PubMed Health⌕ Search

Biomedical subjects

M Herlyn

Publications and source records attributed to M Herlyn.

At least 19 recordsLinked to original sources
PubMed2006

AKT induces senescence in primary esophageal epithelial cells but is permissive for differentiation as revealed in organotypic culture.

Epidermal growth factor receptor (EGFR) overexpression and activation is critical in the initiation and progression of cancers, especially those of epithelial origin. EGFR activation is associated with the induction of divergent signal transduction pathways and a gamut of cellular processes; however, the cell-type and tissue-type specificity conferred by certain pathways remains to be elucidated. In the context of the esophageal epithelium, a prototype stratified squamous epithelium, EGFR overexpression is relevant in the earliest events of carcinogenesis as modeled in a three-dimensional organotypic culture system. We demonstrate that the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, and not the MEK/MAPK (mitogen-activated protein kinase) pathway, is preferentially activated in EGFR-mediated esophageal epithelial hyperplasia, a premalignant lesion. The hyperplasia was abolished with direct inhibition of PI3K and of AKT but not with inhibition of the MAPK pathway. With the introduction of an inducible AKT vector in both primary and immortalized esophageal epithelial cells, we find that AKT overexpression and activation is permissive for complete epithelial formation in organotypic culture, but imposes a growth constraint in cells grown in monolayer. In organotypic culture, AKT mediates changes related to cell shape and size with an expansion of the differentiated compartment.

Cell Differentiation↗
PubMed2006

Oncogenic BRAF regulates beta-Trcp expression and NF-kappaB activity in human melanoma cells.

Mutational activation of BRAF is a frequent event in human malignant melanomas suggesting that BRAF-dependent signaling is conducive to melanoma cell growth and survival. Previously published work reported that melanoma cells exhibit constitutive anti-apoptotic nuclear factor kappaB (NF-kappaB) transcription factor activation triggered by proteolysis of its inhibitor IkappaB. IkappaB degradation is dependent upon its phosphorylation by the IkappaB kinase (IKK) complex and subsequent ubiquitination facilitated by beta-Trcp E3 ubiquitin ligase. Here, we report that melanocytes expressing a conditionally oncogenic form of BRAF(V600E) exhibit enhanced beta-Trcp expression, increased IKK activity and a concomitant increase in the rate of IkappaBalpha degradation. Conversely, inhibition of BRAF signaling using either a broad-spectrum Raf inhibitor (BAY 43-9006) or by selective knock-down of BRAF(V600E) expression by RNA interference in human melanoma cells leads to decreased IKK activity and beta-Trcp expression, stabilization of IkappaB, inhibition of NF-kappaB transcriptional activity and sensitization of these cells to apoptosis. Taken together, these data support a model in which mutational activation of BRAF in human melanomas contributes to constitutive induction of NF-kappaB activity and to increased survival of melanoma cells.

Cell Line, Tumor↗
PubMed2006

Overexpression of IL-8 in the cornea induces ulcer formation in the SCID mouse.

AIMS: Although interleukin 8 (IL-8) is not produced in the normal cornea, it has been detected there in several pathological conditions. In this study, the direct effects of IL-8 overexpression on the cornea was examined. METHODS: The corneal surface of severe combined immunodeficiency mice was infected by the adenovirus vector encoding human IL-8 (IL-8/Ad5) and clinical and pathological changes were observed at various time points. RESULTS: Clinically, marked angiogenesis and ulcer formation in the cornea were observed by 12 hours and 24 hours, respectively. Histologically, prominent angiogenesis was observed in the corneal stroma at 12 hours. Cleft formation between the corneal epithelium and stroma, and neutrophil infiltration into the corneal stroma were seen at 16 hours. By 24 hours after the infection with IL-8/Ad5, a shallow ulcer was formed in the cornea. In contrast, infection with the control adenovirus carrying the beta galactosidase gene (LacZ) showed neither corneal ulceration nor neutrophil infiltration. Immunohistochemical analysis showed that infection with IL-8/Ad5 resulted in the production of IL-8 by corneal and conjunctival stromal cells. CONCLUSION: Our results indicate that IL-8 overexpression in corneal tissue causes ulcer formation in the cornea through chemoattraction of neutrophils, suggesting the aetiological role of IL-8 in some types of corneal ulcers.

Adenoviridae↗
PubMed2006

Monocyte chemoattractant protein-1 (MCP-1) inhibits the intestinal-like differentiation of monocytes.

Monocytes (MO) migrating into normal, non-inflamed intestinal mucosa undergo a specific differentiation resulting in a non-reactive, tolerogenic intestinal macrophage (IMAC). Recently we demonstrated the differentiation of MO into an intestinal-like macrophage (MAC) phenotype in vitro in a three-dimensional cell culture model (multi-cellular spheroid or MCS model). In the mucosa of patients with inflammatory bowel disease (IBD) in addition to normal IMAC, a reactive MAC population as well as increased levels of monocyte chemoattractant protein 1 (MCP-1) is found. The aim of this study was to investigate the influence of MCP-1 on the differentiation of MO into IMAC. MCS were generated from adenovirally transfected HT-29 cells overexpressing MCP-1, macrophage inflammatory protein 3 alpha (MIP-3alpha) or non-transfected controls and co-cultured with freshly elutriated blood MO. After 7 days of co-culture MCS were harvested, and expression of the surface antigens CD33 and CD14 as well as the intracellular MAC marker CD68 was determined by flow-cytometry or immunohistochemistry. MCP-1 and MIP-3alpha expression by HT-29 cells in the MCS was increased by transfection at the time of MCS formation. In contrast to MIP-3alpha, MCP-1 overexpression induced a massive migration of MO into the three-dimensional aggregates. Differentiation of IMAC was disturbed in MCP-1-transfected MCS compared to experiments with non-transfected control aggregates, or the MIP-3alpha-transfected MCS, as indicated by high CD14 expression of MO/IMAC cultured inside the MCP-1-transfected MCS, as shown by immunohistochemistry and FACS analysis. Neutralization of MCP-1 was followed by an almost complete absence of monocyte migration into the MCS. MCP-1 induced migration of MO into three-dimensional spheroids generated from HT-29 cells and inhibited intestinal-like differentiation of blood MO into IMAC. It may be speculated that MCP-1 could play a role in the disturbed IMAC differentiation in IBD mucosa.

Antigens, CD↗
PubMed2006

Towards the targeted therapy of melanoma.

Novel anti-cancer treatments use knowledge about the underlying biology of the tumor to find suitable molecular targets. The recent years have seen great advances in understanding the biology of melanoma. In the current review we discuss the most promising molecular targets for melanoma and suggest possible strategies for overcoming resistance.

Antineoplastic Agents↗
PubMed2005

Microarray analysis of phosphatase gene expression in human melanoma.

BACKGROUND: Tyrosine phosphate is abnormally elevated in malignant melanoma, and this has been interpreted to reflect the activity of oncogenic protein tyrosine kinases. However, elevation may also arise due to decreased protein tyrosine phosphatase (PTP) expression. OBJECTIVES: To survey phosphatase gene expression in melanoma cell lines, a benign naevus and normal melanocytes: we searched for downregulation of phosphatase gene expression in malignant cells that may indicate a role as melanoma suppressor genes. METHODS: Microarray analysis was used to compare gene expression for 133 phosphatase genes, comprising 39 PTPs, 16 dual-specificity phosphatases (DSPs), 47 serine/threonine phosphatases and 31 acid/alkaline and lipid-based phosphatases. Northern blotting analysis was used to study gene expression in human melanoma biopsies. RESULTS: There was decreased expression of four DSP genes (including PTEN); eight receptor PTP genes were downregulated in melanoma, among which were PTP-KAPPA and PTP-PI (consistent with our previous data). In addition, PTP-RF/LAR was downregulated in 13 of 22 metastatic melanomas. CONCLUSIONS: The expression of multiple PTP receptors is decreased in melanoma; this may be a mechanism which stimulates autonomous growth in advanced melanoma.

Blotting, Northern↗
PubMed2004

Absence of BRAF mutations in UV-protected mucosal melanomas.

BACKGROUND: Mutations in BRAF have recently been identified in a significant percentage of primary and metastatic cutaneous malignant melanomas. As ultraviolet (UV) exposure may play a role in the development of cutaneous melanoma lesions with BRAF mutations, BRAF mutation frequency in melanomas arising in sites protected from sun exposure may be lower than those from sun-exposed areas. Thus, we determined the BRAF mutation frequency in a panel of 13 mucosal melanomas and compared those data with data from all currently published series of cutaneous melanomas. METHODS: BRAF exon 15 DNA from 13 archival primary mucosal melanomas (eight vulvar, four anorectal, and one laryngeal) was sequenced using intron-based primers. As archival DNA occasionally produces poor-quality template, results were confirmed with a TspRI restriction fragment length polymorphism (RFLP) that distinguishes wild-type BRAF from the common mutant form V599E. A binomial test was used to compare the mutation frequency in the mucosal melanomas with the published mutation frequency in cutaneous melanomas. RESULTS: None of the 13 mucosal melanomas in this series had an exon 15 BRAF mutation, as compared to 54/165 (33%) primary cutaneous melanomas with BRAF mutations in a compilation of all current published studies (p = 0.006). DISCUSSION: These data suggest that UV exposure, plays a role in the genesis of BRAF mutations in cutaneous melanoma, despite the absence of the characteristic C>T or CC>TT mutation signature associated with UV exposure, and suggests mechanisms other than pyrimidine dimer formation are important in UV-induced mutagenesis.

DNA Mutational Analysis↗
PubMed2003

All-trans retinoic acid (atRA) differentially induces apoptosis in matched primary and metastatic melanoma cells -- a speculation on damage effect of atRA via mitochondrial dysfunction and cell cycle redistribution.

All-trans retinoic acid (atRA) has been suggested to exert its cytotoxicity via apoptosis but the mechanisms behind the damage effects have not been fully understood. In this study, we investigated the cytotoxic effects of atRA in eleven primary and matched metastatic cutaneous melanoma cell lines. All the primary and metastatic melanoma cell lines examined expressed the retinoic acid receptors. The cultured melanoma cells treated with atRA showed dysfunction of mitochondria and altered cell cycle distribution, inhibited cell proliferation and apoptosis. The cytotoxic effects of atRA were dose- and time-dependent. The dysfunction of mitochondria and induction of apoptosis were more pronounced in the primary tumor cells than in the metastatic cell lines from the same patients. The data indicate that the cytotoxic effect of atRA was mediated through dysfunction of mitochondria, alterations in cell cycle and induction of apoptosis. Melanoma in early stage may have better response to atRA adjuvant therapy than the melanoma in late stage, suggesting the early utility of atRA in melanoma chemotherapy.

Apoptosis↗
PubMed2002

Emerging concepts and technologies in melanoma research.

Melanoma research has made great advances in recent years. Particularly in the field of immunology, melanoma researchers have opened new avenues for basic and translational cancer research overall. Emerging research areas such as molecular epidemiology promise to develop a similar leadership role. On the other hand, research in biology, genetics or experimental therapy of melanoma has remained confined to few laboratories and entire research areas are not covered due to lack of researchers and resources. New developments in defining stem cells in skin and bone marrow enable us to develop new concepts for melanoma development and progression. New technologies allow rapid progress but they require close cooperation between laboratories. The field has to better bridge experimental with clinical research and increase communication. Corroboration with advocacy groups should activate the public for increased awareness and funding.

Animals↗
PubMed2002

A versatile method for the removal of melanin from ribonucleic acids in melanocytic cells.

Melanin pigments often co-purify during preparation of nucleic acids from cells or tissues of melanocytic origin. Contaminating melanin can severely impede subsequent analyses of RNA. We attempted to eliminate melanin in RNA preparations using selected gel matrices. We show here that co-purified melanin pigments can be largely eliminated from RNA samples after passing through polyacrylamide-based beads (Bio-Gel P-60). After isolation from the pigment-containing cells or tissues, RNA was subsequently processed through batch or column purification under acidic pH conditions. The resulting RNA was devoid of contaminating melanin pigments and amenable to molecular reactions such as polymerase chain reaction and cDNA synthesis by reverse transcriptase. Although the process results in some loss of input RNA, this purification procedure is simple, robust and can easily be adopted in any laboratory for the molecular analysis of RNA that requires removal of melanin contamination.

Acrylic Resins↗
PubMed2002

Dynamics of cell interactions and communications during melanoma development.

Melanoma development not only involves genetic and epigenetic changes that take place within the cell, but also involves processes determined collectively by micro-environmental factors, including cell-cell interactions and communications. During the transition from normal cells to benign and malignant lesions, and subsequently to metastatic cancer, stepwise changes in intercellular communications provide tumor cells with the ability to overcome cell-cell adhesion and micro-environmental controls from the host and to invade surrounding tissues and disperse to distant locations. Cadherins are major cell-cell adhesion molecules involved in the development and maintenance of skin. E-cadherin expressed in normal melanocytes mediates growth and invasion control by keratinocytes. Progressive loss of E-cadherin and gain of N-cadherin during melanoma development not only free melanoma cells from control by keratinocytes, but also provide new adhesion properties, resulting in switched partnerships with fibroblasts and vascular endothelial cells. The cadherin subtype switching also dictates gap junctional specificity in melanocytic cells during tumor development. This selective intercellular communication may contribute to the regulation of cell growth, differentiation, apoptosis, and migration of melanocytic cells in both physiologic and pathologic conditions. Abnormal up-regulation of the immunoglobin repeat-containing cell adhesion molecules Mel-CAM and L1-CAM potentiates invasion and migration of melanoma. Thus, abnormal expression of intercellular adhesion receptors and dysregulated intercellular communication underlies melanoma development and progression.

CD146 Antigen↗
PubMed2001

Downregulation of E-cadherin and Desmoglein 1 by autocrine hepatocyte growth factor during melanoma development.

During melanoma development, transformed cells evade keratinocyte-mediated control by downregulating cell adhesion molecules. This study investigated the regulation of cell adhesion by hepatocyte growth factor (HGF) in melanoma. Melanocytes and two melanoma lines, WM164 and WM35, expressed normal level E-cadherin and Desmoglein 1, whereas most melanomas (18 out of 20) expressed no E-cadherin and significantly reduced Desmoglein 1. Overexpression of dominant negative E-cadherin and Desmoglein in melanocytes demonstrated that both molecules contribute to adhesion between melanocytes and keratinocytes. In contrast to melanocytes, most melanomas expressed HGF. All melanocytic cells expressed the HGF receptor c-Met, and autocrine HGF caused constitutive activation of c-Met, MAPK and PI3K. When autocrine activation was induced with HGF-expressing adenovirus, E-cadherin and Desmoglein 1 were decreased in melanocytes, WM164 and WM35. MAPK inhibitor PD98059 and PI3K inhibitor wortmannin partially blocked the downregulation, suggesting that both pathways are involved in this process. c-Met was coimmunoprecipitated with E-cadherin, Desmoglein 1 and Plakoglobin, suggesting that they form a complex (es) that acts to regulate intercellular adhesion. Together, the results indicate that autocrine HGF decouples melanomas from keratinocytes by downregulating E-cadherin and Desmoglein 1, therefore frees melanoma cells from the control by keratinocytes and allows dissemination of the tumor mass.

Autocrine Communication↗
PubMed2001

Transforming growth factor-beta1 increases survival of human melanoma through stroma remodeling.

Transforming growth factor (TGF)-beta is growth inhibitory for normal epithelial cells and melanocytes but can stimulate mesenchymal cells. Resistance to its inhibitory effects is characteristic of human melanoma, the growth of which may instead be promoted by TGF-beta, because its production is increased with melanoma progression. Whether TGF-beta has an autocrine function for melanoma cells or is important for paracrine stimulation of the tumor stroma is not known. In this study, TGF-beta1 was expressed in melanoma cells via adenoviral gene transfer, and tumor growth was analyzed in vitro, in human skin grafts, and in mixtures with fibroblasts that were injected s.c. into immunodeficient mice. The TGF-beta1 produced by the melanoma cells activated the fibroblasts to produce matrix within and around the tumor mass, whereas control tumors showed less stroma and more cell death. High expression of collagen, fibronectin, tenascin, and alpha2 integrin was detected in the TGF-beta1-expressing tumors by immunohistochemistry. Number and size of lung metastases were significantly increased. cDNA expression array analysis of TGF-beta1-transduced fibroblasts embedded in type I collagen and of TGF-beta1-transduced melanoma cells demonstrated induction of types XV, XVIII, and VI collagens, tenascin, plasminogen activator inhibitor-I, vascular endothelial growth factor, cysteine-rich fibroblast growth factor receptor-1, and platelet-derived growth factor receptor-beta, which could be linked to promotion of growth and survival in melanoma. These data suggest that remodeling of the neighboring stroma, which provides a supporting scaffolding and a positive feedback stimulation of tumor growth, is an important function of TGF-beta1 in melanoma.

Animals↗
PubMed2001

Insulin-like growth factor-1 induces survival and growth of biologically early melanoma cells through both the mitogen-activated protein kinase and beta-catenin pathways.

Melanoma cells produce growth factors for autocrine growth control and for stimulating fibroblasts and endothelial cells in the tumor stroma. Activated stromal fibroblasts can in turn secrete growth factors that support tumor growth. We studied this feedback from fibroblasts to melanoma cells by overexpressing insulin-like growth factor 1 (IGF-1) with an adenoviral vector. Melanoma cells do not produce IGF-1. IGF-1 enhanced survival, migration, and growth of cells from biologically early lesions, but not from biologically late primary or metastatic lesions. Early melanoma cells were activated by IGF-1 to phosphorylate Erk1 and -2 of the mitogen-activated protein kinase pathway. IGF-1 also activated Akt, inhibited its down-stream effector GSK3-beta, and stabilized beta-catenin. Late primary and metastatic melanoma cells did not respond to growth stimulation by IGF-1 because of a constitutive activation of the mitogen-activated protein kinase pathway and a higher level of stabilized beta-catenin. These studies demonstrate that fibroblast-derived growth factors from the tumor environment can provide the malignant cells with a positive feedback through multiple mechanisms but that this stimulation is required only for cells from early and not late stages of tumor progression.

Cell Division↗
PubMed2001

Mel-CAM-specific genetic suppressor elements inhibit melanoma growth and invasion through loss of gap junctional communication.

Normal human melanocytes are interspersed singly among keratinocytes along the basement membrane of the epidermis, whereas melanoma cells readily adhere to each other during invasion of the dermis or distant organs. The tumorigenic and metastatic phenotype of melanoma cells often correlates with increased expression of cell-cell and cell-matrix adhesion receptors. Mel-CAM (MCAM, MUC 18, CD146) is a cell-cell adhesion receptor highly expressed by melanoma cells but not normal melanocytes. We show here that inhibition of Mel-CAM expression in metastatic melanoma cells using genetic suppressor elements of Mel-CAM cDNA leads to inhibition of adhesion between melanoma cells and to downregulation of the tumorigenic phenotype. Growth was not inhibited in genetic suppressor elements-transduced melanoma cells cultured in monolayers but was inhibited when cells were maintained anchorage-independently in soft agar and greatly reduced in immunodeficient mice. A three-dimensional epidermal skin equivalent model demonstrated that Mel-CAM allows melanoma cells to separate from the epidermis and invade the basement membrane zone and dermis. However, melanoma cells with little or no Mel-CAM were poorly invasive, possibly due to their loss of gap junctional communication. These results suggest the multifunctional role of a melanoma-associated cell-cell adhesion receptor in tumor progression.

Animals↗
PubMed2001

Low-level monocyte chemoattractant protein-1 stimulation of monocytes leads to tumor formation in nontumorigenic melanoma cells.

Tumors commonly produce chemokines for recruitment of host cells, but the biological significance of tumor-infiltrating inflammatory cells, such as monocytes/macrophages, for disease outcome is not clear. Here, we show that all of 30 melanoma cell lines secreted monocyte chemoattractant protein-1 (MCP-1), whereas normal melanocytes did not. When low MCP-1-producing melanoma cells from a biologically early, nontumorigenic stage were transduced to overexpress the MCP-1 gene, tumor formation depended on the level of chemokine secretion and monocyte infiltration; low-level MCP-1 secretion with modest monocyte infiltration resulted in tumor formation, whereas high secretion was associated with massive monocyte/macrophage infiltration into the tumor mass, leading to its destruction within a few days after injection into mice. Tumor growth stimulated by monocytes/macrophages was due to increased angiogenesis. Vessel formation in vitro was inhibited with mAbs against TNF-alpha, which, when secreted by cocultures of melanoma cells with human monocytes, induced endothelial cells under collagen gels to form branching, tubular structures. These studies demonstrate that the biological effects of tumor-derived MCP-1 are biphasic, depending on the level of secretion. This correlates with the degree of monocytic cell infiltration, which results in increased tumor vascularization and TNF-alpha production.

Animals↗
PubMed2001

Fibroblast growth factor-binding protein expression changes with disease progression in clinical and experimental human squamous epithelium.

Basic fibroblast growth factor (bFGF) is synthesized by a wide variety of normal and malignant cells. However, bFGF cannot exert its effects unless it gets outside of the cell. Since it lacks a signal sequence to direct secretion, the method by which cells release it remains unclear. A 17 kDa secreted binding protein for bFGF (FGF-BP, HBp-17) is expressed at high levels in squamous cell carcinoma (SCC) and transformed keratinocytes and may act as a chaperone to transport bFGF outside of the cell. In our study, FGF-BP mRNA expression in normal keratinocytes was higher than in 5/5 SCCs. Using a new monoclonal antibody, we demonstrate that FGF-BP can dimerize. Immunoassays demonstrate that normal keratinocytes have a higher level of FGF-BP than SCCs. In normal human squamous epithelium, we observed diffuse, moderate to intense cytoplasmic and membranous expression of FGF-BP. Expression decreased and became focal with disease progression to invasive cancer. Injection of immortalized but non-tumorigenic HaCaT cells transduced with FGF-BP into normal human skin xenografts failed to result in tumors. Transfection of FGF-BP into the SCCs Det 562 and FaDu did not promote tumor growth more than controls, and peri-tumoral microvessel density was lower in FGF-BP-transfected than in control tumors. Taken together, these data suggest that FGF-BP expression in squamous epithelium does not play an important role in progression to invasive carcinoma.

Antibodies, Monoclonal↗