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At least 703 records · Page 39Linked to original sources
PubMed2002

Differential effects of DNA tumor virus genes on the expression profiles, differentiation, and morphogenetic reprogramming potential of epithelial cells.

The availability of cell lines that retain their differentiation programs is important for the study of differentiated cell types and the development of cell therapies. DNA tumor virus genes are often used to establish cell lines from primary culture for the analysis of cell-specific functions. To ascertain whether viral immortalizing or transforming genes differed in their effects on cellular differentiation programs, the E1A 12S (WT12S) gene of adenovirus and the large T antigen (LT) gene of SV40 were used to derive stable cell lines from primary kidney. The resultant cell types exhibited very different morphologies, growth and behavior patterns, differentiation states, and plasticities. Renal cells immortalized by LT exhibited branching tubulogenesis in response to Matrigel. This was in contrast to their behavior under normal culture conditions, wherein they were less differentiated, very nonadhesive, very rapidly growing, and transformed. These cells coexpressed adult epithelial (keratin) and embryonic mesenchymal (vimentin, osteopontin, FSP1, PAX-2, and WT1) genes. WT12S-immortalized cells grown on or in Matrigel formed cysts or tubules, consistent with their expression profiles, which consisted of both epithelial and adult kidney markers (E-cadherin, alpha-catenin, circumferential actin filaments (CAF), alkaline phosphatase, aminopeptidase M, BMP7, or podocalyxin), but not embryonic/mesenchymal markers (PAX-2 or WT1). The WT12S-expressing cells were well differentiated, adhesive, slow growing, and nontransformed. Thus, cells expressing WT12S maintained their original differentiation status and were less sensitive to reprogramming, while cells expressing LT were dedifferentiated, but had the potential for reprogramming by exogenous factors.

Animals↗
PubMed2000

Suppression of human microvascular endothelial cell invasion and morphogenesis with synthetic matrixin inhibitors. Targeting angiogenesis with MMP inhibitors.

Matrix metalloproteinases (MMPs, matrixins) are a family of zinc proteinases that digest extracellular matrix and play a very important role in normal development and pathological conditions such as cardiovascular diseases and cancer metastasis. Type IV collagenases (gelatinase A/MMP-2 and gelatinase B/MMP-9) may be critical in the early steps of angiogenesis, the digestion of basement membrane and the migration of endothelial cells from the existing blood vessels. Human dermal microvascular endothelial cells were cultured on type I collagen, type IV collagen, and reconstituted basement membrane Matrigel and differentiation was examined in the presence of potent synthetic inhibitors of MMPs. The thiol inhibitor MAG-283 had IC50 values of 480 nM and 3 nM against human interstitial collagenase (MMP-1) and MMP-2, respectively, and KI value of 2.2 nM against MMP-9. The sulfodiimine inhibitor YLL-224 had IC50 values of 180 nM, 63 nM, and 44 nM against MMP-1, -2, and -9, respectively. These inhibitors at very low micromolar concentrations inhibited cell-mediated type I collagen degradation and partially blocked cell invasion through type IV collagen. These inhibitors also suppressed endothelial differentiation, i.e., formation of capillary-like tubes on Matrigel and on type I collagen. These results suggest that collagen-degrading MMPs play an important role during the initiation of angiogenesis.

Amides↗
PubMed1998

On the possible role of reactive oxygen species in angiogenesis.

Human microvascular endothelial cells grown on a 3-D reconstituted extracellular matrix (Matrigel) spontaneously and rapidly form a capillary network of tubular structures, thus modeling part of the angiogenic cascade. Exposure of the cells at the time of plating onto Matrigel to a brief episode of hypoxia (40-60) min and subsequent reoxygenation, significantly accelerated (up to 3-fold) the rate of tubular morphogenesis, as determined by computer-aided morphometry. This effect was not dependent on activation of PKC or upregulation/release of angiogenic growth factors. Rather, hypoxia/reoxygenation (H/R), but not hypoxia alone, caused the formation of reactive oxygen species (ROS) and the activation of the nuclear transcription factor NF kappa B, both of which were inhibited by ROS-scavengers, such as pyrollidine dithiocarbamate. Tube formation was inhibited, also under normoxic conditions, by diverse ROS antagonists in a dose-dependent fashion. Our results indicate that angiogenesis is accompanied by and/or requires generation of ROS. We hypothesize that in the clinical setting of hypoxia/reoxygenation during ischemic pre-conditioning, enhanced activation of ROS-dependent intracellular signaling may accelerate the rate of neovascularization also in vivo, thus contributing to the alleviation of certain ischemic lesions.

Cell Hypoxia↗
PubMed1995

Angiogenesis as a component of epithelial-mesenchymal interactions.

Here we review the role of angiogenesis as it pertains to the interactions between the epithelium and the mesenchyme, especially during tumor growth and metastasis. We illustrate and discuss several models of angiogenesis including endothelial tube formation on Matrigel. Finally, we examine angiogenic factors using the Matrigel model and investigate several other matrix molecules for their importance in angiogenesis and epithelial/stromal interactions.

Animals↗
PubMed1996

Urokinase-type plasminogen activation in three human breast cancer cell lines correlates with their in vitro invasiveness.

In order to invade and spread cancer cells must degrade extracellular matrix proteins. This degradation is catalysed by the concerted action of several enzymes, including the serine protease plasmin. Several experimental studies have shown that inhibition of plasmin formation reduces cancer cell invasion and metastasis, indicating a critical role of this proteolytic pathway in these processes. In order to further study the role of plasmin in cancer progression, we have characterized urokinase-type plasminogen activator (uPA) mediated plasmin formation in three human breast cancer cell lines. Using monoclonal antibodies against uPA and its receptor uPAR, we have investigated the contribution of uPA and uPAR to invasive capacity in an in vitro invasion assay. MDA-MB-231 BAG cells were found to express high protein levels of uPA, uPAR and PAI-1. MDA-MB 435 BAG cells produced low amounts of uPA, PAI-1 and moderate amounts of uPAR, whereas MCF-7 BAG cells showed low levels of uPA, uPAR and PAI-1 protein. In a plasmin generation assay MDA-MB-231 BAG cells were highly active in mediating plasmin formation, which could be abolished by adding either an anticatalytic monoclonal antibody to uPA (clone 5) or an anti-uPAR monoclonal antibody (clone R3), which blocks binding of uPA to uPAR. The two other cell lines lacked the capacity to mediate plasmin formation. In the Matrigel invasion assay the cells showed activity in this order: MCF-7 BAG < MDA-MB-435 BAG < MDA-MB-231 BAG. Testing MDA-MB-231 BAG cells in the Matrigel invasion assay revealed that invasion could be inhibited in a dose-dependent manner either by the clone 5 uPA antibody or by the clone R3 uPAR antibody, suggesting that the cell surface uPA system is actively involved in this invasive process. It is concluded that these three cell lines constitute a valuable model system for in vitro studies of the role of cell surface uPA in cancer cell invasion and has application in the search for novel compounds which inhibit mechanisms involved in uPA-mediated plasmin generation on cancer cells.

Antibodies, Monoclonal↗
PubMedDate not supplied

The effect of butyric acid and retinoic acid on invasion and experimental metastasis of murine melanoma cells.

The effect of butyric acid (BA) and all trans-retinoic acid (RA) on murine melanoma cells was investigated in vitro and in vivo. The in vitro assays included 3H-IdUR incorporation, adhesion, migration and invasion experiments. Butyric acid decreased 3H-IdUR cellular incorporation within 24 h and increased adhesion as measured by trypsin release of 3H-IdUR labelled cells from either polycarbonate (p.c.) or Matrigel-coated p.c. membranes. Migration and invasion rates after 72 h were quantified by scanning electron microscopy (SEM). The invasion barrier consisted of Matrigel-coated p.c. membranes. Butyric acid significantly enhanced migration and invasion of B16a cells, while RA significantly decreased migration and invasion of B16a and K-1735 cells. Subcutaneous administration of either BA or RA pellets significantly decreased the number of lung nodules in the experimental metastatic assay. The experimental metastatic assay is defined as a tail vein inoculation protocol followed by subsequent lung evaluation.

Animals↗
PubMed1993

U-77,863: a novel cinnanamide isolated from Streptomyces griseoluteus that inhibits cancer invasion and metastasis.

Several cinnamoyl compounds have been shown to have antitumor activities, but not specifically anti-invasive or antimetastatic effects. U-77,863 (o-methyl cinnanamide) was originally isolated from a fermentation beer of Streptomyces griseoluteus and recently synthesized (Harper, DE and Welch DR. Journal of Antibiotics, in press). Based upon some differential activities of cinnanamides, in general, and U-77,863, specifically, we tested the hypothesis that U-77,863 could inhibit invasion and metastasis of human malignant melanoma cell lines C8161 and A375M. Pretreatment of melanoma cells in vitro with nontoxic doses of U-77,863 caused a dose-, and time-dependent, reversible reduction (IC50 = 12.5 micrograms/ml) of invasion through Matrigel-coated polycarbonate filters in the Membrane Invasion Culture System (MICS). Likewise, lung colonization was significantly (P < 0.05) inhibited when tumor cells were pretreated in vitro with U-77,863 prior to intravenous injection. Structure-activity analysis revealed that the acrylamide side-chain alone and cinnanamide were only slightly less potent than U-77,863, whereas cinnamic acid analogs did not inhibit tumor cell invasion at doses < or = 100 micrograms/ml. U-77,863 inhibits invasion and metastasis without decreasing growth rates or clonogenic potential. Adhesion to endothelial monolayers or extracellular matrices (Matrigel) is not affected by exposure to U-77,863. U-77,863 presumably inhibits metastasis by inhibiting tumor cell extravasation (invasion). U-77,863 is a lead compound for developing a novel class of anti-invasive/anti-metastatic drugs.

Animals↗
PubMed1995

Expression of antisense epidermal growth factor receptor RNA downmodulates the malignant behavior of human colon cancer cells.

Human colon cancer (Moser) cells produce and secrete epidermal growth factor (EGF) and respond to EGF via an autocrine/paracrine mode through the cell surface EGF receptor (EGFR). In this report we show that EGF promotes the malignant behavior of the Moser cells in vitro in terms of growth in soft agarose and invasion of Matrigel-coated porous membranes. Expressing antisense EGFR RNA in the Moser cells (through transfection with an inducible antisense EGFR expression vector) downmodulated the expression of cell surface EGFR and EGFR mRNA with a concurrent inhibition of growth in soft agarose and invasion of Matrigel-coated membranes. In addition, the ability of exogenously applied EGF in promoting the malignant behavior of these cells was circumvented. We conclude that antisense EGFR RNA was a potent agent in circumventing the in vitro malignant properties of the Moser cells.

Cell Division↗
PubMed1995

Urokinase plasminogen activator expression by primary and HPV 16-transformed keratinocytes.

The molecular events underlying progression of human papillomavirus (HPV) 16-associated intraepithelial neoplasia to invasive cancer have not been studied in detail. Penetration of the basement membrane is an early, but poorly understood step in this process and probably involves the action of one or more metallo- and serine proteinases. Urokinase-type plasminogen activator (uPA) is a serine proteinase that has been implicated in the pathogenesis of several epithelial tumors, but its role in HPV-associated tumors is not known. To examine uPA expression by HPV 16-transformed keratinocytes in vitro, primary foreskin keratinocyte cultures were transfected by HPV 16 DNA. The primary parental cells and the HPV 16-transformed keratinocytes were studied using substrate gel zymography, Western blot analysis and an in vitro assay measuring penetration of a Matrigel artificial basement membrane. Both uPA and its inhibitor, plasminogen activator inhibitor-1 (PAI-1), were overexpressed in the HPV 16-transformed cells relative to the parental cell line. The transformed cells, but not the parental cells, were able to degrade and penetrate the Matrigel membrane and penetration was blocked by both PAI-1 and by antibodies to uPA. Our data suggest that HPV 16-induced transformation of keratinocytes is associated with upregulation of uPA expression. In conjunction with other proteinases, uPA plays an important role in the ability of HPV 16-transformed keratinocytes to penetrate artificial basement membranes.

Basement Membrane↗
PubMed1992

Quantitative evaluation of the growth of established cell lines on the surface of collagen, collagen composite and reconstituted basement membrane.

As a step in the development of a system for assessing growth of human urothelium and transitional cell carcinoma, the growth of two established cell lines on collagen-based membranes has been evaluated. HT1080 (metastatic human fibrosarcoma) and WI38 VA13 (virus-transformed human fibroblasts) were grown on substrates of collagen, collagen/hyaluronic acid or chondroitin sulphate and reconstituted basement membrane (Matrigel). Cell growth was quantified using a new fluorimetric assay utilizing carboxyfluorescein diacetate. There were differences in morphology between cells grown on collagen and those grown on polystyrene. There were, however, no differences in growth of the WI38 VA13 cells on collagen compared with polystyrene, but growth of the HT1080 cells was increased on membranes of collagen/2.5% hyaluronic acid and collagen/5% chondroitin sulphate, and decreased on Matrigel. Adequate growth on collagen substrates is dependent on cell line. The fluorimetric assay used was suitable for quantifying cell growth on such substrates.

Basement Membrane↗
PubMed1994

Differentiation state and invasiveness of human breast cancer cell lines.

Eighteen breast cancer cell lines were examined for expression of markers of epithelial and fibroblastic differentiation: E-cadherin, desmoplakins, ZO-1, vimentin, keratin and beta 1 and beta 4 integrins. The cell lines were distributed along a spectrum of differentiation from epithelial to fibroblastic phenotypes. The most well-differentiated, epithelioid cell lines contained proteins characteristic of desmosomal, adherens and tight junctions, were adherent to one another on plastic and in the basement membrane matrix Matrigel and were keratin-positive and vimentin-negative. These cell lines were all weakly invasive in an in vitro chemoinvasion assay. The most poorly-differentiated, fibroblastic cell lines were E-cadherin-, desmoplakin- and ZO-1-negative and formed branching structures in Matrigel. They were vimentin-positive, contained only low levels of keratins and were highly invasive in the in vitro chemoinvasion assay. Of all of the markers analyzed, vimentin expression correlated best with in vitro invasive ability and fibroblastic differentiation. In a cell line with unstable expression of vimentin, T47DCO, the cells that were invasive were of the fibroblastic type. The differentiation markers described here may be useful for analysis of clinical specimens and could potentially provide a more precise measure of differentiation grade yielding more power for predicting prognosis.

3T3 Cells↗
PubMed1994

Collagen induced MMP-2 activation in human breast cancer.

Matrix metalloproteinase-2 (MMP-2), a zymogen requiring proteolytic activation for catalytic activity, has been implicated broadly in the invasion and metastasis of many cancer model systems, including human breast cancer (HBC). MMP-2 has been immunolocalized to carcinomatous human breast, where the degree of activation of MMP-2 correlates well with tumor grade and patient prognosis. Using Matrigel assays, we have stratified HBC cell lines for invasiveness in vitro, and compared this to their potential for metastatic spread in nude mice. HBC cell lines expressing the mesenchymal marker protein vimentin were found to be highly invasive in vitro, and tended to form metastases in nude mice. We have further discovered that culture on collagen-I gels (Vitrogen; Vg) induces MMP-2-activator in highly invasive but not poorly invasive HBC cell lines. As seen for other MMP-2-activator inducing regimens, this induction requires protein synthesis and an intact MMP-2 hemopexin-like domain, appears to be mediated by a cell surface activity, and can be inhibited by metalloproteinase inhibitors. The induction is highly specific to collagen I, and is not seen with thin coatings of collagen I, collagen IV, laminin, or fibronectin, or with 3-dimensional gels of laminin, Matrigel, or gelatin. This review focuses on collagen I and MMP-2, their localization and source in HBC, and their relationship(s) to MMP-2 activation and HBC metastasis. The relevance of collagen I in activation of MMP-2 in vivo is discussed in terms of stromal cell: tumor cell interaction for collagen I deposition, MMP-2 production, and MMP-2-activation. Such cooperativity may exist in vivo for MMP-2 participation in HBC dissemination. A more complete understanding of the regulation of MMP-2-activator by type I collagen may provide new avenues for improved diagnosis and prognosis of human breast cancer.

Amino Acid Sequence↗
PubMed1995

Methylmercury-injury effect on tube formation by cultured human vascular endothelial cells.

The effect of methylmercury chloride (MeHg) on growth and tube formation by cultured human umbilical vein endothelial cells (HUVECs) was investigated. HUVECs were collected by enzymatic digestion with collagenase. Precultivation of HUVECs with MeHg at concentrations of 1.0-50.0 mumol/L exerted negligible effects on the viable cell number, while the viable cell number was slightly reduced at 100 mumol/L and fell to zero at concentrations exceeding 500.0 mumol/L MeHg. The viable cell number was depressed in a concentration-dependent manner. Tube formation was studied by culturing the cells on gelled basement membrane matrix (Matrigel). Treatment of HUVECs with 0.1-5.0 mumol/L MeHg for 24 h inhibited tube formation dose-dependently. Fetal bovine serum (FBS) increased tube formation in a dose-dependent manner, with half-maximum stimulation of tube formation at approximately 3.4% FBS. The length of tube formation decreased time-dependently at concentrations of 0.1 and 1.0 mumol/L MeHg. Pretreatment of Matrigel with 1 mumol/L MeHg before the cell seeding reduced the tube formation by HUVECs. These results suggest that the growth and tube formation by HUVECs is susceptible to MeHg cytotoxicity, and that MeHg could be injurious to endothelial cell function.

Cell Count↗
PubMed1997

Unstimulated human acute myelogenous leukemia blasts secrete matrix metalloproteinases.

PURPOSE: The secretion of metalloproteinases was examined, especially the 92-kDa and 72-kDa type IV collagenases/gelatinases, and their role in the degradation of reconstituted basement membrane (Matrigel) by leukemic blasts. METHODS AND RESULTS: Leukemic blasts were obtained from the peripheral blood of 11 patients diagnosed with acute myelogenous leukemia (AML). After incubation of the AML blasts in serum-free cultures, conditioned media were collected and examined by zymography. The 92-kDa gelatinase was detected in ten AML patients after 2 h and 24 h of incubation, and in five samples its activated form (83 kDa) was observed. 72-kDa gelatinase was detected in cell-conditioned media from four patients after 2 h and in media from ten patients after 24 h. Its activated forms (64-60 kDa) were observed in one of four samples after 2 h and in five of ten after 24 h. By contrast, normal peripheral mononuclear cells from healthy donors secreted only 92-kDa gelatinase after 24 h; the 72 kDa enzyme was not detectable. A specific inhibitor of metalloproteinases, 1,10-phenanthroline, significantly reduced the in vitro invasion of AML blasts in a Matrigel assay and completely inhibited gelatinolytic activity in zymography. CONCLUSIONS: We concluded that primary, unstimulated peripheral-blood AML blasts secrete metalloproteinases, which may contribute to the in vitro degradation of reconstituted basement membrane.

Adult↗
PubMed1994

Heterogeneities of attachment, chemotaxis, and protease production among clones with different metastatic potentials from a human pancreatic cancer cell line.

The present study extends our investigations into the metastatic heterogeneity among four clonal cell lines (S2-007:H, S2-013:M1, S2-020:M2, and S2-028:L) from a human pancreatic cancer cell line (SUIT-2), and extends our discussion the positive correlation between metastatic potential and the type I collagenase activity of the cells, focusing on their interaction with extracellular matrix. Ability to attach to the reconstituted basement membrane (Matrigel) was higher for clone H than clone L during an observation period of 30-60 min, whereas clones M1 and M2 were found to be intermediate in ability. In densitometric and radioactive studies, clone L exhibited the lowest collagenolytic activity against mouse and human type IV collagen, while clone H exhibited the highest activity in the densitometric study and clone M1 was the highest in the radioactive study. The production of urinary-type plasminogen activator was highest in clone L and lowest in clone H. On the other hand, tissue-type plasminogen activator was highest in clone M2 and low in both clones H and L. Clone M2 exhibited the highest chemotactic activity toward diluted Matrigel, whereas clone L had the lowest activity. On the whole, these clones showed heterogenous interactions with an extracellular matrix. It is suggested that the attachment activity to basement membrane and the type IV collagenolytic activity of the cells may be positively correlated with their metastatic potential, whereas the production of urinary-type plasminogen activator was negatively correlated, but confirmation of these findings awaits further study.

Cell Adhesion↗
PubMedDate not supplied

Long-term culture of hepatocytes from human adults.

A long-term primary human hepatocyte culture retraining liver-specific functions is important and essential for basic research and for the future development of hepatocyte-based applications. We established a normal hepatocyte culture system from excess normal tissues obtained from adult liver cancer patients who received partial liver resection. Hepatocytes were isolated after perfusion and enzymatic disaggregation, and were first maintained in hormonally defined media on a Matrigel matrix, and then transferred to collagen sandwich gel. The hepatocytes formed clusters on the Matrigel matrix and increased in size and numbers with time of culture and eventually grew into spheroids of variable sizes. After being transferred to collagen gel, the cells migrated outward from spheroids to form a monolayer with cuboidal or polygonal cell shapes with granular cytoplasm and continued to proliferate. Cellular functions specific for hepatocytes were analyzed using immunoblot assay for proteins specifically secreted by the liver cells on different days of culture. The cells secreted albumin, transferrin and alpha-fetoprotein consistently for more than 100 days, to a maximum of 150 days. Thus, we have established a long-term culture of hepatocytes from human adults, which will be useful for basic studies of liver physiology such as metabolism and morphogenesis, as well as for other applications in the study of infectious hepatitis, pharmacology, pharmacokinetics, and toxicology.

Adult↗
PubMedDate not supplied

Interleukin-6 increases vascular endothelial growth factor and angiogenesis in gastric carcinoma.

Interleukin-6 (IL-6) is a proinflammatory cytokine associated with the disease status of gastric carcinoma (GC). Vascular endothelial growth factor (VEGF) is a potent tumor angiogenic factor in GC. In this study, we attempted to clarify whether IL-6 can regulate VEGF and angiogenesis in GC. GC samples from 54 surgical specimens were subjected to immunohistochemical examination of IL-6, VEGF, and tumor microvessels, and results showed that IL-6 was positively correlated with VEGF expression and tumor vasculature. We determined VEGF expression in four GC cell lines by ELISA, revealing that GC cells can produce significant amount of VEGF with increasing dose and duration of IL-6 stimulation. Next, a luciferase reporter gene assay was employed to determine the signaling pathway driving the VEGF promoter by IL-6, which showed that the JAK/STAT pathway is involved in the stimulation of VEGF gene expression. The effects of IL-6 on angiogenesis in vitro and in vivo were evaluated by HUVEC studies and the Matrigel plug assay, respectively. Results showed that IL-6 effectively promoted HUVEC proliferation and tube formation in vitro and Matrigel plug vascularization in vivo, primarily by inducing VEGF in GC. This study provides evidence that the multifunctional cytokine, IL-6, may induce VEGF expression which increases angiogenesis in gastric carcinogenesis.

Cell Proliferation↗
PubMed1997

The invasion-MTT assay as a predictor of liver metastasis using human gastrointestinal carcinomas transplanted in nude mice.

The invasion-3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay, which evaluates invasive potential into the reconstituted basement membrane, Matrigel, was performed on 49 human gastrointestinal carcinomas transplanted in nude mice. There were 19 colorectal carcinomas, 10 pancreatic carcinomas, 10 gastric carcinomas, 8 esophageal carcinomas, and 2 bile duct carcinomas. The percent invasion (PI) value of each tumor by the invasion-MTT assay expresses the invasive rate of tumor cells into the Matrigel as a percentage. There were no significant differences in correlations between the PI values and primary tumor site, clinicopathological findings, tumor doubling time, or DNA index; however, the PI values of primary tumors and lymph nodes with liver metastases were significantly higher than those of primary tumors without liver metastasis (P < 0.05). Furthermore, the primary tumors with synchronous (P < 0.05) or asynchronous (P < 0.01) liver metastases showed significantly higher PI values compared with the primary tumors without liver metastases. These results suggest that PI is not only an independent factor to predict liver metastasis, but it also correlates closely with liver metastasis. Thus, the invasion-MTT assay for primary tumors might be clinically useful to predict liver metastasis in patients following surgery for gastrointestinal carcinomas.

Animals↗