sensitive cell lines Me-180 and MCF-7, the resistant cell line TCC-Sup, and the intermediate line 5637 were used. After a one day treatment, the transit through all phases of the cell cycle slowed down and after 3 days stopped completely, as measured with the BrdU-assay and flow cytometry. During the same time however, the levels of c-Myc and Ki-67 expression and the number of cells becoming apoptotic increased. Combined treatment with TNF-alpha and IFN-gamma augmented both the effects on the cell cycle and on apoptosis in the sensitive lines, and had only a minor additional effect on the resistant cell line as compared to single TNF-alpha treatment. The cells becoming apoptotic detached from the culture flask bottom and floated in the medium. Several apoptosis assays were used to prove that the floating cells were indeed apoptotic. As a subsidiary result of receptor measurement, we observed complexes of TNF-alpha receptors I with TNF-alpha receptors II using the related blocking antibodies and I125-TNF-alpha as ligand. The association of proliferative parameters and apoptosis became obvious by plotting the levels of c-Myc expression versus remaining live cells after apoptotic cells were detached. Our data revealed a good linear correlation indicating that high levels of c-Myc render cells sensitive to apoptosis, independent of the treatment, TNF-alpha alone or TNF-alpha in combination with IFN-gamma. The quantitative linear correlation may point to a threshold mechanism. 相似文献
20.
We examined the effects of berberrubine, a protoberberine alkaloid, on interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) expression in a human retinal pigment epithelial cell line (ARPE-19) stimulated with interleukin-1beta (IL-1beta) or tumor necrosis factor alpha (TNF-alpha). ARPE-19 cells were cultured to confluence. Berberrubine and IL-1beta or TNF-alpha were added to the medium. IL-8 and MCP-1 protein concentrations were measured using enzyme-linked immunosorbent assay. IL-8 and MCP-1 mRNA were measured by real time polymerase chain reaction. Nuclear factor kappaB (NF-kappaB) translocation was examined by immunofluorescent staining/microscopy. Berberrubine dose-dependently inhibited IL-8 and MCP-1 protein levels in the media and mRNA expression of the cells stimulated with IL-1beta or TNF-alpha. Immunofluorescent staining/microscopy of NF-kappaB in the nucleus of unstimulated cells was faint (51+/-14 arbitrary units). Fluorescein was dense (215+/-42 or 170+/-24 arbitrary units, respectively) 30 min after stimulation with IL-1beta or TNF-alpha and was decreased to 62+/-18 or 47+/-16 arbitrary units, respectively, by berberrubine. Berberrubine dose-dependently inhibited IL-8 and MCP-1 expression and protein secretion induced by IL-1beta or TNF-alpha. Possibly, the effect on chemotactic factors may be via suppression of NF-kappaB translocation. 相似文献