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PubMed2005

The anti-angiogenic effect of sinomenine.

Sinomenine is an alkaloid extracted from the Chinese medicinal plant, Sinomenium acutum, which has been utilized to treat rheumatoid arthritis (RA) in China for over 2000 years. Sinomenine has been shown to mediate a wide range of pharmacological actions which includes anti-inflammatory and anti-rheumatic effects. RA has been classified as a chronic immune-mediated disease that exhibits overlapping manifestation of inflammatory, abnormal cellular and hormonal immune responses with synovial hyperplasia. Since, angiogenesis is recognized to play a critical role in the development of RA and anti-angiogenic therapy has been proposed as a new therapeutic strategy for treatment of RA, we would like to see if sinomenine possesses anti-angiogenic property. In this study, sinomenine inhibited bFGF-induced proliferation of human umbilical vein endothelial cells (HUVEC) and arrested its cell cycle in G1 phase. Sinomenine disrupted tube formation of HUVEC on Matrigel and suppressed the chemotaxis of HUVEC. In addition, sinomenine reduced neovascularization in Matrigel plug assay as well as microvascular outgrowth in rat aorta ring sprouting assay. These results suggest that sinomenine inhibited bFGF-induced angiogenesis in vitro and in vivo. As the leukocytes-endothelial adhesive interactions also play an important role in inflammation, we found that sinomenine reduced the transmigration of granulocytic differentiated HL60 cells across IL-1beta activated HUVEC monolayer. Therefore, the inhibition of leukocytes migration across blood vessel walls and the anti-angiogenic effect of sinomenine may contribute towards its therapeutic mechanisms in alleviating the pathogenesis of RA.

Angiogenesis Inhibitors↗
PubMed2006

A bone-derived mixture of TGF beta-superfamily members forms a more mature vascular network than bFGF or TGF-beta 2 in vivo.

Clinical trials of therapeutic angiogenesis for the treatment of cardiovascular ischemia have failed to meet the expectations with the use of single growth factors, namely VEGF and bFGF. We show here that a bovine bone-derived growth factor mixture (GFM) of TGFbetas, BMPs, and no more than 0.1% aFGF can initiate a dose-dependent angiogenic response in subcutaneously implanted Growth Factor Reduced Matrigel plugs that includes abundant smooth muscle actin positive (SMA+) tubes and functional CD31+, red blood cell filled, capillaries. Tube forming activity of the single factors, recombinant bFGF and bone-derived TGF-beta2, were comparable to GFM, but only the bone-derived factors were able to create a larger fraction of SMA+ tubes than Matrigel alone at an equal dose. Basic FGF formed a greater number of RBC-filled capillaries within the plugs than GFM or TGF-beta2 at the highest doses, although GFM created RBC-filled capillaries that penetrated deeper into the plugs than bFGF. However, bFGF showed the greatest number of non-cell-lined, RBC-filled pools, suggestive of vessel rupture, and the largest number of plugs showing signs of fluid accumulation in the form of large, cell-lined clefts in the implants. TGF-beta2 showed less RBC-filled pools, but a significant number of implants with signs of fluid accumulation. At high doses of GFM penetration by blood vessels and mesenchymal cells was obstructed by cartilage development within the plugs accompanied by a prominent band of SMA+ granulation tissue with abundant RBC-filled capillaries encapsulating the implants. Thus, GFM is also capable of dramatically remodeling the vascular system in the interstitial space surrounding the plug. These results show that GFM is capable of inducing the formation of a more mature vascular system than that formed by the single factors bFGF and TGFbeta-2. Natural mixtures of TGFbetas, BMPs, and FGFs may have superior clinical utility in therapeutic angiogenesis applications.

Actins↗
PubMed2006

Telomerase immortalization of human mammary epithelial cells derived from a BRCA2 mutation carrier.

A novel human mammary epithelial cell line, HME348, was established from benign breast tissue from a 44-year-old germ-line BRCA2 mutation carrier with a history of stage 1 breast cancer. Mutation analysis showed that the patient had a known 6872del4 BRCA2 heterozygous mutation. The human mammary epithelial cells passaged in culture exhibited cellular replicative aging as evidenced by telomere shortening, lack of telomerase activity, and senescence. Ectopic expression of telomerase (hTERT) reconstituted telomerase activity in these cells and led to the immortalization of the cells. When grown on glass, the majority of immortalized HME348 cells expressed ESA and p63 with a small population also expressing EMA. In three-dimensional Matrigel culture, HME348 cells formed complex branching acini structures that expressed luminal (EMA, CK18) and myoepithelial (p63, CALLA, CK14) markers. Three clones derived from this culture were also p63(+)/ESA(+)/EMA(+/-) on glass but formed similar acinar structures with both luminal and myoepithelial cell differentiation in Matrigel confirming the mammary progenitor nature of these cells. Additionally, the experimentally immortalized HME348 cells formed acini in cleared mammary fat pads in vivo. As this is the first report establishing and characterizing a benign human mammary epithelial cell line derived from a BRCA2 patient without the use of viral oncogenes, these cells may be useful for the study of BRCA2 function in breast morphogenesis and carcinogenesis.

Animals↗
PubMed2006

Novel in vitro model barriers for evaluation of the permeability of antitumor compounds, thioxanthones.

Anticancer drugs have to cross many layers of cells and the extracellular matrix to reach the tumor cells and elicit their pharmacological action. The three dimensional structure (micro-environment) of tumors poses a penetration barrier to antitumor drugs resulting in poor response. Two in vitro model barriers representing the extracellular matrix and multilayered structure of tumors were used in this study to evaluate the permeability of four structurally related antitumor compounds, thioxanthones. Matrigel was used to represent the tumor extracellular matrix and multilayered Caco-2 cells were used to represent the multilayered confluence of solid tumor. The in vitro permeability characteristics of the thioxanthones across the model barriers were correlated to their in vivo distribution. The apparent permeability coefficients of thioxanthone analogs are in the range of 2.9 x 10(-6) to 11.8 x 10(-6) cm/s across matrigel and 12.6 x 10(-6) to 24.5 x 10(-6) cm/s across Caco-2 multilayers. This high in vitro permeability of thioxanthones across the model barriers suggested their good tissue distribution in vivo. Therefore, the use of in vitro model barriers was found to predict in vivo tissue distribution for thioxanthones.

Antineoplastic Agents↗
PubMed2006

Collagen binding alpha2beta1 and alpha1beta1 integrins play contrasting roles in regulation of Ets-1 expression in human liver myofibroblasts.

Activation of hepatic stellate cells from quiescence to myofibroblast-like cells (MFBs) is a pivotal event in hepatic fibrogenesis. Plastic-cultured stellate cells (an established in vitro model of the activated phenotype) recultured on Matrigel revert to quiescence. In the present study we analyzed the molecular mechanism underlying this process, focusing on the effect of collagen receptors alpha(2)beta(1) and alpha(1)beta(1) integrin signaling on the expression of Ets-1 transcription factor and its target gene MMP1 in cultured human MFBs. Cells grown in 3-dimensional (3D) substrates (Matrigel) or collagen type I gel) markedly upregulated Ets-1 and MMP1 messages, in comparison to cells cultured on plastic. A similar effect but less intense was mimicked by stimulation of alpha(2)beta(1) or blocking of alpha(1)beta(1) integrin in cells grown on plastic. We observed increased expression of MMP1 transcripts with parallel changes in MMP1 promoter activity, and in mRNA and protein levels of upstream transcription factors Ets-1 and c-Jun. Interference with alpha(2)beta(1) and alpha(1)beta(1) integrin function in cells cultured in a 3D collagen substrate resulted in an even greater effect. Morphologically, stimulation of alpha(2)beta(1) integrin resulted in formation of multicellular networks, probably by facilitation of cell migration. Thus, we report the novel observation that in cultured human MFBs reverting to quiescence, the expression of transcription factor Ets-1 and its downstream target MMP1 can be modulated by changes in the microenvironment, which are mediated, at least in part, by the balance between collagen receptor integrin alpha(2)beta(1) and alpha(1)beta(1) activities.

Cell Differentiation↗
PubMed2006

De-differentiation of primary human hepatocytes depends on the composition of specialized liver basement membrane.

Basement membrane (BM) is a highly specialized extracellular matrix (ECM), which is associated with epithelia and endothelia. BM provide epithelia with structural support and also regulate cell behavior. The liver contains a unique ECM within the space of Disse, which consists of basement membrane constituents as well as fibrillar ECM molecules. Changes in composition of this ECM are considered detrimental for viability of hepatocytes during progression of liver disease. Mouse tumor-derived BM preparations, such as Matrigel, which are commonly used as a model for BM in vitro, differ significantly in their composition from liver BM present in vivo. In order to gain further insights into the role of BM in the regulation of hepatocyte behavior in health and disease, we generated a liver-derived basement membrane matrix (LBLM). LBLM allowed investigation of BM-hepatocyte interactions in vitro. Here we report a novel approach of generating a liver-derived basement membrane matrix by separate isolation of type IV collagen, laminin, nidogen, and heparan sulfate proteoglycans, and subsequent reconstitution into a matrix-like gel. Adhesion of primary human hepatocytes to LBLM was increased and the rate of de-differentiation was decreased compared to hepatocyte cultivation on Matrigel or type I collagen matrix. Primary human hepatocytes maintained their differentiated epithelial phenotype on LBLM isolated from normal human livers for more than 21 days, whereas they de-differentiated rapidly on LBLM isolated from cirrhotic human livers. Normal human LBLM contains a unique isoform composition of type IV collagen, namely alpha1 (IV), alpha2(IV), alpha4(IV), and alpha6(IV) chains, whereas cirrhotic LBLM contains only alpha1(IV) and alpha2(IV) isoforms, albeit present in increased amounts. These findings suggest that the composition of liver basement membrane is important for the maintenance of hepatocyte viability and provide anti-de-differentiation clues.

Aged↗
PubMed2005

Inhibition of cell invasion by indomethacin on glioma cell lines: in vitro study.

Malignant glioma invasion into the surrounding brain tissue is still a major problem for any therapeutical methods. Matrix metalloproteinases (MMPs) have been implicated as important factors in this pathological process. In this study, one of the non-steroidal anti-inflammatory drugs (NSAIDs) indomethacin was employed to investigate the effect of inhibition of cell invasion mediated by MMP-2 and MMP-9 in human malignant glioma cell lines, A172, U87MG, U251MG, and U373MG in vitro. MTT assay was firstly examined to determine non-cytotoxic dose range, then gelatin zymography, matrigel invasion assay, migration assay and MMP-2 activity assay for 24 h exposure in indomethacin were employed to assess the inhibitory effect of indomethacin. MTT assay revealed that dose with 0, 50, and 500 microM/ml were non-cytotoxic. Zymography demonstrated: (a) expression of MMP-2 and MMP-9 activity was downregulated along with elevated dose of indomethacin. (b) MMP-2 activity that changed from pro-MMP-2 to active form of MMP-2 in supernatants of cell lines could not be inhibited by indomethacin. Invasion assay disclosed that the number of invading cells through the matrigel were significantly decreased in a dose dependent manner. Migration assay indicated indomethacin did not affect cells migration. MMP-2 activity assay showed the total and active MMP-2 secretion was suppressed by 500 microM/ml of indomethacin. Our present study is the first report on inhibitive effect of indomethacin mediated by MMP-2 and MMP-9 in invasion assay of glioma cell lines. The current study suggested that non-cytotoxic level of indomethacin was able to reduce the cell invasion of malignant gliomas mediated by MMP-2 and MMP-9, but it did not affected on cell motility. It also lowered down the activity of MMP-2 and MMP-9, and could reduce of MMP-2 secretion of cell lines. Thus, high concentration of indomethacin within non-cytotoxic dose might offer a new therapeutic strategy to impair cell invasion of gliomas.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2006

Antiangiogenic effect of bile acid acylated heparin derivative.

PURPOSE: Chemically modified heparin-DOCA was prepared and found to have markedly lower anticoagulant activity than heparin. In the present study, we elucidated the antiangiogenic and antitumoral activities of heparin-DOCA derivative. METHODS: To evaluate the antiangiogenic and antitumoral effects of heparin-DOCA, capillary-like tube formation assay, Matrigel plug assay in vivo, western blotting for FGFR phosphorylation, ERK and p38 MAPK activities, tumor growth of SCC in vivo and immunostaining of blood vessels in tumor tissues were performed. RESULTS: Heparin-DOCA inhibited capillary-like tubular structures of endothelial cells and bFGF-induced neovascularizations in Matrigel plug assays. Signaling experiments showed that heparin-DOCA significantly inhibited angiogenesis by suppressing the phosphorylation of FGFR and its downstream signal pathways (ERK and p38 MAPK activities). The antiangiogenic activity of this heparin derivative was found to be closely associated with antitumoral activity in a mouse model. In addition, histological evaluations supported the inhibitory effect of heparin-DOCA on blood vessel formation in tumor tissues. CONCLUSION: Heparin-DOCA derivative exerted a significant antitumoral effect by inhibiting angiogenesis resulting from the disruption of FGF/FGFR and its downstream signal pathways, and could be applied to treat various angiogenic diseases.

Angiogenesis Inhibitors↗
PubMedDate not supplied

Enhanced myogenic differentiation by extracellular matrix is regulated at the early stages of myogenesis.

Myogenic cell lines have been used extensively in the study of skeletal muscle development, regeneration, and homeostasis. To induce myogenic differentiation, culture media composed of a wide variety of growth factors and other additives have been used. Because the diversity in these components may modulate the differentiation process differentially, we describe a differentiation protocol that does not require the introduction of any factors to the differentiation media (DM) other than those present in the growth media. By culturing C2C12 skeletal myocytes on a coating of diluted Matrigel, a soluble basement membrane, consisting of collagen IV, laminin, heparan sulfate proteoglycans, and entactin, myogenic differentiation was accomplished by mere serum reduction. Assessment of myotube formation, creatine kinase activity, myosin heavy chain-fast, and myogenin demonstrated that the kinetics and extent of myogenic differentiation were superior using this protocol, compared with a commonly used differentiation protocol, in which an extracellular matrix is not provided and the DM contains horse serum. In addition, the elevated transactivation of a troponin-I promoter reporter construct suggested that myogenesis was enhanced at the transcriptional level. Finally, assessment of genomic deoxyribonucleic acid content revealed that the Matrigel differentiation protocol resulted in lowered proliferation. This protocol may aid studies aimed at elucidating mechanisms of myogenic differentiation, where a homogeneous population of myotubes is preferred.

Animals↗
PubMedDate not supplied

Differential effects of cellular fibronectin and plasma fibronectin on ovarian cancer cell adhesion, migration, and invasion.

The main form of fibronectin (FN) encountered by tumor cells in vivo is cellular FN (cFN), which differs structurally and functionally from the commonly used plasma FN (pFN). We compared the effects of cFN and pFN on the ovarian carcinoma lines OVCAR-3 and SKOV-3 and on cultures of normal ovarian surface epithelium, which is the precursor of the epithelial ovarian carcinomas. Ovarian surface epithelial cells and SKOV-3 cells attached and spread faster on cFN than on pFN. On cFN, SKOV-3 migration was enhanced compared with pFN or plastic. In a matrigel transfilter assay, cFN strongly inhibited SKOV-3 invasion, whereas pFN did not. In contrast to SKOV-3, OVCAR-3 cells adhered faster on FN than on plastic but did not discriminate between cFN and pFN, and they did not migrate or invade matrigel either with or without FN. In both carcinoma lines, proliferation was unaffected by either FN. The results show profound differences in the responses to cFN and pFN by two invasive ovarian carcinoma lines. Because cFN is the main type that cancer cells encounter in vivo, extrapolations from culture data to in vivo events should preferably be based on studies using this form of FN.

Cell Adhesion↗
PubMed1991

Expression of extracellular matrix-degrading metalloproteinases by cultured human cytotrophoblast cells: effects of cell adhesion and immunopurification.

In vitro, invasion of basement membrane by human trophoblast can be blocked by metalloproteinase inhibitors. The purpose of our study was to characterize these enzymes by zymography, to define their cellular origin. First-trimester cytotrophoblast cells were prepared according to the method of Kliman et al. Half of the cell suspension was further purified with an antibody to leukocyte common antigen (CD45). Cytotrophoblast cells (immunopurified or not) were incubated in Dulbecco's modified Eagle's medium on different matrices. Progesterone, total human chorionic gonadotropin, and free beta-human chorionic gonadotropin were measured in the supernatant by radioimmunoassay or enzyme immunoassays. Secreted (in the medium) and cell-bound proteases were characterized by zymography on sodium dodecyl sulfate-polyacrylamide gels containing gelatin. Cytotrophoblast cell preparations contained 12% to 34% leukocyte common antigen-positive cells before and 0% after immunopurification. Large zones of digested matrices were observed after 48 hours of culture on Matrigel or rat tail collagen but not on agarose. Cells secreted progesterone, human chorionic gonadotropin, and free beta-human chorionic gonadotropin in vitro, but no difference was observed among cells grown on different matrices or between immunopurified and nonimmunopurified cells. By zymography, seven gelatin-degrading enzymes were seen in culture supernatants and five of them were present in cell lysates. The molecular weights of these proteases ranged from 59 to 230 kd. Immunopurification eliminated three of these enzymes, so they were clearly produced by bone marrow-derived cells (leukocyte common antigen positive) contaminating the cytotrophoblast cell preparation. Cells grown on Matrigel express a unique 59 kd gelatinase that was not seen in the supernatants of cells grown on other matrices. Zymography in the presence of inhibitors showed that these enzymes were neutral metalloproteinases, which might be responsible for the observed extracellular matrix degradation.

Cell Adhesion↗
PubMed1992

Motility of IL-2-stimulated lymphocytes in neutral and acidified extracellular matrix.

The migration of lymphocytes through extracellular matrix (ECM) is an essential feature of the infiltration process. In the course of their extravasation into poorly perfused neoplastic lesions, lymphocytes often encounter regions of acidified ECM. This study was designed to determine whether lymphocyte adherence and motility in ECM are influenced by ambient pH. Murine splenic lymphocytes, activated by culture with high-titer IL-2, were allowed to migrate into three-dimensional gels of Type I collagen, a major component of interstitial stroma, or into Matrigel, a basement membrane model. After 18 hr at pH 7.1, the leading cell front traveled a mean distance of approx 475 microM into Type I collagen gel. Approx 50% of the cells remained nonadherent, 25% adhered to the gel surface, and 25% were motile (penetrated beneath the surface). At pH 6.7, the leading-front distance increased significantly, by a factor of 1.4X, but there was little change in the proportion of cells exhibiting nonadherence, surface adherence, or motility. The relative motilities of CD3+ and AsGM1+ subsets were also unaltered. It therefore appears that acidification of collagen matrix increases the locomotory activity of motile lymphocytes, but causes little recruitment of nonmotile lymphocytes into the motile pool. Similar results were obtained in experiments with Matrigel. The increased motility observed at pH 6.7 did not reflect breakdown or relaxation of matrix lattices, as measured by the passive diffusion of latex beads of defined diameter. Preincubation of lymphocytes at pH 6.7 did not alter their subsequent motility in pH 7.1 gels. The findings establish ambient pH as a microenvironmental variable which can influence lymphocyte migration through ECM. The weak acidity characteristic of certain tumor microenvironments may be a factor which encourages lymphocyte infiltration through tissue matrix. Treatments which alter intratumor pH could potentially be used to manipulate the infiltration process for immunotherapeutic benefit.

Animals↗
PubMed1992

Effects of the extracellular matrix on fetal choroid plexus epithelial cells: changes in morphology and multicellular organization do not affect gene expression.

We have developed a primary culture system for fetal mouse choroid plexus epithelial cells which maintains their differentiated phenotype. When grown on a reconstituted basement membrane substrate (Matrigel) epithelial cells formed aggregates which became embedded in the matrix and developed into characteristic and highly reproducible multicellular vesicular structures. These vesicles consisted of a squamous layer of epithelial cells with extensive attachment to the matrix substrate, surrounding a fluid-filled lumen. Electron microscopy showed that cells comprising these vesicles had a high degree of membrane specialization and polarized morphology which in many respects mimicked the in vivo morphology. Biochemical analyses demonstrated that under these culture conditions the tissue-specific pattern of gene expression of fetal choroid plexus epithelium was maintained. After 6 days in culture these cells contained approximately the same amount of transthyretin mRNA as the 12.5-day choroid plexus in vivo, and the level of total RNA per cell, which is proportional to the protein synthetic capability of the cells, was also maintained. The pattern of protein secretion was also very similar to that generated by fetal mouse choroid plexus cells in vivo. In contrast choroid plexus epithelial cells attached poorly to collagen I gels. Heterogeneous aggregates were formed in which cell-cell interactions were more extensive than cell-substrate interactions, and in no cases was a central lumen observed. Cells on the surface of large aggregates showed some evidence of membrane polarization, while the majority of cells in the cultures exhibited little evidence of polarized morphology. Despite the striking difference in morphology and multicellular organization these cells still expressed high levels of transthyretin mRNA and maintained the same pattern of protein synthesis as cells cultured on Matrigel. These results indicate that the basement membrane is important for the organization of choroid plexus epithelial cells into a functional epithelium in vitro and thus presumably the maintenance of the integrity of the blood-brain barrier in vivo. In contrast to several other epithelial systems which have been studied, the type of extracellular matrix does not appear to directly influence tissue-specific gene expression by choroid plexus epithelial cells. Thus the level of gene expression is not dependent on the cytoarchitecture and multicellular organization of this cell type.

Animals↗
PubMed1994

Expression of the integrin alpha 4 beta 1 on melanoma cells can inhibit the invasive stage of metastasis formation.

Among a series of adhesion molecules, expression of integrin alpha 4 beta 1 showed a unique inverse correlation with the invasive potential of B16 melanoma cell lines. When an alpha 4 cDNA was introduced into an alpha 4-beta 1+ highly invasive melanoma line, alpha 4 beta 1 heterodimers were expressed on the surface. Matrigel invasion by the alpha 4+ beta 1+ cells was reduced. Pulmonary metastasis was also suppressed when the transfectants were placed subcutaneously, but not when injected intravenously. Expression of alpha 4 beta 1 promoted homotypic intercellular adhesion. The homotypic adhesion was abrogated, and the alpha 4+ beta 1+ (less invasive cell lines) increased matrigel invasion following the anti-alpha 4 MAb treatment. These results suggest that integrin alpha 4 beta 1 could play a role in controlling melanoma cell metastasis at the invasive stage.

Animals↗
PubMed1991

Establishment of endometrial glandular epithelial cell subculture in a serum-free, hormonally defined medium, on a basement membrane matrix.

Epithelial glands were isolated from guinea-pig endometrium. In order to reduce the requirement for a serum supplement and the contamination by non epithelial cells in primary culture, various coatings of the culture dishes were tested using serum-free Ham's F12 containing defined chemicals including 17 beta-estradiol. While epithelial glands seeded on culture dishes coated with Matrigel, a basement membrane matrix-failed to spread, they formed on poly-D-lysine plus serum-coated dishes, a subconfluent monolayer (5-7 days) enriched in cytokeratin-immunostained cells (78%). Cells from subconfluent primary cultures, obtained on poly-D-lysine plus serum-coated dishes in serum-free hormonally defined medium, were passaged on Matrigel-coated dishes in serum-free hormonally defined medium. These subcultures contained, at confluence (4-5 days), a high percentage (greater than 95%) of cytokeratin-immunostained cells. These monolayers consisted of well-differentiated cells which exhibited ultrastructural features characteristic of endometrial epithelial cells. Moreover, these confluent cells contained 50% immunostained nuclei for progesterone receptors. Progesterone receptor amounts decreased in confluent subcultures treated with progesterone and became undetectable after long-term treatment, suggesting responsiveness of these cells to progesterone. This culture system provides a well-defined model for the study of protein synthesis and secretion by endometrial glandular epithelial cells under hormonal control.

Animals↗
PubMed1994

Different cells and cell lines produce factors that modulate Sertoli cell function.

Peritubular myoid cells derived from immature rat testes produce factors that modulate Sertoli cell function (P-Mod-S). The secretion of these factors is controlled in part by androgens. Cultured prostatic stromal cells strongly resemble peritubular myoid cells and produce mediators with similar activity. Here we investigated whether myoid cell lines can be used as a source of P-Mod-S-like factors. Rat kidney fibroblast (NRK) and mouse fibroblast (3T3) cell lines were used as non-myoid controls. Surprisingly, serum-free media conditioned by all cell lines studied modulated Sertoli cell function in a similar fashion as media conditioned by peritubular cells (PTCM) or stromal cells (STCM). Using Sertoli cell transferrin secretion as an endpoint for P-Mod-S-like activity, the nature of the active principles involved was further explored. The observed activity could not be explained by residual contamination with fetal calf serum. Moreover, the effects of the conditioned media could not be mimicked by classical growth factors (IGF-I, bFGF, EGF, TGF-beta, NGF, PDGF-BB) added singly or in combination with submaximally effective concentrations of PTCM. Finally, the possibility that conditioned media might indirectly enhance Sertoli cell function by promoting the production or deposition of extracellular matrix elements was made unlikely by the demonstration that the observed effects were not mimicked by Matrigel and were unaffected when Sertoli cells were seeded on Matrigel. Superdex 75 chromatography after analytical reversed-phase chromatography indicates that the factors from different origin have a similar size (45-50 kDa). It is concluded that mediators with P-Mod-S-like activity are produced by various cells and cell lines both with and without smooth muscle cell characteristics. Whether the active principles involved are really identical requires further investigation.

Animals↗
PubMed1995

Enhanced effect of epidermal growth factor on pulmonary metastasis and in vitro invasion of rat mammary carcinoma cells.

We examined the effects of epidermal growth factor (EGF) on metastatic and in vitro invasive capacity of weakly malignant ER-1 cells derived from a rat mammary carcinoma cell line, c-SST-2. EGF enhanced the metastatic capacity and in vitro invasiveness to reconstituted basement membrane, Matrigel, of ER-1 cells in a dose-dependent fashion. EGF-stimulated invasiveness was inhibited by anti-EGF antibody, which is able to neutralize the binding of EGF to EGF receptor, in the invasion assay system. EGF stimulated chemotactic migration toward fibronectin, laminin or newborn rat fibroblast-conditioned medium which was used as a chemoattractant in the in vitro invasion assay, but showed neither adhesion to Matrigel nor production of gelatinase and plasminogen activators. These results suggested that the increased metastatic and invasive capacity of ER-1 cells by EGF might be due to the increase in cell motility.

Animals↗
PubMed1996

Expression of a developmentally regulated, phosphorylated isoform of microtubule-associated protein 1B in sprouting and regenerating axons in vitro.

We have developed a novel culture system for studying axonal regeneration. Short lengths of spinal nerves with their attached dorsal root ganglia were removed from adult mice, explanted into Matrigel and maintained in serum-free medium for up to eight days. Profuse outgrowth of unfasciculated, naked axons occurred within 6 h from the cut ends of the peripheral nerve, dorsal roots and eventually from the ganglion itself, and continued to grow throughout the observation period. Some axons were entirely smooth, whilst others showed prominent varicosities. The former stained with antibody RT97, a marker for large-calibre, myelinated axons, whilst the latter stained with antibodies to calcitonin gene-related peptide, predominantly a marker for unmyelinated and small-diameter myelinated sensory axons. All axons stained with a monoclonal antibody (150) that recognizes a developmentally regulated phosphorylated isoform of the microtubule-associated protein 1B [Gordon-Weeks P. R. et al. (1993) Eur. J. Neurosci. 5, 1302-1311]. Monoclonal antibody 150 staining was observed along the entire length of all axons growing out of the explant; the proximal regions of these axons within the explant itself did not stain. The staining extended to the growth cones, which had elaborate morphologies. Other antibodies (e.g. to growth-associated protein 43) labelled axons within the nerve, as well as those growing in Matrigel. In preparations where the peripheral nerve had been crushed half-way along its length at the time of explantation, monoclonal antibody 150 staining was absent from axons in the nerve proximal to the crush, but present in axons which had regenerated within the nerve distal to the crush. The results indicate that re-expression during axonal regeneration of the phosphorylated isoform of microtubule-associated protein 1B recognized by monoclonal antibody 150 is restricted to the newly formed lengths of regenerated axons. The correlation between its expression and axonal growth during development and regeneration suggests that it may play a role in axonal extension. Our observations also demonstrate the usefulness of these explant cultures for studying axonal regeneration.

Animals↗