heparitin-sulfate and Skin-Neoplasms

Merkel cell polyomavirus (MCPyV) is a small, nonenveloped tumor virus associated with an aggressive form of skin cancer, Merkel cell carcinoma (MCC). MCPyV infections are highly prevalent in the human population, with MCPyV virions being continuously shed from human skin. However, the precise host cell tropism(s) of MCPyV remains unclear: MCPyV is able to replicate within a subset of dermal fibroblasts, but MCPyV DNA has also been detected in a variety of other tissues. However, MCPyV appears different from other polyomaviruses, as it requires sulfated polysaccharides, such as heparan sulfates and/or chondroitin sulfates, for initial attachment. Like other polyomaviruses, MCPyV engages sialic acid as a (co)receptor. To explore the infectious entry process of MCPyV, we analyzed the cell biological determinants of MCPyV entry into A549 cells, a highly transducible lung carcinoma cell line, in comparison to well-studied simian virus 40 and a number of other viruses. Our results indicate that MCPyV enters cells via caveolar/lipid raft-mediated endocytosis but not macropinocytosis, clathrin-mediated endocytosis, or glycosphingolipid-enriched carriers. The viruses were internalized in small endocytic pits that led the virus to endosomes and from there to the endoplasmic reticulum (ER). Similar to other polyomaviruses, trafficking required microtubular transport, acidification of endosomes, and a functional redox environment. To our surprise, the virus was found to acquire a membrane envelope within endosomes, a phenomenon not reported for other viruses. Only minor amounts of viruses reached the ER, while the majority was retained in endosomal compartments, suggesting that endosome-to-ER trafficking is a bottleneck during infectious entry.

Topics: A549 Cells; Antigens, Viral, Tumor; Carcinoma, Merkel Cell; Cell Line; Cell Line, Tumor; Cell Movement; Fibroblasts; HEK293 Cells; HeLa Cells; Heparitin Sulfate; Humans; Merkel cell polyomavirus; N-Acetylneuraminic Acid; Polyomavirus Infections; Skin; Skin Neoplasms; Tumor Virus Infections; Viral Tropism

Our previous report suggested the potential role of the exchange protein directly activated by cyclic AMP (Epac) in melanoma metastasis via heparan sulfate (HS)-mediated cell migration. In order to obtain conclusive evidence that Epac1 plays a critical role in modification of HS and melanoma metastasis, we extensively investigated expression and function of Epac1 in human melanoma samples and cell lines. We have found that, in human melanoma tissue microarray, protein expression of Epac1 was higher in metastatic melanoma than in primary melanoma. In addition, expression of Epac1 positively correlated with that of N-sulfated HS, and N-deacetylase/N-sulfotransferase-1 (NDST-1), an enzyme that increases N-sulfation of HS. Further, an Epac agonist increased, but ablation of Epac1 decreased, expressions of NDST-1, N-sulfated HS, and cell migration in various melanoma cell lines. Finally, C8161 cells with stable knockdown of Epac1 showed a decrease in cell migration, and metastasis in mice. These data suggest that Epac1 plays a critical role in melanoma metastasis presumably because of modification of HS.

Topics: Animals; Cell Line, Tumor; Cell Movement; Gene Deletion; Guanine Nucleotide Exchange Factors; Heparitin Sulfate; Humans; Melanoma; Mice; Neoplasm Metastasis; Skin Neoplasms; Staining and Labeling; Sulfotransferases

Germline mutations in the Exostoses-1 gene (EXT1) are found in hereditary multiple exostoses syndrome, which is characterized by the formation of osteochondromas and an increased risk of chondrosarcomas and osteosarcomas. However, despite its putative tumor-suppressor function, little is known of the contribution of EXT1 to human sporadic malignancies. Here, we report that EXT1 function is abrogated in human cancer cells by transcriptional silencing associated with CpG island promoter hypermethylation. We also show that, at the biochemical and cellular levels, the epigenetic inactivation of EXT1, a glycosyltransferase, leads to the loss of heparan sulfate (HS) synthesis. Reduced HS production can be reversed by the use of a DNA demethylating agent. Furthermore, the re-introduction of EXT1 into cancer cell lines displaying methylation-dependent silencing of EXT1 induces tumor-suppressor-like features, e.g. reduced colony formation density and tumor growth in nude mouse xenograft models. Screening a large collection of human cancer cell lines (n=79) and primary tumors (n=454) from different cell types, we found that EXT1 CpG island hypermethylation was common in leukemia, especially acute promyelocytic leukemia and acute lymphoblastic leukemia, and non-melanoma skin cancer. These findings highlight the importance of EXT1 epigenetic inactivation, leading to an abrogation of HS biosynthesis, in the processes of tumor onset and progression.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; CpG Islands; DNA Methylation; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Tumor Suppressor; Heparitin Sulfate; Humans; Leukemia, Promyelocytic, Acute; Mice; Mice, Nude; Mutation, Missense; N-Acetylglucosaminyltransferases; Neoplasms, Experimental; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Promoter Regions, Genetic; Skin Neoplasms; Transplantation, Heterologous; Tretinoin

Glycosaminoglycans (GAGs) are anionic polysaccharides present on cells and in the extracellular matrix (ECM). They likely play a role in tumor formation because of their capacity to bind and modulate a variety of proteins including growth factors, cytokines, and proteases. Using a panel of (human) phage display-derived anti-GAG antibodies, the location and expression of GAG epitopes in human cutaneous melanocytic lesions was studied. Antibodies EW4E1 and EW4G2 identified a melanoma-associated chondroitin sulfate/heparan sulfate epitope, whereas antibody EW4B7 recognized a melanoma-associated heparan sulfate epitope. These antibodies showed a high reactivity with blood vessels and ECM in cutaneous melanoma tumors, whereas their reactivity with nevi was very low. Using a set of defined oligosaccharides it was established that sulfate groups are of main importance in the binding to the antibodies and that glycomimetics can mimic natural oligosaccharides. In xenografts of melanoma cell line MeL57, a strong association of GAG epitopes with an injected fluorescent fluid flow tracer was observed. In uveal melanoma antibody, EW4E1 proved to be a sensitive probe for the detection of the geometry of ECM structures, known to have prognostic value. Taken together, data indicate that in melanoma a defined set and location of GAG epitopes are present with possible functional significance.

Topics: Animals; Antibodies; Chondroitin; Epitopes; Heparitin Sulfate; Humans; Melanoma; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Oligosaccharides; Peptide Library; Rats; Rats, Wistar; Skin Neoplasms; Transplantation, Heterologous; Uveal Neoplasms

Heparan sulphate (HS) represents a heterogeneous class of molecules on cell membranes and extracellular matrices. These molecules are involved in a variety of biological processes, including immune responses, through their binding and functional modulation of proteins. Recently a panel of HS-epitope-specific, human single chain antibodies have been generated by phage display, facilitating analysis of the structural heterogeneity of HS in relation to pathological conditions. In a pilot study a heterogeneous staining pattern in melanoma metastases was observed with one of the clones (EW4G1). Using a double-staining technique, the expression of this epitope was studied in 12 metastatic melanoma lesions in relation to the presence of a CD3(+) cell infiltrate. Different staining patterns with EW4G1 were observed in the different lesions. The different staining patterns were associated with the presence and pattern of inflammation with CD3(+) cells. A pronounced staining pattern of blood vessels with EW4G1 was associated with a more or less brisk presence of CD3(+) cells, while a pronounced staining of tumour cells or tumour cell matrix or absence of staining with EW4G1 was associated with absence of CD3(+) cells. These results suggest a dualistic role for HS in the recruitment and intratumoural migration of CD3(+) cells, depending on the location of expression of its epitope recognized by EW4G1. Further characterization of the structural diversity of HS and its function in T-cell recruitment and migration is therefore warranted, since detailed understanding of this relation may provide new targets for therapeutic intervention, such that better homing and migration of T cells (in)to tumours might be achieved in immunologically based treatment strategies.

Topics: Antibody Affinity; Antibody Specificity; CD3 Complex; Epitopes; Heparitin Sulfate; Humans; Immunohistochemistry; Melanoma; Peptide Fragments; Pilot Projects; Skin Neoplasms

Previous in vitro studies have shown CD44 isoforms containing the alternatively spliced exon v3 (CD44v3) to be modified with heparan sulphate (HS) and to bind HS-binding basic fibroblast growth factor (bFGF). Here, we demonstrate that exogenously added bFGF is also bound in vivo by CD44v3-positive keratinocytes in normal skin and by tumour cells in basal cell carcinoma and squamous cell carcinoma (SCC), two skin cancers of keratinocyte origin. bFGF binding and CD44v3 expression were colocalized in cultured human normal keratinocytes (HNK) and on the SCC cell line A431. By contrast, benign or malignant tumours of melanocyte origin failed to express CD44v3 and bound no bFGF. The bFGF binding to normal or transformed keratinocytes in vivo and in vitro was dependent on HS modification, as it was completely eliminated by pretreatment with heparitinase or by blocking with free heparin, whereas chondroitinase had no effect. In addition, specific removal of CD44v3 by antibody-induced shedding also diminished bFGF binding to keratinocytes. Furthermore, bFGF stimulated the proliferation of CD44v3-positive HNK and A431 in a dose-dependent fashion. This bFGF effect was again completely abolished by heparitinase or free heparin, but not by chondroitinase. In aggregate, our results suggest that a function of HS-modified CD44 isoforms such as CD44v3 in skin is to present the HS-binding growth factor bFGF, thereby stimulating the proliferation of normal or transformed keratinocytes.

Topics: Antigens, Neoplasm; Carcinoma, Squamous Cell; Cell Culture Techniques; Cell Division; Fibroblast Growth Factor 2; Heparitin Sulfate; Humans; Hyaluronan Receptors; Infant, Newborn; Keratinocytes; Male; Skin; Skin Neoplasms; Tumor Cells, Cultured

The epidemiology of atypical nevi (AN) is currently obscure; however the diagnosis must be made early in order to follow these individuals and treat any melanomas that may arise at an early stage, thus preventing premature death.. Following the guidelines of the NIH on clinical and histologic features of ANS, 38 adult members in 8 families were investigated. Twenty-seven were physically examined and 25 biopsied. Biopsies from ANS and junctional nevi from unrelated persons were also stained with antibodies against heparan sulfate proteoglycan (HSPG).. At least 21 of 38 members had ANS. Staining with HSPG antibodies did not differentiate between ANS and benign junctional nevi, all showing slightly irregular staining. In seven of eight families, two or more family members were affected by ANS.. Although it is not known whether or not HSPG plays a role in melanomas becoming invasive, or the potential of melanoma developing in ANS there were no differentiating features of staining in ANS, and junctional nevi to help in the differential-diagnosis of the two.

Topics: Adult; Antibodies; Diagnosis, Differential; Family; Fluorescent Antibody Technique, Direct; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Melanoma; Nevus; Proteoglycans; Skin Neoplasms

Recent cell biologic studies have emphasized the importance of the basement membrane (BM) and its molecular components in angiogenesis. We immunostained 60 angioproliferative lesions (angiosarcoma, sclerosing hemangioma of skin, pyogenic granuloma, capillary hemangioma, lymphangioma, glomangioma and granulation tissue) and 23 cases of Kaposi's sarcoma (KS) for the major macromolecular components laminin, collagen type IV, fibronectin and heparan sulfate proteoglycan (HSPG). Normal structures served as aggregate controls in each group, and semiquantitative scoring reflected the degree of consistency of staining about blood and lymphatic endothelium and vascular sheath (pericyte/smooth muscle) within and peripheral to each lesion. Benign and reactive vasoproliferations consistently maintained immunoreactivity for each BM component around endothelium and sheath components of blood vessels. Angiosarcoma showed from 20 to more than 60% less consistent immunoreactivity by comparison, although the score variances were greater than for non-malignant lesions. Staining about blood vessel endothelium was both strong and consistent among histologic stages in KS with the exception of HSPG, which was weakly immunoreactive in all stages. Marked selective HSPG loss was characteristic only of KS and normal lymphendothelium, and in the light of evidence for a role for HSPG in the assembly and maintenance of BM, suggests that reduced HSPG may be responsible for the loss of ultrastructural integrity of perivascular BM in both.

Topics: Basement Membrane; Collagen; Fibronectins; Granuloma, Pyogenic; Hemangiosarcoma; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Immunohistochemistry; Laminin; Proteoglycans; Sarcoma, Kaposi; Skin Diseases; Skin Neoplasms

Abnormal expression of proteoglycans has been implicated in cancer and metastasis primarily because these macromolecules are involved in the control of cell growth and matrix assembly. In this report, we have investigated the expression and immunolocalization of perlecan, a major heparan sulfate proteoglycan of basement membranes and pericellular matrices, in human metastatic melanomas. Twenty-six of the 27 tumor samples showed a significant increase (up to 15-fold) in the perlecan mRNA levels when compared with normal tissue. This change correlated with a vast deposition of perlecan protein core in the pericellular matrix of metastatic melanomas. Furthermore, we have established a relationship between perlecan expression in clonal melanoma cells (70W) stimulated with neurotrophins and their increased invasiveness. Interestingly, perlecan mRNA levels were up-regulated within 10 min of neurotrophin stimulation, indicating that perlecan is an early response gene. This upregulation also occurred prior to heparanase production, suggesting that perlecan expression and its regulation might play a pivotal role in the initial onset of invasion.

Topics: Blotting, Northern; Cell Communication; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Melanoma; Neoplasm Invasiveness; Nerve Growth Factors; Neurotrophin 3; Proteoglycans; RNA, Messenger; Skin Neoplasms; Tumor Cells, Cultured; Up-Regulation

Topics: Antibodies, Monoclonal; Basement Membrane; Carcinoma, Squamous Cell; Chondroitin Sulfates; Chondroitinases and Chondroitin Lyases; Collagen; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Immunohistochemistry; Keratosis; Laminin; Proteoglycans; Skin Neoplasms

An immunohistochemical study using a comprehensive panel of antibodies to Schwann cell markers, intermediate filaments and extracellular matrix components has been performed on three cases of plexiform schwannoma. All tumour cells expressed S 100 protein, Leu 7-HNK 1 antigen and vimentin; glial fibrillary acidic protein was detected in many tumour cells. In addition, expression of cytokeratin was also demonstrated in one case. The associated extracellular matrix was found to be reactive with antibodies to laminin, heparan sulfate proteoglycan, fibronectin, type I, III, IV and VI collagen. It is concluded that Schwann cells producing their own extracellular matrix are the main components of these tumours. The significance of the cytokeratin expression and the possible role of the extracellular matrix in regulating Schwann cells' proliferation in peripheral nerve tumours are discussed.

Topics: Adult; Antigens, Differentiation; Biomarkers, Tumor; Cell Division; Child, Preschool; Collagen; Extracellular Matrix Proteins; Fibronectins; Glial Fibrillary Acidic Protein; Heparitin Sulfate; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratins; Laminin; Male; Neurilemmoma; S100 Proteins; Schwann Cells; Skin Neoplasms; Vimentin

Levels of laminin and heparan sulfate proteoglycan (basal membrane components) were studied immunohistochemically in samples prepared from 18 basal-cell carcinomas using indirect immunoperoxidase technique. In the majority of cases, the basal membrane surrounding tumor foci remained unaltered. Some areas of solid basaliomas revealed defects in the membrane as well as deposits of laminin (but not heparan sulfate proteoglycan) in tumor tissue. It was suggested that areas of basalioma are surrounded by chemically defective membrane.

Topics: Aged; Antibodies, Monoclonal; Carcinoma, Basal Cell; Female; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Immunoenzyme Techniques; Laminin; Male; Middle Aged; Proteoglycans; Skin; Skin Neoplasms

To investigate alterations in the basement membrane (BM) in squamous cell carcinoma (SCC), we investigated 20 tumors. Four had the cytologic characteristics of Bowen's disease (SCC-BD) and 16 did not have them (SCC-NB). Tumors were studied immunohistochemically by double immunofluorescent staining by using mouse monoclonal antibodies to the core protein of heparan sulfate proteoglycan (HSPG) and chondroitin 6-sulfate glycosaminoglycan (Ch6S) as well as rabbit antiserum to laminin (LN) and type IV collagen (C-4). In well-differentiated and highly keratinized SCC-NB, LN, C-4, and HSPG could be detected in the tumor nest BM and showed no loss of continuity, but they were largely lost in poorly differentiated and poorly keratinized SCC-NB. This suggests that poorly differentiated SCC-NB cause greater enzymatic degradation of BM components than well-differentiated SCC-NB. Ch6S was detected in parts of the BM of SCC-BD, but it was absent in all SCC-NB examined. It appears that SCC-NB have lost the ability to synthesize Ch6S, and that SCC-BD degrade Ch6S although they continue to produce it. Thus, it appears that in SCC the BM is qualitatively different from that of normal epidermis, and that SCC-BD can be distinguished from SCC-NB by the Ch6S content of the BM.

Topics: Antibodies; Antibodies, Monoclonal; Basement Membrane; Bowen's Disease; Carcinoma, Squamous Cell; Chondroitin Sulfate Proteoglycans; Collagen; Fluorescent Antibody Technique; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Laminin; Reference Values; Skin; Skin Neoplasms

The heparan sulfates synthesized in vitro by three cell lines were isolated by proteolysis and preparative anion exchange chromatography and purified free of other glycosaminoglycans by selective enzymatic degradation. The isolates from the medium of BALB/c 3T3 fibroblasts, B16.F10 melanoma cells, and a cutaneous fibrosarcoma line, along with that from the detergent-extracted cell layer of the fibroblasts, were affinity-fractionated on columns of matrix-immobilized human antithrombin III. Each heparan sulfate contained subfractions with high affinity for the proteinase inhibitor, ranging from 3-34% of the starting material. The high affinity species possessed measurable anticoagulant activities by a clotting assay (6 to 30 units/mg). Since none of the lines were derived from cell types having any known biological role in vascular homeostasis, we suggest that anticoagulant activity of the glycosaminoglycan is a random property of its primary structure.

Topics: Animals; Antithrombin III; Blood Coagulation; Cell Line; Chromatography, Affinity; Chromatography, Ion Exchange; Fibroblasts; Fibrosarcoma; Glycosaminoglycans; Heparitin Sulfate; Melanoma; Mice; Mice, Inbred BALB C; Skin Neoplasms; Tumor Cells, Cultured

A case of multiple clear cell acanthomas is described with a review of the literature. This benign glycogen-rich epidermal tumor is generally unable to accept melanin and lacks phosphorylase or cytochrome oxidase activity. However, the basement membrane zone appears antigenically normal, as documented by the presence of epidermolysis bullosa acquisita antigen, bullous pemphigoid antigen, heparan sulfate proteoglycan, type IV collagen, and laminin by immunofluorescence microscopy.

Topics: Antigens; Autoantigens; Basement Membrane; Carrier Proteins; Chondroitin Sulfate Proteoglycans; Collagen; Collagen Type XVII; Cytoskeletal Proteins; Dystonin; Fluorescent Antibody Technique; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Laminin; Male; Middle Aged; Neoplasms, Multiple Primary; Nerve Tissue Proteins; Non-Fibrillar Collagens; Papilloma; Skin Neoplasms

Specific antibodies against basement membrane associated, connective tissue components: type IV and V collagens, laminin, fibronectin and heparan sulphate proteoglycan were used to study the basement membrane-like structures in cylindroma lesions. All these components were immunohistochemically demonstrated as a band surrounding islands of epithelial cells and all except fibronectin also inside the islands. Antibodies to keratin filaments stained most of the cells inside the epithelial islands confirming the epithelial origin of the cells.

Topics: Basement Membrane; Carcinoma, Adenoid Cystic; Chondroitin Sulfate Proteoglycans; Collagen; Female; Fibronectins; Fluorescent Antibody Technique; Genes, Dominant; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Keratins; Laminin; Middle Aged; Skin Neoplasms

Glycosaminoglycan synthesis was studied in cell populations of ultraviolet light-induced murine cutaneous fibrosarcoma cells under conditions of varying growth rates in vitro. After labeling with the precursors, 3H-glucosamine and 35SO4, sulfated glycosaminoglycans recoverable by direct proteolysis of the culture monolayers increased approximately 5-fold on a per cell basis from sparsely populated, exponential cell cultures (greater than 85% of cells in S, G2, or M phases) to stationary cultures inhibited by high cell density (greater than 50% of cells in G1). Within this cell surface-associated material, the relative ratio of heparan sulfate to the chondroitin sulfates was approximately 60/40% under conditions of exponential growth; in the growth-arrested cultures, the reverse ratio was found. The substratum attached material, obtained from the flask surface after ethyl glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA)-mediated detachment of the monolayers, contained relatively more hyaluronic acid, heparan sulfate, and chondroitin sulfates in the most actively proliferating cultures compared with the growth-inhibited cell populations. Furthermore, heparan sulfate and the chondroitin sulfates, which were enriched in the substratum material and in the cell pellet of exponential cultures, showed a relative shift to the cell surface-associated compartment (releasable by mild trypsinization after EGTA-mediated cell detachment) and to the compartment loosely associated with the pericellular matrix (i.e., released into the supernatant during detachment of the monolayers in the presence of EGTA). These results demonstrate that a variety of differences in the quantities, relative compositional ratios, and cell compartment distributions of hyaluronic acid and sulfated glycosaminoglycans occur in fibrosarcoma cell populations which vary in their rate of cell growth consequent to cell density in culture.

Topics: Animals; Cell Compartmentation; Cell Count; Cell Division; Cell Line; Chondroitin Sulfates; Fibrosarcoma; Glycosaminoglycans; Heparitin Sulfate; Hyaluronic Acid; Kinetics; Mice; Neoplasms, Radiation-Induced; Skin Neoplasms; Ultraviolet Rays