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PubMed1995

Phenotypic modulation of rat glomerular visceral epithelial cells by culture substratum.

The interaction of cells with their supporting extracellular matrix influences cellular phenotype, cellular proliferation, protein synthetic profile, and specific gene activation. To examine the ability of culture substratum to modulate the phenotype expressed by glomerular epithelial cells (GEC) in culture, GEC were grown on plastic culture plates coated with collagen gels (Type I collagen, Vitrogen) or a complex matrix from the Englebreth-Holm-Swarm tumor (Matrigel). Cultures were examined by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). On untreated plastic, GEC grew in a random pattern. Cells were flat and thin with many filamentous processes. When grown on collagen I gels, GEC grew to confluence as a tight monolayer with typical cobblestone appearance. These cells demonstrated surface microvilli and a central cilium. TEM showed an epithelial appearance with tight junctions. When plated on the surface of Matrigel, GEC formed nests of cells that gradually burrowed into the gel. Proliferation on this matrix was extremely slow. TEM demonstrated that there are surface projections that abut the matrix and that the nests of cells are hollow with a central lumen. SEM demonstrated nests of cells that formed a sphere. Surface microvilli were not as abundant as cells grown on Vitrogen, and cilia were not seen. Cells could be removed from one surface, plated onto another, and would shift phenotype to that observed for subcultures primarily plated onto that surface. Cells on each complex substrate, as well as GEC plated on tissue culture plates coated with individual matrix proteins were biosynthetically labeled with (35S)methionine. The profile and rate of protein synthesis were modified by the plating substrate. These observations demonstrate that rat GEC can be induced to display variable phenotypes in culture that are determined by the plating substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

Hypoxia induces vascular endothelial growth factor gene and protein expression in cultured rat islet cells.

The formation of new microvasculature by capillary sprouting at the site of islet transplantation is crucial for the long-term survival and function of the graft. Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen with potent angiogenic and vascular permeability-inducing properties, may be a key factor in modulating the revascularization of islets after transplantation. In this study, we examined the gene expression of VEGF mRNA in three tumor cell lines and in isolated whole and dispersed rat islets in vitro by Northern blot hybridization in normoxic (5% CO2, 95% humidified air) and hypoxic (1% O2, 5% CO2, 94% N2) culture conditions. Increased expression of VEGF mRNA was observed in beta-TC3, RAW 264.7, and IC-21 tumor cell lines when subjected to hypoxia. With isolated whole islets in normoxic culture, a threefold increase in VEGF mRNA (P < 0.001) was seen at 48 h as compared with freshly isolated islets. This response was similar to the 3.8-fold increase observed with islets subjected to hypoxia. Dispersed rat islet cell clusters cultured on Matrigel for 24 h under hypoxic conditions showed a 3.4-fold increase (P < 0.01) in VEGF mRNA compared with those cultured in normoxia. This correlated with increased VEGF secretion as determined by enzyme-linked immunosorbent assay. Immunohistochemical studies revealed the presence of increased expression of VEGF protein near the center of islets after 24 h of normoxic culture. Islet cell clusters on Matrigel showed intense cellular localization of VEGF in both beta-cells and non-beta-cells. These findings suggest that rat islet cells, when subjected to hypoxia during the first few days after transplantation, may act as a major source of VEGF, thereby initiating revascularization and maintaining the vascular permeability of the grafted islets.

Animals↗
PubMed2006

Pulmonary artery adventitial fibroblasts cooperate with vasa vasorum endothelial cells to regulate vasa vasorum neovascularization: a process mediated by hypoxia and endothelin-1.

The precise cellular and molecular mechanisms regulating adventitial vasa vasorum neovascularization, which occurs in the pulmonary arterial circulation in response to hypoxia, remain unknown. Here, using a technique to isolate and culture adventitial fibroblasts (AdvFBs) and vasa vasorum endothelial cells (VVECs) from the adventitia of pulmonary arteries, we report that hypoxia-activated pulmonary artery AdvFBs exhibited pro-angiogenic properties and influenced the angiogenic phenotype of VVEC, in a process of cell-cell communication involving endothelin-1 (ET-1). We demonstrated that AdvFBs, either via co-culture or conditioned media, stimulated VVEC proliferation and augmented the self-assembly and integrity of cord-like networks that formed when VVECs where cultured on Matrigel. In addition, hypoxia-activated AdvFBs produced ET-1, suggesting a paracrine role for this pro-angiogenic molecule in these processes. When co-cultured on Matrigel, AdvFBs and VVECs self-assembled into heterotypic cord-like networks, a process augmented by hypoxia but attenuated by either selective endothelin receptor antagonists or oligonucleotides targeting prepro-ET-1 mRNA. From these observations, we propose that hypoxia-activated AdvFBs exhibit pro-angiogenic properties and, as such, communicate with VVECs, in a process involving ET-1, to regulate vasa vasorum neovascularization occurring in the adventitia of pulmonary arteries in response to chronic hypoxia.

Animals↗
PubMed1991

Growing human corneal epithelium on collagen shield and subsequent transfer to denuded cornea in vitro.

Three fundamental in vitro experiments have been done in the present report: 1) comparison of three different nutrient media on their abilities to culture and passage the human corneal epithelial cells; 2) evaluation of the ability of extracellular matrix material to promote the growth of cultured human corneal epithelium on collagen corneal shields; and 3) determination of the feasibility of the shield to serve as a carrier for the transfer of cultured cells to allogeneic, denuded corneal surface in vitro. Primary cultures of human corneal epithelium were established from explants which were obtained from limbal and peripheral corneal tissue by three different nutrient media respectively: KGM (Keratinocyte Growth Medium), SHEM (Supplemental Hormonal Epithelial Medium), and one combination of the two media (KGM/SHEM). We found the KGM/SHEM combination to be more favorable because morphology was better preserved, the proliferation rate increased five-fold over the 14 days observed time course, and we were able to subculture the tissue for at least three passages. With this combined medium, a suspension of cultured corneal epithelial cells (5 x 10(5)/ml) was seeded onto either the concave surface of collagen corneal shields or onto shields which had been coated with extracellular matrix materials (Matrigel or type IV collagen). The cells attached readily to all the coated shields (20/20) but to only a few of the uncoated shields (3/10), and formed a stratified tissue (2 to 3 layers) within seven days once the cells attached. However, the cells on the shields coated with Matrigel failed to become confluent under these conditions. The stratified tissue on type IV collagen coated shields could then be subsequently transferred to denuded human corneal stroma in organ culture by placing them together and incubating for 2-7 days. After that, histologic examinations showed that the epithelial cells had attached tightly to the recipient stromal surface, even after the removal of the collagen shield.

Biological Dressings↗
PubMed2003

Increased invasive capacity of connexin43-overexpressing malignant glioma cells.

OBJECT: Malignant glioma cells, similar to astrocytes, express connexin43 (Cx43) universally but at widely varied levels. Data from previous studies have demonstrated that malignant glioma cells form functional gap junction channels among themselves as well as with astrocytes and that such a communication has the potential to modulate the phenotypic characteristics of astrocytes. Recently, gap junctions have been demonstrated to play a role in the invasive phenotype of malignant gliomas. In this study, the authors have further investigated the motility and invasion ability of Cx43-overexpressing and Cx43-deficient malignant glioma cells. METHODS: Using a standard invasion system of a Matrigel transwell invasion chamber, the authors found that the number of Cx43-transfected C6 glioma cells (C6-Cx43 cells) migrating through the Matrigel-coated membrane was similar to that of mock-transfected control cells (C6-mock cells) during the first 24 hours, but increased significantly thereafter. When these cells were cocultured with astrocytes, the number of invading C6-Cx43 cells was more than threefold greater than the number of invading C6-mock cells. Results of an in vitro cell motility assay also demonstrated that C6-Cx43 cells were more motile and scatter-active than C6-mock cells. Furthermore, zymographic analysis of MMPs, an important determinant in glioma invasion, demonstrated that the amounts of MMP-2 and MMP-9 in culture medium collected from C6-Cx43 cells were orders of magnitude higher than those from C6-mock cells. In addition, BB-94, a synthetic MMP inhibitor, significantly inhibited C6-Cx43 cell invasion. CONCLUSIONS: The overexpression of gap junction proteins in glioma cells and the intercellular communication between tumor and nontumor glia cells may play important roles in the facilitation of glioma cell invasion.

Animals↗
PubMed2004

Peripheral nerve regeneration by transplantation of bone marrow stromal cell-derived Schwann cells in adult rats.

OBJECT: Bone marrow stromal cells (BMSCs) can be induced to form Schwann cells by sequentially treating the cells with beta-mercaptoethanol and retinoic acid, followed by forskolin and neurotrophic factors including heregulin. In this study the authors made artificial grafts filled with BMSC-derived Schwann cells (BMSC-DSCs) and transplanted them into the transected sciatic nerve in adult rats to evaluate the potential of BMSCs as a novel alternative method of peripheral nerve regeneration. METHODS: The BMSC-DSCs were suspended in Matrigel and transferred into hollow fibers (12 mm in length), which were transplanted into the transected sciatic nerve in adult Wistar rats. Six months after cell transplantation, electrophysiological evaluation and walking track analysis were performed. Results of these studies showed significant improvement in motor nerve conduction velocity and sciatic nerve functional index in the BMSC-DSC-transplanted group compared with the control group (Matrigel graft only). Immunohistochemical study data demonstrated that transplanted BMSCs labeled with retrovirus green fluorescent protein were positive for P0 and myelin-associated glycoprotein and had reconstructed nodes of Ranvier and remyelinated regenerated nerve axons. The number of regenerated axons in the axial section of the central portion of the graft was significantly greater in the transplanted group. Although BMSCs can differentiate into several types of cells, tumor formation did not occur 6 months after engraftment. CONCLUSIONS: Results in this study indicate that BMSC-DSCs have great potential to promote regeneration of peripheral nerves. The artificial graft made with BMSC-DSCs represents an alternative method for the difficult reconstruction of a long distance gap in a peripheral nerve.

Animals↗
PubMed2003

Axonal regeneration across long gaps in silicone chambers filled with Schwann cells overexpressing high molecular weight FGF-2.

Basic fibroblast growth factor (FGF-2) has been shown to enhance the survival and neurite extension of various types of neurons including spinal ganglion neurons. In addition, endogenous FGF-2 and FGF receptors are upregulated following peripheral nerve lesion in ganglia and at the lesion site. FGF-2 protein is expressed in different isoforms (18 kDa, 21 kDa, 23 kDa) and differentially regulated after nerve injury. In the rat we analyzed the regenerative capacity of the high molecular weight (HMW) FGF-2 isoforms (21/23 kDa) to support the regeneration of the axotomized adult sciatic nerve across long gaps. The nerve stumps were inserted into the opposite ends of a silicone chamber resulting in an interstump gap of 15 mm. Silicone tubes were filled with Matrigel or a mixture of Schwann cells (SC) and Matrigel. SC were prepared from newborn rats and transfected to overexpress HMW FGF-2. Four weeks after the operation procedure, channels were analyzed with regard to tissue cables bridging both nerve stumps and myelinated axons distal to the original proximal nerve stump. Peripheral nerves interposed with HMW Schwann cells displayed significantly enhanced nerve regeneration, with the greatest number of tissue cables containing myelinated axons and the highest number of myelinated axons. These results suggest that a cellular substrate together with a source of a trophic factor could be a promising tool to promote nerve regeneration and, therefore, become useful also for a clinical approach to repair long gaps.

Animals↗
PubMed2003

Effect of adhesion or collagen molecules on cell attachment, insulin secretion, and glucose responsiveness in the cultured adult porcine endocrine pancreas: a preliminary study.

The effect of either adhesion or collagen molecules on cell attachment, insulin secretion, and glucose responsiveness was investigated in adult porcine pancreatic endocrine (PE) cells that were cultured for a longer term in vitro. Six different types of molecules--laminin, fibronectin, poly-L-lysine (PLL), type I collagen, gelatin, and Matrigel--were used. Approximately 2.0 x 10(5) cells per dish of each molecule type were cultured for 4 weeks. In the laminin group, the insulin accumulation was maintained at a significantly higher level than in the control group at 4 weeks of culture, and glucose-stimulated insulin secretion and the insulin-positive rate were also higher than in the control group. In the Matrigel group, islet-like cell clusters were formed, but insulin accumulation rapidly decreased at 3-4 weeks of culture. A large number of PE cells attached tightly and spread in the fibronectin group until the fourth week of culture, but their function was not better than those in the control group. In the PLL and gelatin groups, the PE cell function was not significantly different from that of the control group. In the type I collagen group, insulin secretion was inferior to that of the other groups. The results of this study suggest that laminin is the most suitable extracellular matrix for the long-term culture preservation of PE cells.

Animals↗
PubMed1991

Involvement of urokinase and its receptor in the invasiveness of human prostatic carcinoma cell lines.

We have investigated the role of urokinase (UK) and its cell-surface receptor in determining the invasiveness of prostate cancer cells. Three human cell lines, DU-145, PC-3 and LNCaP, that differ in androgen-responsiveness and growth characteristics, were tested. Analysis of the conditioned medium by an enzyme-linked immunosorbent assay showed secretion of UK by DU-145 (63 ng/mL/10(6) cells/48 hr) and PC-3 (682 ng/mL/10(6) cells/48 hr), but absence of secretion by LNCaP cells. Western blot analysis and enzyme activity assay of the conditioned medium confirmed these results. Scatchard analysis of radioligand binding with acid pretreated cells showed the presence of a single population of high affinity UK receptors on DU-145 cells (93,000 sites/cell, Kd = 0.9 nM) and PC-3 cells (25,000 sites/cell, Kd = 1.0 nM) but not on LNCaP cells. DU-145 and PC-3 cells were found to be highly invasive in in vitro invasion assays: 4.5 +/- 0.5% and 6.5 +/- 0.5%, respectively, of total tumor cells (approximately 2 x 10(5)) had penetrated reconstituted basement membrane (Matrigel) in a 72 hr incubation in serum-free growth medium. Under similar conditions, less than 0.25% LNCaP cells invaded Matrigel. The data indicate that androgen unresponsive, aggressive prostate tumor cells of high metastatic potential, DU-145 and PC-3, secrete UK and display cell-surface UK receptors, fully charged with the protease. Conversely, relatively indolent LNCaP cells of low metastatic potential do not secrete UK nor do they possess its binding sites. UK receptor antagonists, UK 12-32 and UK 6-135, which compete with labeled UK for binding to prostatic cells but do not inhibit cellular proliferation or UK secretion, markedly reduced DU-145 and PC-3 cell invasion (80-85% inhibition), thereby suggesting an important role of receptor-bound UK in prostate tumor cell invasion.

Basement Membrane↗
PubMed1991

Effects of epidermal growth factor on growth response, morphology, and invasive potential of human endometrial carcinoma cell line RL95-2.

Exposure of RL95-2 human endometrial adenosquamous carcinoma cells of early passage (less than 30 passages) and late passage (greater than 250 passages) to epidermal growth factor (EGF) resulted in density- and concentration-dependent effects. At low seeding density, EGF (20 nM) inhibited the growth of early passage cells, whereas at high seeding density, 4.98 nM and 20 nM concentrations of EGF stimulated their growth. Furthermore, the growth of late passage cells was stimulated by 0.0166 nM EGF and inhibited by 4.98 nM and 20 nM EGF at both seeding densities. EGF (20 nM) caused marked morphological changes of both passages at the low seeding density. Inhibition of invasion of both passages through Matrigel-coated filters was seen at low seeding density, while at the high seeding density, EGF enhanced invasiveness. At high seeding density, EGF stimulated an increase in urokinase type plasminogen activator activity, which may have augmented the ability of cells to degrade the extracellular matrix. In addition, the ability of high seeding density cells of both passages to adhere to matrigel after EGF treatment correlated with invasiveness.

Adenocarcinoma↗
PubMed2004

Effects of c9,t11-conjugated linoleic acid on adhesion of human gastric carcinoma cell line SGC-7901.

AIM: To investigate the effect of c9,t11-conjugated linoleic acid (c9,t11-CLA) on the adhesion of human gastric carcinoma cell line (SGC-7901). METHODS: SGC-7901 cells were at first treated with different concentrations (25, 50, 100, 200 micromol/L) of c9,t11-CLA and 1 mL/L ethanol (as a negative control) for 24 h. Using adhesion assay and Western blot, we investigated the ability of SGC-7901 cells to adhere to intracellular matrix and examined the expression of E-cadherin (ECD), alpha-catenin, intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in these cells. RESULTS: The attachment rate to laminin of SGC-7901 cells treated with different concentrations of c9,t11-CLA (0, 25, 50, 100, and 200 micromol/L) was 100.0+/-3.3, 95.7+/-4.0, 89.2+/-4.6, 87.9+/-6.1, and 65.9+/-5.8, respectively. The attachment rate to fibronectin was 100.0+/-4.7, 96.8+/-3.8, 94.5+/-4.1, 76.5+/-4.3, and 61.8+/-4.8, respectively. The attachment rate to Matrigel was 99.9+/-6.6, 91.4+/-6.8, 85.5+/-7.4, 79.3+/-5.6, and 69.6+/-5.1, respectively. Besides, c9,t11-CLA could increase the level of ECD and alpha-catenin, and decrease the level of ICAM-1 and VCAM-1 in SGC-7901 cells. CONCLUSION: c9,t11-CLA can reduce the adhesion of human gastric carcinoma cells to laminin, fibronectin and Matrigel. c9,t11-CLA can increase the level of ECD and alpha-catenin, and decrease the level of ICAM-1 and VCAM-1 in human gastric carcinoma cells.

Animals↗
PubMed2004

Effects of mifepristone on invasive and metastatic potential of human gastric adenocarcinoma cell line MKN-45 in vitro and in vivo.

AIM: To investigate the effects of mifepristone on the invasive and metastatic potential of human gastric adenocarcinoma cell line MKN-45 and its mechanisms. METHODS: After incubation with various concentrations of mifepristone (5, 10, 20 micromol/L), the adhesion to artificial basement membrane, Matrigel, and the migration of MKN-45 cells were assayed using MTT assay and Transwell cell culture chambers, respectively. Enzyme- linked immunoabsorbent assay (ELISA) and flow cytometry were used to determine the expression of vascular endothelial growth factor (VEGF) and integrin beta3 in the cells. After subcutaneous transplantation of MKN-45 cells in nude mice, mifepristone (50 mg/kg.d) was administrated subcutaneously for 8 wk to assess its effects on tumor metastasis. Immunohistochemical analysis was used to detect the expression of VEGF and microvascular density (MVD) in xenografted tumors. RESULTS: Mifepristone dose-dependently inhibited the heterotypic adhesion to Matrigel of MKN-45 cells. The inhibition was accompanied by a significant down-regulation of integrin beta3 expression in the cells. After incubation with 5, 10, 20 micromol/L mifepristone, the number of migrated MKN-45 cells was 72+/-8, 50+/-6, 41+/-5 in experiment group, and 94+/-16 in control group (P<0.01). Meanwhile, secreted VEGF protein of MKN-45 cells in mifepristone-treated group (14.2+/-2.9, 8.9+/-3.1, 5.4+/-2.1 ng/g per liter) was significantly lower than that in control group (22.7+/-4.3 ng/g per liter, P<0.01). In vivo, mifepristone decreased the number of metastatic foci in lungs of nude mice and down-regulated the expression of VEGF and MVD in the xenograted tumors. CONCLUSION: Mifepristone can effectively inhibit the invasive and metastatic potential of human gastric adenocarcinoma cell line MKN-45 in vitro and in vivo through inhibition of heterotypic adhesion to basement membrane, cell migration and angiogenesis.

Adenocarcinoma↗
PubMed2005

Mechanism of metastasis by membrane type 1-matrix metalloproteinase in hepatocellular carcinoma.

AIM: To investigate the precise role of membrane type 1-matrix metalloproteinase (MT1-MMP) in hepatocellular carcinoma (HCC) metastasis. METHODS: Human HCC cells Hep3B with overexpression of MT1-MMP were established by stable transfection, and compared with control cells carrying the empty vector. Cells were examined in vivo for their differences in the metastatic ability of athymic nude mice, and analyzed in vitro for their differences in invasion ability by invasion chamber coated with Matrigel, adhesion towards collagen I and migration through culture chamber. Cell proliferation and apoptosis in adherent and suspension status were evaluated by MTT and flow cytometry analysis. RESULTS: We found that overexpression of MT1-MMP could increase intrahepatic metastasis in nude mice with orthotopic implantation of HCC cells (incidence of 100% [MT1-MMP transfectants] vs 40% [vector control transfectants], P<0.05). MT1-MMP could also enhance cell invasion through Matrigel (107.7 vs 39.3 cells/field, P<0.001), adhesion towards matrix (0.30 vs 0.12 absorbance unit at 540 nm, P<0.001), cell migration (89.3 vs 39.0 cells/field, P<0.001), and cell proliferation (24.3 vs 40.5 h/doubling, P<0.001). We also observed that MT1-MMP supported cell survival (71.4% vs 23.9%, P<0.001) with reduced apoptosis (43.7% vs 51.0%, P<0.05) in an attachment-free environment. CONCLUSION: MT1-MMP overexpression could enhance metastasis. In addition to its active role in matrix degradation during tumor invasion, MT1-MMP enhances tumor cell survival upon challenge of detachment, which is important during metastasis when cells enter the circulation.

Animals↗
PubMed2006

Alphastatin downregulates vascular endothelial cells sphingosine kinase activity and suppresses tumor growth in nude mice bearing human gastric cancer xenografts.

AIM: To investigate whether alphastatin could inhibit human gastric cancer growth and furthermore whether sphingosine kinase (SPK) activity is involved in this process. METHODS: Using migration assay, MTT assay and Matrigel assay, the effect of alphastatin on vascular endothelial cells (ECs) was evaluated in vitro. SPK and endothelial differentiation gene (EDG)-1, -3, -5 mRNAs were detected by reverse transcription-polymerase chain reaction (RT-PCR). SPK activity assay was used to evaluate the effect of alphastatin on ECs. Matrigel plug assay in nude mice was used to investigate the effect of alphastatin on angiogenesis in vivo. Female nude mice were subcutaneously implanted with human gastric cancer cells (BGC823) for the tumor xenografts studies. Micro vessel density was analyzed in Factor VIII-stained tumor sections by the immunohistochemical SP method. RESULTS: In vitro, alphastatin inhibited the migration and tube formation of ECs, but had no effect on proliferation of ECs. RT-PCR analysis demonstrated that ECs expressed SPK and EDG-1, -3, -5 mRNAs. In vivo, alphastatin sufficiently suppressed neovascularization of the tumor in the nude mice. Daily administration of alphastatin produced significant tumor growth suppression. Immunohistochemical studies of tumor tissues revealed decreased micro vessel density in alphastatin-treated animals as compared with controls. CONCLUSION: Downregulating ECs SPK activity may be one of the mechanisms that alphastatin inhibits gastric cancer angiogenesis. Alphastatin might be a useful and relatively nontoxic adjuvant therapy in the treatment of gastric cancer.

Animals↗
PubMed2006

Occurrence of cGMP/nitric oxide-sensitive store-operated calcium entry in fibroblasts and its effect on matrix metalloproteinase secretion.

AIM: To examine the existence of Nitric oxide/cGMP sensitive store-operated Ca(2+) entry in mouse fibroblast NIH/3T3 cells and its influence on matrix metalloproteinase (MMP) production and adhesion ability of fibroblasts. METHODS: NIH/3T3 cells were cultured. Confocal laser scanning microscopy was used to examine the existence of thapsigargin-induced store-operated Ca(2+) entry in fibroblasts. Gelatin zymography and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) were employed to detect the involvement of [Ca(2+)]i and NO/cGMP in MMP secretion. The involvement of NO/cGMP-sensitive Ca(2+) entry in adhesion was determined using matrigel-coated culture plates. RESULTS: 8-bromo-cGMP inhibited the thapsigargin-induced Ca(2+) entry in 3T3 cells. The cGMP-induced inhibition was abolished by an inhibitor of protein kinase G, KT5823 (1 micromol/L). A similar effect on the Ca(2+) entry was observed in 3T3 cells in response to a NO donor, (+/-)-S-nitroso-N-acetylpenicillamine (SNAP). The inhibitory effect of SNAP on the thapsigargin-induced Ca(2+) entry was also observed, indicating NO/cGMP-regulated Ca(2+) entry in 3T3 cells. Results of gelatin zymography assay showed that addition of extracellular Ca(2+) concentration induced MMP release and activation in a dose-dependent manner. RT-PCR also showed that cGMP and SNAP reduced the production of MMP mRNA in 3T3 cells. Experiments investigating adhesion potentials demonstrated that cGMP and SNAP could upgrade 3T3 cell attachment rate to the matrigel-coated culture plates. CONCLUSION: NO/cGMP sensitive store-operated Ca(2+) entry occurs in fibroblasts, and attenuates their adhesion potentials through its influence on MMP secretion.

Animals↗
PubMed2000

Phorbol 12-myristate 13-acetate induces alteration in mucin gene expression and biological properties of colon cancer cells.

Phorbol esters such as phorbol 12-myristate 13-acetate (PMA) have been reported to modulate diverse cellular responses through signal transduction pathways including the protein kinase C (PKC) pathway. In the present study, we sought to determine the effect of PMA on mucin gene expression and on the biological properties of a human colon cancer cell line, HM3. The cells were treated for 8 and 24 h with various concentrations of PMA and total RNA was extracted and Northern and slot blot analyses were carried out using MUC2, MUC3 and MUC5AC mucin cDNA probes to assess the steady state levels of mRNA. Spent media were collected and the level of cancer associated carbohydrate antigens (T, Tn, sialyl Tn, sialyl Lex, and sialyl Lea) and matrix-degrading metalloproteinase (MMPs) activity were examined. Trypsinized cells were used for assessing in vitro invasion, motility and adhesion to matrigel. Our results showed that PMA caused upregulation of steady state mRNA levels of MUC2, MUC3 and MUC5AC which was inhibited after treatment with protein synthesis inhibitors. Calphostin C, a highly specific inhibitor of protein kinase C significantly inhibited the PMA induced induction of mRNA levels of MUC2, MUC3, and MUC5AC. The levels of all cancer-associated mucin carbohydrate antigens examined in the media were increased by PMA treatment. PMA also caused an increase in MMPs activity and in in vitro invasion and motility properties, but did not affect adhesion of HM3 cells to matrigel. Thus, PMA caused a significant increase in the expression of all three mucin genes through signaling pathways involving protein kinase C and increased secretion of mucin associated carbohydrate antigens. These changes were associated with increases in MMP activity as well as by increases in the invasive and motility properties of HM3 colon cancer cells. These data suggest that protein kinase C signaling pathways may be involved in mucin gene regulation and in modulating the invasive and metastatic properties of colon cancer cells.

Adenocarcinoma↗
PubMed2002

In vivo studies of the anti-tumor effects of a human prolactin antagonist, hPRL-G129R.

Previously we demonstrated that a mutated human prolactin (hPRL) with a single amino acid substitution at position 129 (hPRL-G129R) was able to inhibit human breast cancer cell proliferation via the induction of apoptosis. In this study, we report the in vivo anti-tumor effects of hPRL-G129R in nude mice bearing human breast cancer xenografts (T-47D and MCF-7). In an effort to prolong the half-life of the proteins, hPRL or hPRL-G129R were formulated with either growth factor reduced Matrigel or into slow-releasing pellets (custom made 5 mg/5 day release). Initially, nude mice inoculated (s.c.) with T-47D human breast cancer cells were treated with either hPRL or hPRL-G129R formulated with Matrigel. At the end of the 7-week study, it was found that hPRL significantly stimulated the in vivo growth of T-47D xenografts (mean tumor volume, 202 +/- 62 mm(3) as compared to 124 +/- 31 mm(3) in control mice), whereas hPRL-G129R inhibited the tumor growth (mean tumor volume, 79+/-32 mm3). The inhibitory effects of hPRL-G129R were further confirmed in a second experiment using nude mice bearing MCF-7 human breast cancer xenografts and treated with slow-releasing pellets containing hPRL-G129R. Based on these results, we believe that hPRL-G129R can be used to improve the outcome of human breast cancer treatment in the near future.

Animals↗
PubMed2006

Translocation of active heparanase to cell surface regulates degradation of extracellular matrix heparan sulfate upon transmigration of mature monocyte-derived dendritic cells.

After Ag capture and exposure to danger stimuli, maturing dendritic cells (DCs) migrate to regional lymph nodes, where antigenic peptides are presented to T lymphocytes. To migrate from peripheral tissue such as the epidermis to regional lymph nodes, Ag-bearing epidermal Langerhans cells must move through an extracellular matrix (ECM) of various compositions. The nature of their capacity to transmigrate via ECM is not well understood, although MIP-3beta and CCR7 play critical roles. We were interested in verifying whether heparanase, a heparan sulfate-degrading endo-beta-d-glucuronidase that participates in ECM degradation and remodeling, is expressed and functional in monocyte-derived DCs. Using immunohistochemistry, confocal microscopy, RT-PCR, Western blot analysis, assays for heparanase activity, and Matrigel transmigration, we show that heparanase is expressed in both nuclei and cytoplasm of immature DCs, and that gene expression and synthesis take place mainly in monocytes and early immature DCs. We also found that both nuclear and cytoplasm fractions show heparanase activity, and upon LPS-induced maturation, heparanase translocates to the cell surface and degrades ECM heparan sulfate. Matrigel transmigration assays showed a MIP-3beta-comparable role for heparanase. Because heparan sulfate glycosaminoglycans play a key role in the self-assembly, insolubility, and barrier properties of the ECM, the results of this study suggest that heparanase is a key enzyme in DC transmigration through the ECM.

Amino Acid Sequence↗