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Biomedical subjects

E H Danen

Publications and source records attributed to E H Danen.

At least 19 recordsLinked to original sources
PubMed2001

Fibronectin, integrins, and growth control.

Cell proliferation is controlled not only by soluble mitogens but also by components of the extracellular matrix (ECM) such as fibronectin, to which cells adhere via the integrin family of transmembrane receptors. Input from both growth factor receptors and integrins is required to stimulate progression through the G1 phase of the cell cycle, via induction of G1 cyclins and suppression of inhibitors of the G1 cyclin-dependent kinases. Extensive crosstalk takes place between integrin and growth factor receptor signaling pathways, and mitogenic signaling is weak and transient in the absence of integrin-mediated cell adhesion. In normal untransformed cells, all of the important mitogenic signal transduction cascades, namely those downstream of the Ras and Rho family small GTPases and the phosphoinositide 3-OH kinase-PKB/Akt pathway, are regulated by integrin-mediated cell adhesion. As a result, these cells are anchorage-dependent for growth. In contrast, constitutive activity of each of these pathways has been reported in cancer cells, which not only reduces their mitogen dependence but also allows these cells to grow in an anchorage-independent fashion.

Animals↗
PubMed2000

Dual stimulation of Ras/mitogen-activated protein kinase and RhoA by cell adhesion to fibronectin supports growth factor-stimulated cell cycle progression.

In cellular transformation, activated forms of the small GTPases Ras and RhoA can cooperate to drive cells through the G1 phase of the cell cycle. Here, we show that a similar but substrate-regulated mechanism is involved in the anchorage-dependent proliferation of untransformed NIH-3T3 cells. Among several extracellular matrix components tested, only fibronectin supported growth factor-induced, E2F-dependent S phase entry. Although all substrates supported the mitogen-activated protein kinase (MAPK) response to growth factors, RhoA activity was specifically enhanced on fibronectin. Moreover, induction of cyclin D1 and suppression of p21(Cip/Waf) occurred specifically, in a Rho-dependent fashion, in cells attached to fibronectin. This ability of fibronectin to stimulate both Ras/MAPK- and RhoA-dependent signaling can explain its potent cooperation with growth factors in the stimulation of cell cycle progression.

3T3 Cells↗
PubMed1999

Shc and FAK differentially regulate cell motility and directionality modulated by PTEN.

Cell migration is modulated by regulatory molecules such as growth factors, oncogenes, and the tumor suppressor PTEN. We previously described inhibition of cell migration by PTEN and restoration of motility by focal adhesion kinase (FAK) and p130 Crk-associated substrate (p130(Cas)). We now report a novel pathway regulating random cell motility involving Shc and mitogen-activated protein (MAP) kinase, which is downmodulated by PTEN and additive to a FAK pathway regulating directional migration. Overexpression of Shc or constitutively activated MEK1 in PTEN- reconstituted U87-MG cells stimulated integrin- mediated MAP kinase activation and cell migration. Conversely, overexpression of dominant negative Shc inhibited cell migration; Akt appeared uninvolved. PTEN directly dephosphorylated Shc. The migration induced by FAK or p130(Cas) was directionally persistent and involved extensive organization of actin microfilaments and focal adhesions. In contrast, Shc or MEK1 induced a random type of motility associated with less actin cytoskeletal and focal adhesion organization. These results identify two distinct, additive pathways regulating cell migration that are downregulated by tumor suppressor PTEN: one involves Shc, a MAP kinase pathway, and random migration, whereas the other involves FAK, p130(Cas), more extensive actin cytoskeletal organization, focal contacts, and directionally persistent cell motility. Integration of these pathways provides an intracellular mechanism for regulating the speed and the directionality of cell migration.

Actins↗
PubMed1999

PTEN interactions with focal adhesion kinase and suppression of the extracellular matrix-dependent phosphatidylinositol 3-kinase/Akt cell survival pathway.

The tumor suppressor PTEN is a phosphatase with sequence homology to tensin. PTEN dephosphorylates phosphatidylinositol 3,4, 5-trisphosphate (PIP3) and focal adhesion kinase (FAK), and it can inhibit cell growth, invasion, migration, and focal adhesions. We investigated molecular interactions of PTEN and FAK in glioblastoma and breast cancer cells lacking PTEN. The PTEN trapping mutant D92A bound wild-type FAK, requiring FAK autophosphorylation site Tyr397. In PTEN-mutated cancer cells, FAK phosphorylation was retained even in suspension after detachment from extracellular matrix, accompanied by enhanced PI 3-K association with FAK and sustained PI 3-K activity, PIP3 levels, and Akt phosphorylation; expression of exogenous PTEN suppressed all five properties. PTEN-mutated cells were resistant to apoptosis in suspension, but most of the cells entered apoptosis after expression of exogenous PTEN or wortmannin treatment. Moreover, overexpression of FAK in PTEN-transfected cells reversed the decreased FAK phosphorylation and PI 3-K activity, and it partially rescued PIP3 levels, Akt phosphorylation, and PTEN-induced apoptosis. Our results show that FAK Tyr397 is important in PTEN interactions with FAK, that PTEN regulates FAK phosphorylation and molecular associations after detachment from matrix, and that PTEN negatively regulates the extracellular matrix-dependent PI 3-K/Akt cell survival pathway in a process that can include FAK.

Cell Adhesion Molecules↗
PubMed1998

The disintegrin eristostatin interferes with integrin alpha 4 beta 1 function and with experimental metastasis of human melanoma cells.

Peptides containing the integrin recognition sequence, RGD, can inhibit experimental metastasis of mouse melanoma cells, but the integrin(s) affected in these experiments is unknown. Besides "classical" RGD-binding integrins such as alpha 5 beta 1 and alpha v beta 3, RGD has been reported to bind alpha 4 beta 1, and mAbs to alpha 4 beta 1 can inhibit melanoma metastasis. We investigated the mode of action of the disintegrin eristostatin, an RGD-containing peptide isolated from snake venom, in a human melanoma experimental metastasis model. Lung colonization following i.v. injection of MV3 cells in nude mice was strongly inhibited by eristostatin. MV3 cells bound FITC-eristostatin and adhered to eristostatin-coated wells. This adhesion was partially inhibited by a GRGDSP peptide and by alpha 4 mAb. Binding of FITC-eristostatin to Jurkat cells and adhesion of Jurkat (but not K562) cells to eristostatin-coated wells further suggested that eristostatin binds alpha 4 beta 1, even though, again, alpha 4 mAb only partially inhibited adhesion. Expression of alpha 4 beta 1 was enhanced in metastatic melanoma cells compared to normal melanocytes and nonmetastatic melanoma cells. Finally, eristostatin inhibited adhesion of both MV3 and CHO alpha 4 cells to the alpha 4 beta 1-ligand VCAM-1, while adhesion to other ligands via other integrins was not affected. These findings demonstrate that inhibition of melanoma cell metastasis by RGD-containing peptides such as eristostatin, may be due to interference with alpha 4 beta 1-VCAM binding, in addition to inhibition of the classical RGD-binding integrins.

Animals↗
PubMed1998

Integrin signaling: cytoskeletal complexes, MAP kinase activation, and regulation of gene expression.

Members of the integrin family of adhesion receptors mediate interactions of cells with the extracellular matrix. Besides their role in tissue morphogenesis by anchorage of cells to basement membranes and migration along extracellular matrix proteins, integrins are thought to play a key role in mediating the control of gene expression by the extracellular matrix. Studies over the past 10 years have shown that integrin-mediated cell adhesion can trigger signal transduction cascades involving translocation of proteins and protein tyrosine phosphorylation events. In this review, we discuss approaches used in our lab to study early events in integrin signalling as well as further downstream changes.

Animals↗
PubMed1996

Integrin beta 3 cDNA transfection into a highly metastatic alpha v beta 3-negative human melanoma cell line inhibits invasion and experimental metastasis.

Even though integrin alpha v beta 3 is thought to play a role in invasive growth of melanomas, some metastatic melanoma cell lines lack alpha v beta 3, and downmodulation of alpha v beta 3, expression can enhance the invasive capacity of certain melanoma cells. To further investigate this apparent dualistic role of alpha v beta 3, we transfected beta 3 cDNA into the highly metastatic, beta 3-negative human melanoma cell line MV3. MV3 cells adhered to fibronectin but not to fibrinogen or a synthetic RGD peptide, while MV3-beta 3 adhered to all three RGD-containing adhesive ligands, and this adhesion was inhibited by LM609 alpha v beta 3 mAb. Expression of alpha v beta 3 did not affect MV3 in vitro proliferation or in vivo tumorigenicity upon subcutaneous inoculation into nude mice. In contrast, it strongly reduced invasion in matrigel and lung colonization in nude mice of MV3 cells. Thus, certain melanoma cell lines have adopted a metastatic strategy in the absence of alpha v beta 3, and in such cells expression of this integrin leads to a less aggressive phenotype.

Animals↗
PubMed1996

Expression of CD44 and the pattern of CD44 alternative splicing in uveal melanoma.

In cutaneous melanoma, the standard CD44 molecule is abundantly expressed, whereas the expression of certain splice variants is related to tumour progression and to the metastatic potential of the cell line. In the present study we have investigated the expression of CD44 and the pattern of CD44 alternative splicing in uveal melanoma in relation to the cell type, diameter and invasiveness of the tumour. All uveal melanomas strongly stained with antibodies to the standard portion of CD44. No expression of the CD44 variant (v) exon CD44v7 was found, whereas v5, v6 and v10 were expressed (in 2/12, 5/12 and 8/12 cases, respectively). No correlation was observed between expression of particular splice variants and cell type, tumour diameter or invasion of the sclera or Bruch's membrane. All three uveal melanoma cell lines tested were strongly CD44 positive and expressed low levels of v6-containing isoforms at the cell surface, but v5, v7 and v10 were absent. Our results show that CD44 is strongly expressed in uveal melanoma and that the pattern of CD44 alternative splicing is similar to that observed in cutaneous melanoma. However, in uveal melanoma this alternative splicing does not appear to be related to prognostic parameters.

Alternative Splicing↗
PubMed1996

E-cadherin expression in human melanoma.

Loss of expression of E-cadherin, the major cell-cell adhesion receptor on keratinocytes, has been linked to tumour progression in various carcinomas. As E-cadherin has been reported to be expressed in cultured human melanocytes, we questioned whether loss of E-cadherin expression may also be related to melanocytic tumour progression. Flowcytometrical analysis demonstrated that E-cadherin was expressed on cultured normal melanocytes and naevus cells. Two non-invasive, non-metastatic melanoma cell lines showed low expression, and four invasive, metastatic melanoma cell lines did not express E-cadherin. Immunohistochemistry on frozen sections of human melanocytic lesions resulted in diffuse staining of 1/23 common naevocellular naevi and 1/13 dysplastic naevi, with no staining in any of seven early primary melanomas (< or = 1.5 mm). Staining was observed in 4/20 advanced primary melanomas (> 1.5 mm) and 5/35 melanoma metastases. Thus, even though E-cadherin is expressed in cultured melanocytes and naevus cells and lost in invasive, metastatic melanoma cells in vitro, it is rarely found in early stages of melanocytic tumour progression in situ and, surprisingly, some expression can be observed in 10-20% of lesions of advanced stages.

Animals↗
PubMed1996

Loss of adhesion to basement membrane components but not to keratinocytes in proliferating melanocytes.

We studied the adhesive characteristics of melanocytes, cultured either in the presence of the mitogen phorbol 12-myristate 13-acetate (PMA) that keeps them in a proliferative state, or in the absence of PMA allowing them to differentiate. On proliferating melanocytes, several integrins, ICAM-1, E-cadherin, and CD44 were expressed. In the absence of PMA, proliferation was arrested, melanin synthesis increased, and the morphology of the melanocytes became more spreaded. Under these conditions, expression of integrins alpha 3 beta 1 and alpha 5 beta 1 decreased, whereas expression of alpha 2 beta 1, alpha 4 beta 1, and alpha 6 beta 1 increased. No changes were observed for any of the other adhesion molecules. Immunoprecipitations from metabolically labeled cells confirmed the shift in integrin expression at the level of biosynthesis. The increased surface expression of alpha 2 beta 1 and alpha 6 beta 1 in the absence of PMA was accompanied by an induction of adhesion to basement membrane components collagen and laminin through these integrins. Integrin alpha 5 beta 1/alpha v beta 3-mediated adhesion to fibronectin, CD44-mediated adhesion to hyaluronate, and E-cadherin/beta 1-integrin-mediated adhesion to keratinocytes were not affected by PMA. These findings indicate that by selective modulation of the expression of adhesion molecules, adhesion to components of the basement membrane is reduced in proliferating melanocytes, whereas adhesion to keratinocytes is maintained. Similar events may be involved in melanocyte proliferation and migration during wound healing and initial steps of melanocytic tumor progression.

Basement Membrane↗
PubMed1995

Generation of antimelanoma cytotoxic T lymphocytes from healthy donors after presentation of melanoma-associated antigen-derived epitopes by dendritic cells in vitro.

MHC class I-restricted CTLs specific for antigens expressed by malignant cells are an important component of immune responses against human cancer. Recently, in melanoma a number of melanocyte differentiation antigens have been identified as potential tumor rejection antigens. In the present study, we show that by applying peptide-loaded dendritic cells, induced by granulocyte-macrophage colony-stimulating factor and interleukin 4 from peripheral blood monocytes of healthy donors, we were able to elicit melanoma-associated antigen-specific CTL in vitro. We demonstrate the induction of CTLs directed against HLA-A2.1 presented epitopes derived from tyrosinase, gp100, and Melan A/MART-1. Apart from lysis of peptide-loaded target cells, these CTLs displayed reactivity with HLA-A2.1+ melanoma tumor cell lines and cultured normal melanocytes endogenously expressing the target antigen. These data indicate that these CTLs recognize naturally processed and presented epitopes and that precursor CTLs against melanocyte differentiation antigens are present in healthy individuals. The ability to generate tumor-specific CTLs in vitro, using granulocyte-macrophage colony-stimulating factor/interleukin 4-induced dendritic cells, illustrates the potential use of this type of antigen-presenting cells for vaccination protocols in human cancer.

Amino Acid Sequence↗
PubMed1995

Requirement for the synergy site for cell adhesion to fibronectin depends on the activation state of integrin alpha 5 beta 1.

We investigated the influence of the activation state of integrin alpha 5 beta 1 on its dependence on the PHSRN synergy site for binding to RGD in fibronectin. K562 and MV3 cells lacked alpha v beta 3 expression and adhered to fibronectin through alpha 5 beta 1. Mel57 cells adhered through alpha v beta 3 and alpha 5 beta 1. A recombinant fibronectin polypeptide, containing five type III repeats from the central cell binding domain 3Fn6-10, and a mutated polypeptide lacking the synergy site were equally effective in promoting Mel57 adhesion. For K562 and MV3, the mutated polypeptide was not or poorly active compared to the control polypeptide. Expression of alpha v beta 3 in MV3 induced strong adhesion to the mutated polypeptide. TS2/16 stimulatory beta 1-integrin antibodies or Mn2+ induced alpha 5 beta 1-mediated adhesion of K562 and MV3 to GRGDSP. In the presence of TS2/16 or Mn2+, alpha 5 beta 1-mediated MV3 adhesion to the mutated polypeptide was equally strong as adhesion to the control polypeptide. Mn2+ or TS2/16 induced weak K562 binding to the mutated polypeptide, and in the presence of a combination of phorbol 12-myristate 13-acetate, Mn2+, and TS2/16, alpha 5 beta 1-mediated K562 adhesion to the mutated and control polypeptide was equally strong. Our findings demonstrate that requirement for the PHSRN synergy site for alpha 5 beta 1-mediated adhesion to RGD in fibronectin depends on the activation state of the integrin.

Amino Acid Sequence↗
PubMed1995

Establishment and characterization of an uveal-melanoma cell line.

A human uveal melanoma cell line (92-1) was established from a primary uveal melanoma, and has now been maintained in culture for over 2 1/2 years. Light microscopy of the cultured cells demonstrated extremely pleiomorphic cells with large prominent nucleoli. Cell proliferation was determined with a non-radioactive propidium-iodide assay and indicated an in vitro doubling time of approximately 58 hr. Furthermore, the cell line was characterized by cytogenetic analysis, electron microscopy, immunocytochemistry and Northern blotting for HLA and c-myc-mRNA analysis. Cytogenetic analysis revealed numerical abnormalities of chromosome 8 and structural abnormalities of chromosome 6. By electron microscopy, different stages of melanosome development were observed. Immunocytochemical analysis demonstrated expression of the melanoma-associated antigen gp 100. Expression analysis of HLA antigens revealed a very low level of, in particular, the HLA-B locus products, which could be induced by interferon-alpha or -gamma treatment. Likewise, Northern-blot experiments revealed decreased levels of HLA-B mRNA as compared with HLA-A. In addition, high levels of c-myc expression were observed. The phenotypic characteristics of the cultured cells indicate that we have established an uveal melanoma cell line. This now well-characterized uveal melanoma cell line can be used in future studies.

Aged↗
PubMed1995

Expression of CD44 splice variants in human cutaneous melanoma and melanoma cell lines is related to tumor progression and metastatic potential.

Expression of CD44, particularly of certain splice variants, has been linked to tumor progression and metastasis formation in a number of different animal and human cancers. Because human cutaneous melanoma is among the most aggressive human cancers, we explored expression of CD44 isoforms (CD44v) in lesions of melanocytic tumor progression. In addition, by RT-PCR and FACS analysis we assessed CD44v RNA species and cell surface expression of CD44v in cultured melanocytes isolated from human foreskin and in a panel of 2 non-, 2 sporadically and 2 highly metastatic human melanoma cell lines. We observed that all melanocytic lesions examined showed strong uniform expression of standard CD44 (CD44s) epitopes. We did not detect CD44v6 expression in the melanocytic lesions. However, CD44 isoforms containing v5 or v10 were differentially expressed. V5 was expressed in 16%, 0%, 20%, 67% and 58% of common nevi, atypical nevi, early primary melanomas (< or = 1.5 mm), advanced primary melanomas (> 1.5 mm) and metastases, respectively, and hence was related to tumor progression. In contrast, CD44v10 was expressed in all common nevi, whereas part of the atypical nevi and most primary melanomas and metastases lacked v10. CD44v RNA patterns were closely similar in cultured melanocytes and all melanoma cell lines. Melanocytes expressed high levels of CD44s but no CD44v, whereas all melanoma cell lines expressed CD44v at the surface. Interestingly, expression of v5 was strongly increased in the highly metastatic cell lines. Our results suggest a role for CD44 variant domains, particularly v5 and v10, in human melanocytic tumor progression.

Base Sequence↗
PubMed1995

Alpha v-integrins in human melanoma: gain of alpha v beta 3 and loss of alpha v beta 5 are related to tumor progression in situ but not to metastatic capacity of cell lines in nude mice.

We investigated the expression of alpha v-integrins in different stages of human cutaneous melanocytic tumor progression. We observed that alpha v beta 5 was the alpha v-integrin expressed in all common nevocellular nevi, in 78% of dysplastic nevi, in 63% of early primary melanomas, in 43% of advanced primary melanomas, and in 33% of melanoma metastases. Hence, loss of alpha v beta 5 expression was related to melanocytic tumor progression. In line with earlier reports, alpha v beta 3 was exclusively detected in advanced primary melanomas and metastases (24% and 50% respectively). Staining with anti-alpha v monoclonal antibodies (MAbs) in lesions where both alpha v beta 3 and alpha v beta 5 were absent showed that alternative alpha v-integrins were expressed in advanced primary melanomas and metastases. By FACS analysis, we determined expression of alpha v beta 5 and alpha v beta 3 in 4 human melanoma cell lines with different metastatic capacities after s.c. inoculation into nude mice. One of the non-metastatic and both highly metastatic cell lines expressed alpha v beta 5 at their surface. Surprisingly, alpha v beta 3 was detected exclusively in the non-metastatic cell lines. Absence of alpha v beta 3 in the highly metastatic cell lines was confirmed by lack of immunoprecipitation from 35S-methionine-labeled cells and by absence of immunohistochemical staining on primary and metastatic xenograft lesions. Our findings indicate that alpha v beta 5 expression is often lost in advanced stages of melanocytic tumor progression in situ, while alpha v beta 3 is acquired, but that a decrease in alpha v beta 5 and an increase in alpha v beta 3 expression are not necessarily related to the metastatic behavior of human melanoma cells in nude mice.

Animals↗
PubMed1995

Glycoconjugate profile and CD44 expression in human melanoma cell lines with different metastatic capacity.

Changes in glycoconjugate production have been reported for tumor cells. In this study, we investigated the glycoconjugate expression pattern in normal human melanocytes and in a panel of 6 human melanoma cell lines with different metastatic capacity after s.c. inoculation into nude mice. Glycoconjugates were labeled in vitro with [35S] sulphate and [3H] glucosamine, purified from cells and culture medium by column chromatography and identified by treatment with specific glycosidases. Characterization of the purified glycoconjugate fractions as well as alcian-blue staining of xenograft lesions revealed that hyaluronic acid (HA) is the main glycoconjugate produced by all cell lines. Highly metastatic cell lines expressed higher levels of HA than melanocytes and than weakly metastatic or non-metastatic cell lines. In addition, a shift in dominance from chondroitin-sulphate proteoglycan to heparan-sulphate proteoglycan was observed with increasing metastatic capacity. We also studied the expression and binding activity of the HA receptor CD44. Immunoprecipitation experiments indicated high CD44 synthesis only in highly metastatic cell lines, but FACS analysis demonstrated approximately the same surface expression in melanocytes as in all cell lines. Adhesion assays to immobilized HA showed that CD44 can be present in an inactive or an active conformation. Our data suggest that a combination of increased HA production and the expression of CD44 on the cell surface may be associated with high metastatic potential of human melanoma cell lines in nude mice.

Animals↗
PubMed1995

Properties of metastasizing and nonmetastasizing human melanoma cells.

Dissemination of tumor cells includes several steps, such as: (a) detachment of tumor cells from the primary tumor, (b) traversement of the basement membrane, and (c) migration into the extracellular matrix. In these processes, at least two important categories of proteins are involved: proteases and adhesion molecules. In this contribution we describe the expression and function of components of the plasminogen activator (PA) system (proteases) and of integrins (cell-matrix adhesion molecules) in a panel of four human melanoma cell lines with different invasive and metastatic capacity. Regarding the components of the PA system, we found differences in expression of urokinase-type PA (uPA) and type 1 and 2 PA inhibitors (PAI-1 and -2) between metastasizing and nonmetastasizing cell lines. Both components were exclusively expressed in the highly invasive and metastatic cell lines. Interestingly, studies on the expression of PA components in fresh human melanocytic lesions, showed expression of these components exclusively in advanced primary melanomas and melanoma metastases. Regarding integrin expression we found elevated levels of VLA-2 and VLA-6 in the highly invasive and metastatic cell lines compared with normal cultured melanocytes and nonmetastatic melanoma cell lines. In addition, increased adhesion of the highly metastatic cell lines to laminin (LM) and collagen (COLL) was observed. Furthermore, reduced adhesion of normal melanocytes and nonmetastatic melanoma cells to LM and CO was mainly due to the fact that the integrins involved in adhesion to these matrix components were present in an inactive state. Finally, differences were observed in expression of integrins involved in adhesion to fibronectin.

Amino Acid Sequence↗