epidermal-growth-factor and Melanoma

Emerging evidence showed that the common polymorphism (+61A>G, rs4444903) in the promoter region of epidermal growth factor (EGF) gene might be associated with melanoma susceptibility in humans. But individually published results are inconclusive. The aim of this Human Genome Epidemiology (HuGE) review and meta-analysis is to derive a more precise estimation of the association between EGF +61A>G polymorphism and melanoma risk. The PubMed, Embase, Web of Science and CBM databases were searched for all articles published up to July 1st, 2012. Seven case-control studies were included with a total of 2367 melanoma cases and 4184 healthy controls. Meta-analysis results showed that there was no significant relationship between EGF +61A>G polymorphism and the risk of melanoma (G vs A: odds ratio [OR]=1.08, 95% confidence interval [CI]: 0.91-1.28, P=0.386; GG+AG vs AA: OR=1.05, 95%CI: 0.88-1.26, P=0.580; GG vs AA+AG: OR=1.10, 95%CI: 0.81-1.49, P=0.552; GG vs AA: OR=1.06, 95%CI: 0.80-1.41, P=0.700; GG vs AG: OR=1.12, 95%CI: 0.81-1.56, P=0.494). Further subgroup analyses based on source of controls, country, detection samples, genotype methods, and Breslow thickness of tumor, we also found no significant association between EGF +61A>G polymorphism and melanoma risk. In conclusion, this meta-analysis indicates that EGF +61A>G polymorphism might not be a primary determinant in melanoma development and progression; EGF gene might be expected to interact with other genes in different signaling pathways to initiate and promote the carcinogenic process.

Topics: Case-Control Studies; Epidermal Growth Factor; Genetic Association Studies; Genetic Predisposition to Disease; Humans; Melanoma; Polymorphism, Single Nucleotide; Promoter Regions, Genetic; Risk; Tumor Burden; White People

Topics: Epidermal Growth Factor; Genetic Predisposition to Disease; Humans; Melanoma; Polymorphism, Genetic; Skin Neoplasms

Melanoma is the leading cause of death from skin tumors worldwide, with an annual increase in incidence over the past decade. The molecular mechanisms involved in melanoma pathogenesis are beginning to be unraveled. While a family history of melanoma and exposure to ultraviolet irradiation have been known for years as risk factors in melanoma development, the precise genes involved in inherited predisposition were defined only in the past decade. Germline mutations in two genes that play a pivotal role in controlling cell cycle and division--CDKN2A and cyclin-dependent kinase 4 (CDK4)--have been detected in autosomal, dominant, high penetrance familial melanoma cases. In addition to these two highly penetrant genes, germline mutations and polymorphisms in a few low penetrance genes have been reported in familial melanoma cases: melanocortin-1 receptor, epidermal growth factor, glutathione s-transferase M1, cytochrome p450 debrisoquine hydroxylase locus (CYP2D6) and vitamin D receptor.

Topics: Cytochrome P-450 CYP2D6; Epidermal Growth Factor; Genes, p16; Genetic Predisposition to Disease; Glutathione Transferase; Humans; Melanoma; Penetrance; Receptor, Melanocortin, Type 1; Receptors, Calcitriol

Predisposition to melanoma is genetically heterogeneous. Two high penetrance susceptibility genes, CDKN2A and CDK4, have so far been identified and mapping is ongoing to localize and identify others. With the advent of a catalogue of millions of potential DNA polymorphisms, attention is now also being focused on identification of genes that confer a more modest contribution to melanoma risk, such as those encoding proteins involved in pigmentation, DNA repair, cell growth and differentiation or detoxification of metabolites. One such pigmentation gene, MC1R, has not only been found to be a low penetrance melanoma gene but has also been shown to act as a genetic modifier of melanoma risk in individuals carrying CDKN2A mutations. Most recently, an environmental agent, ultraviolet radiation, has also been established as a modifier of melanoma risk in CDKN2A mutation carriers. Hence, melanoma is turning out to be an excellent paradigm for studying gene-gene and gene-environment interactions.

Topics: Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Cytochrome P-450 CYP2D6; Epidermal Growth Factor; Genes, p16; Genetic Predisposition to Disease; Glutathione Transferase; Humans; Melanoma; Mutation; Penetrance; Polymorphism, Genetic; Proto-Oncogene Proteins; Receptors, Calcitriol; Receptors, Corticotropin; Receptors, Melanocortin; Skin Pigmentation; Tumor Suppressor Protein p14ARF; Ultraviolet Rays

Topics: Animals; Brain Neoplasms; Breast Neoplasms; Cell Division; Cell Transformation, Neoplastic; Digestive System Neoplasms; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Growth Substances; Head and Neck Neoplasms; Humans; Lung Neoplasms; Melanoma; Neoplasm Proteins; Neoplasms; Neoplasms, Experimental; Oncogenes; Rats; Receptor Protein-Tyrosine Kinases; Receptors, Growth Factor; Signal Transduction; Urogenital Neoplasms

Topics: Animals; Cytokines; Epidermal Growth Factor; Fibroblast Growth Factors; Growth Substances; Humans; Melanoma; Peptides; Platelet-Derived Growth Factor; Skin Neoplasms; Transforming Growth Factors

Topics: Aminoacridines; Amsacrine; Animals; Anthracenes; Antineoplastic Agents; Blood Physiological Phenomena; Bone Marrow; Breast Neoplasms; Colony-Forming Units Assay; Drug Evaluation; Drug Evaluation, Preclinical; Drug Stability; Epidermal Growth Factor; Female; Head and Neck Neoplasms; Humans; Interferons; Karyotyping; Leukemia; Lung Neoplasms; Melanoma; Mice; Mice, Nude; Multiple Myeloma; Neoplasm Transplantation; Neoplasms; Ovarian Neoplasms; Platelet-Derived Growth Factor; Suspensions; Transplantation, Heterologous; Tumor Stem Cell Assay

Melanoma is one of the leading causes of cancer death. Kinesin Family member 22 (KIF22) is essential for the invasion of melanoma cells, but the role and mechanism of KIF22 in the proliferation and glycolysis in melanoma remains unknown.. KIF22 expression in melanoma tissues and the relationship between KIF22 high expression and overall survival rate in patients with melanoma were analyzed using the Tnmplot database. KIF22 expression in melanoma cells was examined by western blot. Then, KIF22 was silenced and CCK-8 assay, EDU staining and flow cytometry analysis were adopted for assessing cell proliferation and apoptosis. In addition, the glycolysis metabolism of melanoma cells was reflected by detecting Extracellular Acidification Rates (ECAR) and Oxygen Consumption Rates (OCR). The expression of proteins related to apoptosis, glycolysis and EGFR/STAT3 signaling was tested by western blot. Subsequently, melanoma cells were treated with EGF or Colivelin to further elucidate the regulatory effect of KIF22 on EGFR/STAT3 signaling.. KIF22 expression was notably upregulated in melanoma tissues and cells, and KIF22 high expression was associated with a poor prognosis. Moreover, KIF22 insufficiency suppressed proliferation and accelerated apoptosis of melanoma cells. Additionally, glycolysis was reduced by KIF22 depletion, evidenced by the decreased ECAR and increased OCR, accompanied by the downregulated expression of HK2, PKM2 and LDHA. Importantly, the impacts of KIF22 depletion on the progression of melanoma were partially attenuated after EGF or Colivelin treatment.. Collectively, KIF22 knockdown suppressed the proliferation and glycolysis and facilitated the apoptosis of melanoma cells by inactivating EGFR/STAT3 signaling.

Topics: Cell Line, Tumor; Cell Proliferation; DNA-Binding Proteins; Epidermal Growth Factor; ErbB Receptors; Glycolysis; Humans; Kinesins; Melanoma; STAT3 Transcription Factor

Drug resistance mechanisms still characterize metastatic melanoma, despite the new treatments that have been recently developed. Targeting of the cGMP/protein kinase G pathway is emerging as a therapeutic approach in cancer research. In this study, we evaluated the anticancer effects of two polymeric-linked dimeric cGMP analogs able to bind and activate protein kinase G, called protein kinase G activators (PAs) 4 and 5. PA5 was identified as the most effective compound on melanoma cell lines as well as on patient-derived metastatic melanoma cells cultured as three-dimensional spheroids and in a zebrafish melanoma model. PA5 was able to significantly reduce cell viability, size, and invasion of melanoma spheroids. Importantly, PA5 showed a tumor-specific outcome because no toxic effect was observed in healthy melanocytes exposed to the cGMP analog. We defined that by triggering protein kinase G, PA5 interfered with the EGF pathway as shown by lower EGFR phosphorylation and reduction of activated, phosphorylated forms of protein kinase B and extracellular signal‒regulated kinase 1/2 in melanoma cells. Finally, PA5 significantly reduced the metastatic process in zebrafish. These studies open future perspectives for the cGMP analog PA5 as a potential therapeutic strategy for melanoma.

Topics: Animals; Antineoplastic Agents; Cell Death; Cell Line, Tumor; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Humans; Melanocytes; Melanoma; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Skin Neoplasms; Zebrafish

Hotspot mutations of the oncogenes BRAF and NRas are the most common genetic alterations in cutaneous melanoma. Still, the nanoscale organization and signal coupling of these proteins remain incompletely understood, particularly upon expression of oncogenic NRas mutants. Here we employed single-molecule localization microscopy to study the nanoscale organization of NRas and BRAF at the plasma membrane (PM) of melanoma cells. NRas and BRAF resided in self-clusters that did not associate well in resting cells. In EGF-activated cells, NRas clusters became more diffused while overall protein levels at the PM increased; thus allowing enhanced association of NRas and BRAF and downstream signaling. In multiple melanoma cell lines, mutant NRas resided in more pronounced self-clusters relative to wild-type (WT) NRas yet associated more with the clustered and more abundant BRAF. In cells resistant to trametinib, a clinical MEK inhibitor (MEKi), a similar coclustering of NRas and BRAF was observed upon EGF activation. Strikingly, treatment of cells expressing mutant NRas with trametinib reversed the effect of mutant NRas expression by restoring the nonoverlapping self-clusters of NRas and BRAF and by reducing their PM levels and elevated pERK levels caused by mutant NRas. Our results indicate a new mechanism for signal regulation of NRas in melanoma through its nanoscale dynamic organization and a new mechanism for MEKi function in melanoma cells carrying NRas mutations but lacking MEK mutations. SIGNIFICANCE: Nanoscale dynamic organization of WT and mutant NRas relative to BRAF serves as a regulatory mechanism for NRas signaling and may be a viable therapeutic target for its sensitivity to MEKi.

Topics: Cell Line, Tumor; Cell Membrane; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; GTP Phosphohydrolases; Humans; MAP Kinase Kinase 1; Melanoma; Melanoma, Cutaneous Malignant; Membrane Proteins; Mutation; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Pyridones; Pyrimidinones; Signal Transduction; Single Molecule Imaging; Skin Neoplasms

Cell migration is a critical process involved in morphogenesis, inflammation, and cancer metastasis. Wound healing assay is a simple, non-expensive, and highly reproducible method to study cancer cell migration in vitro. It is based on the observation that cells growing in a monolayer migrate to re-establish cell contacts after the development of an artificial wound. The assay involves creation of a wound in a monolayer, image acquisition during wound closure, and comparison of migrated area at initial and final time points.

Topics: Cell Culture Techniques; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Humans; Melanoma; Wound Healing

The global impact of cancer emphasizes the importance of developing innovative, effective and minimally invasive therapies. In the context of superficial cancers, the development of a multifunctional nanoparticle-based system and its in vitro and in vivo safety and efficacy characterization are, herein, proposed as a proof-of-concept. This multifunctional system consists of gold nanoparticles coated with hyaluronic and oleic acids, and functionalized with epidermal growth factor for greater specificity towards cutaneous melanoma cells. This nanoparticle system is activated by a near-infrared laser. The characterization of this nanoparticle system included several phases, with in vitro assays being firstly performed to assess the safety of gold nanoparticles without laser irradiation. Then, hairless immunocompromised mice were selected for a xenograft model upon inoculation of A375 human melanoma cells. Treatment with near-infrared laser irradiation for five minutes combined with in situ administration of the nanoparticles showed a tumor volume reduction of approximately 80% and, in some cases, led to the formation of several necrotic foci, observed histologically. No significant skin erythema at the irradiation zone was verified, nor other harmful effects on the excised organs. In conclusion, these assays suggest that this system is safe and shows promising results for the treatment of superficial melanoma.

Topics: Animals; Cell Line, Tumor; Epidermal Growth Factor; Gold; Humans; Low-Level Light Therapy; Male; Melanoma; Metal Nanoparticles; Mice, SCID; Multifunctional Nanoparticles; Oleic Acid; Proof of Concept Study; Skin Neoplasms; Xenograft Model Antitumor Assays

Topics: Antineoplastic Agents, Immunological; Cetuximab; Colorectal Neoplasms; Epidermal Growth Factor; ErbB Receptors; Humans; Male; Melanoma; Middle Aged; Nevus, Pigmented; Skin Neoplasms

We present a strategy for the fabrication of biomimetic nanoarrays, based on the use of DNA origami, that permits the multivalent investigation of ligand-receptor molecule interactions in cancer cell spreading, with nanoscale spatial resolution and single-molecule control. We employed DNA origami to control the nanoscale spatial organization of integrin- and epidermal growth factor (EGF)-binding ligands that modulate epidermal cancer cell behavior. By organizing these multivalent DNA nanostructures in nanoarray configurations on nanopatterned surfaces, we demonstrated the cooperative behavior of integrin and EGF ligands in the spreading of human cutaneous melanoma cells: this cooperation was shown to depend on both the number and ratio of the selective ligands employed. Notably, the multivalent biochips we have developed allowed for this cooperative effect to be demonstrated with single-molecule control and nanoscale spatial resolution. By and large, the platform presented here is of general applicability for the study, with molecular control, of different multivalent interactions governing biological processes from the function of cell-surface receptors to protein-ligand binding and pathogen inhibition.

Topics: Biomimetic Materials; Cell Line, Tumor; DNA; Epidermal Cells; Epidermal Growth Factor; Humans; Integrins; Ligands; Melanoma; Microarray Analysis; Nanotechnology; Protein Binding; Single-Cell Analysis

Uveal melanoma (UM) is the major intraocular malignancy in adults, of which the molecular biology is still unknown. Therefore, this study was designed to determine the aqueous concentrations of angiogenic, inflammatory, and chemotactic cytokines in eyes with UM.Aqueous humor samples were collected from 38 patients with UM and 22 patients undergoing cataract surgery. Interleukin 6, 8 (IL-6, IL-8, respectively), interferon-inducible protein-10 (IP-10), placental growth factor1 (PIGF1), regulated on activation, normal T Cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), nerve growth factor-beta (NGF-β), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and vascular endothelia growth factor A (VEGF-A) were assessed by multiplex bead assay.In the study group, significantly higher concentrations of IL-6 (P = .006), IL-8 (P = .018), IP-10 (P = .004), RANTES (P = .008), MCP-1 (P = .02), NGF-β (P = .013), EGF (P < .001), PIGF1 (P = .01), bFGF (P = .016), and VEGF (P = .017) were measured, when compared with the control group.Several angiogenic, inflammatory, and chemotactic cytokines are highly expressed in the aqueous humor of the UM eyes, which provides new insights into the pathophysiology of UM and could be potential targets for treatment.

Topics: Adult; Aged; Aqueous Humor; Biomarkers, Tumor; Cataract Extraction; Cytokines; Epidermal Growth Factor; Female; Humans; Inflammation Mediators; Male; Melanoma; Middle Aged; Uveal Neoplasms; Vascular Endothelial Growth Factor A

Malignant melanoma remains a challenge for clinical practice and novel therapeutic strategies are urgently needed. Herbacetin, a natural flavonoid compound that has multiple pharmacological activities, exerts anticancer effects on several human tumors. In this study, the anti-angiogenesis effect of Herbacetin in human malignant melanoma was investigated. The results indicated that Herbacetin treatment significantly suppressed tumor growth and angiogenesis of malignant melanoma both in vitro and in vivo. In melanoma A375 and Hs294T cells, Herbacetin treatment suppressed both EGF-induced and constitutive phosphorylation of EGFR, accelerated the internalization and degradation of EGFR, and subsequently suppressed the activation of the downstream kinases (AKT and ERK). Moreover, MMP9 was determined as a key angiogenic factor in Herbacetin treated melanoma cells. Knockdown of MMP9 suppressed the in vitro angiogenesis while overexpression of MMP9 in Herbacetin treated melanoma cells restored the angiogenesis ability. We concluded that Herbacetin suppressed melanoma angiogenesis through blocking EGFR-ERK/AKT-MMP9 signaling pathway and Herbacetin may be developed as a potential drug for melanoma treatment.

Topics: Angiogenesis Inhibitors; Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Flavonoids; Humans; MAP Kinase Signaling System; Matrix Metalloproteinase 9; Melanoma; Melanoma, Cutaneous Malignant; Mice; Mice, Nude; Neovascularization, Pathologic; Skin Neoplasms; Xenograft Model Antitumor Assays

Recent studies suggest that the β-blocker drug carvedilol prevents skin carcinogenesis but the mechanism is unknown. Carvedilol is one of a few β-blockers identified as biased agonist based on an ability to promote β-arrestin-mediated processes such as ERK phosphorylation. To understand the role of phosphoproteomic signaling in carvedilol's anticancer activity, the mouse epidermal JB6 P+ cells treated with EGF, carvedilol, or their combination were analyzed using the Phospho Explorer Antibody Array containing 1318 site-specific and phospho-specific antibodies of over 30 signaling pathways. The array data indicated that both EGF and carvedilol increased phosphorylation of ERK's cytosolic target P70S6 K while its nuclear target ELK-1 were activated only by EGF; Furthermore, EGF-induced phosphorylation of ELK-1 and c-Jun was attenuated by carvedilol. Subcellular fractionation analysis indicated that ERK nuclear translocation induced by EGF was blocked by co-treatment with carvedilol. Western blot and luciferase reporter assays confirmed that the biased β-blockers carvedilol and alprenolol blocked EGF-induced phosphorylation and activation of c-Jun/AP-1 and ELK-1. Consistently, both carvedilol and alprenolol strongly prevented EGF-induced neoplastic transformation of JB6 P+ cells. Remarkably, oral carvedilol treatment significantly inhibited the growth of A375 melanoma xenograft in SCID mice. As nuclear translocation of ERK is a key step in carcinogenesis, inhibition of this event is proposed as a novel anticancer mechanism for biased β-blockers such as carvedilol.

Topics: Adrenergic beta-Antagonists; Animals; Anticarcinogenic Agents; Carcinogenesis; Carvedilol; Epidermal Growth Factor; HEK293 Cells; Humans; Male; Melanoma; Mice, Inbred NOD; Mice, SCID; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphorylation; Proteome; Proto-Oncogene Proteins c-jun

Melanoma represents one of the most aggressive forms of cancer. With the rapid increases in the incidence of melanoma in the United States, Australia and Europe over the last decades, melanoma has been considered an epidemic cancer in these areas. The aim of our study was to evaluate the utility of osteoprotegerin (OPG), osteopontin (OPN), epidermal growth factor (EGF) and vascular endothelial growth factor VEGF for the diagnosis and prognosis of melanoma.. Overall, 322 individuals were assessed: 183 melanoma patients and 139 healthy individuals. Melanoma patients were divided into four subgroups according to the Breslow score. OPN, OPG, EGF, and VEGF were determined in each plasma sample.. The serum levels of the following biomarkers were statistically significantly higher in the melanoma group compared to the control group: OPG and, OPN (p<0.0001), EGF (p=0.0379). In the first stage, OPG (p=0.0236) and OPN (p=0.0327) showed a statistically significant increase. Concerning positive and negative sentinel node metastases a statistically significant change was observed in: OPN (p<0.0001), EGF (p=0.0114), VEGF (p=0.0114).. OPG and OPN are promising biomarkers of early-stage melanoma. EGF and VEGF appear to be prognostic biomarkers.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Epidermal Growth Factor; Female; Humans; Lymphatic Metastasis; Male; Melanoma; Melanoma, Cutaneous Malignant; Middle Aged; Neoplasm Staging; Osteopontin; Osteoprotegerin; Prognosis; Skin Neoplasms; Vascular Endothelial Growth Factor A; Young Adult

It has been shown that the response of V600EBRAF melanoma cells to targeted therapeutics is affected by growth factors. We have investigated the influence of three different growth factors, bFGF, EGF and HGF used either alone or in combination, on the response of V600EBRAF melanoma cell populations established from surgical specimens to vemurafenib and trametinib, targeting V600EBRAF and MEK1/2, respectively. We report that proliferation and phenotype of V600EBRAF melanoma cell populations were not detectably influenced by exogenous growth factors. Neither cell distribution in cell cycle and CCND1 expression nor activity of signaling pathways crucial for melanoma development and maintenance, including the RAF/MEK/ERK pathway, WNT/β-catenin pathway and NF-κB signaling, were affected by the presence of different growth factors. We furthermore show that vemurafenib and trametinib abrogated the activity of ERK1/2, arrested cells in G0/G1 cell cycle phase, triggered apoptosis, induced changes in the expression of CXCL8, CCND1 and CTGF and the frequency of Ki-67high and CD271high cells. These effects were, however, similar in the presence of different growth factors. Interestingly, comparable results were also obtained for melanoma cells grown without exogenous growth factors bFGF, EGF and HGF for a period as long as 4 months prior the drug treatment. We conclude that the composition or lack of exogenous growth factors bFGF, EGF and HGF do not markedly influence viability and phenotype of V600EBRAF melanoma cells and their response to vemurafenib and trametinib in vitro. Our results question the necessity of these growth factors in the medium that is used for culturing V600EBRAF melanoma cells.

Topics: Antineoplastic Agents; Blotting, Western; Epidermal Growth Factor; Fibroblast Growth Factor 2; Flow Cytometry; Hepatocyte Growth Factor; Humans; Immunophenotyping; In Vitro Techniques; Indoles; Melanoma; Microscopy, Fluorescence; Proto-Oncogene Proteins B-raf; Pyridones; Pyrimidinones; Sulfonamides; Tumor Cells, Cultured; Vemurafenib

This case-control study aims to investigate the correlations of EGF G1380A, bFGF C754G and VEGF T460C polymorphisms with the susceptibility and prognosis of malignant melanoma. A total of 153 patients with multiple primary melanomas were collected as the case group and another 170 healthy individuals were selected as the control group. ELISA and PCR-RFLP were performed to test the serum level of VEGF and to analyze the genotype as well as allele frequencies of VEGF T460C, EGF G1380A, and bFGF C754G, respectively. The patients were assigned into complete remission (CR), partial remission (PR) and non-remission groups after treatment. HE and CD34 staining were conducted in tissue samples of CR and PR patients. Event-free survival (EFS) and overall survival (OS) were measured. AA genotype of EGF G1380A and GG genotype of bFGF C754G had higher frequency distribution in the case group than the control group. Patients with AA genotype of EGF G1380 and GG genotype of bFGF C754G had an elevated VEGF level in comparison to other genotypes. Patients with GA+GG genotypes of EGF G1380A and CG+CC genotypes of bFGF C754G had higher EFS and OS than those with AA genotype and those with GG genotype, respectively. According to the haplotype analysis, the case group had a notably higher frequency of TAG and CAG along with while lower frequency of TGG and CGC compared with the control group. Logistic regression analysis revealed that the polymorphisms of EGF G1380A and bFGF C754G as well as the haploid TAG increased the susceptibility of malignant melanoma. The results indicated that EGF G1380A and bFGF C754G gene polymorphisms were associated with the susceptibility and prognosis of malignant melanoma, and that the polymorphisms of EGF G1380A and bFGF C754G as well as the haploid TAG increased the susceptibility of malignant melanoma.

Topics: Aged; Case-Control Studies; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Haplotypes; Humans; Male; Melanoma; Middle Aged; Polymorphism, Single Nucleotide; Survival Analysis; Vascular Endothelial Growth Factor A

Cyclin-dependent kinase 2 (CDK2) is a known regulator in the cell cycle control of the G1/S and S/G2 transitions. However, the role of CDK2 in tumorigenesis is controversial. Evidence from knockout mice as well as colon cancer cell lines indicated that CDK2 is dispensable for cell proliferation. In this study, we found that ectopic CDK2 enhances Ras (G12V)-induced foci formation and knocking down CDK2 expression markedly decreases epidermal growth factor (EGF)-induced cell transformation mediated through the downregulation of c-fos expression. Interestingly, CDK2 directly phosphorylates ELK4 at Thr194 and Ser387 and regulates the ELK4 transcriptional activity, which serves as a mechanism to regulate c-fos expression. In addition, ELK4 is overexpressed in melanoma and knocking down the ELK4 or CDK2 expression significantly attenuated the malignant phenotype of melanoma cells. Taken together, our study reveals a novel function of CDK2 in EGF-induced cell transformation and the associated signal transduction pathways. This indicates that CDK2 is a useful molecular target for the chemoprevention and therapy against skin cancer.

Topics: Animals; Cell Cycle; Cell Line; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase 2; Epidermal Growth Factor; ets-Domain Protein Elk-4; Humans; Melanoma; Mice; Phosphorylation; Proto-Oncogene Proteins c-fos; Transcriptional Activation

Activating mutations in neuroblastoma RAS viral oncogene homolog (NRAS) are frequent driver events in cutaneous melanoma. NRAS is a guanosine triphosphate-binding protein whose most well-characterized downstream effector is RAF, leading to activation of mitogen-activated protein kinase (MEK)-extracellular signal-regulated protein kinase 1/2 signaling. Although there are no Food and Drug Administration-approved targeted therapies for melanoma patients with a primary mutation in NRAS, one form of targeted therapy that has been explored is MEK inhibition. In clinical trials, MEK inhibitors have shown disappointing efficacy in mutant NRAS patients, the reasons for which are unclear. To explore the effects of MEK inhibitors in mutant NRAS melanoma, we used a high-throughput reverse-phase protein array platform to identify signaling alterations. Reverse-phase protein array analysis of phospho-proteomic changes in mutant NRAS melanoma in response to trametinib indicated a compensatory increase in v-akt murine thymoma viral oncogene homolog signaling and decreased expression of mitogen-inducible gene 6 (MIG6), a negative regulator of epidermal growth factor receptor/v-erb-b2 erythroblastic leukemia viral oncogene homolog receptors. MIG6 expression did not alter the growth or survival properties of mutant NRAS melanoma cells. Rather, we identified a role for MIG6 as a negative regulator of epidermal growth factor-induced signaling and cell migration and invasion. In MEK-inhibited cells, further depletion of MIG6 increased migration and invasion, whereas MIG6 expression decreased these properties. Therefore, a decrease in MIG6 may promote the migration and invasiveness of MEK-inhibited mutant NRAS melanoma, especially in response to epidermal growth factor stimulation.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Movement; Down-Regulation; Epidermal Growth Factor; GTP Phosphohydrolases; Humans; Immunohistochemistry; MAP Kinase Kinase 1; Melanoma; Membrane Proteins; Mutation; Skin Neoplasms; Tumor Suppressor Proteins

The understanding of melanoma malignancy mechanisms is essential for patient survival, because melanoma is responsible for ca. 75% of deaths related to skin cancers. Enhanced formation of invadopodia and extracellular matrix (ECM) degradation are two important drivers of cell invasion, and actin dynamics facilitate protrusive activity by providing a driving force to push through the ECM. We focused on the influence of epidermal growth factor (EGF), hepatocyte growth factor (HGF) and transforming growth factor β (TGFβ) on melanoma cell invasiveness, since they are observed in the melanoma microenvironment. All three factors stimulated invasion of A375 and WM1341D cells derived from primary tumor sites. In contrast, only EGF and HGF stimulated invasion of WM9 and Hs294T cells isolated from lymph node metastases. Enhanced formation of invadopodia and ECM degradation underlie the increased amount of invasive cells after stimulation with the tested agents. Generally, a rise in invasive potential was accompanied by a decrease in actin polymerization state (F:G ratio). The F:G ratio remained unchanged or was even increased in metastatic cell lines treated with TGFβ. Our findings indicate that the effects of stimulation with EGF, HGF and TGFβ on melanoma cell invasiveness could depend on melanoma cell progression stage.

Topics: Cell Line, Tumor; Cell Membrane Structures; Epidermal Growth Factor; Hepatocyte Growth Factor; Humans; Melanoma; Neoplasm Invasiveness; Transforming Growth Factor beta; Tumor Microenvironment

Cutaneous melanoma (CM) is a malignant skin cancer with the high incidence in whiteskinned populations. Host genetic factors (such as: genes in nucleotide excision repair system or cell proliferation regulation system) interacted with ultraviolet radiation are potential reasons for CM. Previous studies about associations between CM and the rs4444903 (+61A>G) in the Epidermal growth factor gene (EGF) or rs13181 (+35931 A>C) in the xeroderma pigmentosum group D gene (XPD) have produced inconsistent results. To clarify these associations, metaanalyses of available candidate case-control association studies about Caucasians were performed. Data of each study were gathered according to the "Quality-Evaluation Score" (Ver.1.0). Finally, the meta-analysis with 2167 cases/4211 controls showed that the EGF rs4444903 had no significant association with CM (p>0.05), while the analysis with 3,492 cases/5,381 controls indicated the A allele of XPD rs13181 was significantly associated with CM (odds ratio= 0.93, 95% CI: 0.87-0.99; p=0.019). These results are also supported with linkage disequilibrium (LD) structure analysis. The current meta-analyses results suggest that the XPD gene, but not the EGF gene, has contributed to CM susceptibility, and XPD is a possible drug target.

Topics: Epidermal Growth Factor; Humans; Melanoma; Polymorphism, Genetic; Skin Neoplasms; White People; Xeroderma Pigmentosum Group D Protein

FLNa is a ubiquitous cytoskeletal protein that links transmembrane receptors, including integrins, to F-actin and functions as a signalling intermediate. We investigated FLNa's role in the function of integrin-type collagen receptors, EGF-EGFR signalling and regulation of PKB/Akt and ERK1/2. Using FLNa-deficient M2 human melanoma cells, and same cells expressing EGFP-FLNa (M2F) or its Ig-like repeats 1-8+24, 8-15+24 and 16-24, we found that in M2F and M2 8-15+24 cells, EGF induced the increased phosphorylation of PKB/Akt and ERK1/2. In M2F cells EGF induced the localisation of these kinases to cell nucleus and lamellipodia, respectively, and the ERK1/2 phosphorylation-dependent co-immunoprecipitation of FLNa with ERK1/2. Only M2F and M2 8-15+24 cells adhered to and spread on type I collagen whereas on fibronectin all cells behaved similarly. α1β1 and α2β1 were the integrin-type collagen receptors expressed on these cells with primarily α1β1 localising to focal contacts and affecting cell adhesion and migration in a manner dependent on FLNa or its Ig-like repeats 8-15. Our results suggest a role for FLNa repeats 8-15 in the α1-subunit-dependent regulation of integrin α1β1 function, EGF-EGFR signalling to PKB/Akt and ERK1/2, identify ERK1/2 in EGF-induced FLNa-associated protein complexes, and show that the function of different integrins is subjected to differential regulation by FLNa.

Topics: Cell Adhesion; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Filamins; Humans; Integrin alpha1beta1; Melanoma; Phosphorylation; Protein Processing, Post-Translational; Protein Transport; Proto-Oncogene Proteins c-akt

Malignant melanoma has the highest risk of mortality among all types of skin cancer due to its highly metastatic potential. The ocular albinism type 1 (OA1) protein is a pigment cell‑specific glycoprotein, which shares significant structural and functional features with G protein‑coupled receptors. However, the role of OA1 in melanoma has yet to be elucidated. The present study aimed to investigate whether OA1 is involved in melanoma cell migration. OA1 was found to stimulate cell migration in a dose‑dependent manner in cultured human melanoma cells. Furthermore, knockdown of OA1 using small interfering RNA was observed to significantly inhibit melanoma cell migration. In addition, the mechanism underlying OA1‑induced melanoma cell migration was investigated. Stimulation of the RAS/RAF/mitogen activated protein kinase kinase (MEK)/extracellular signal‑regulated kinase (ERK) pathway using growth factors enhanced OA1 expression and melanoma cell migration, whereas inhibition of this pathway using U0126 was observed to markedly decrease OA1 expression and the number of migrated cells. These findings indicate that OA1 is involved in melanoma cell migration and that OA1‑induced melanoma cell migration is mediated through the RAS/RAF/MEK/ERK signaling pathway. Therefore, OA1 may serve as a novel therapeutic target for melanoma.

Topics: Butadienes; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Eye Proteins; Humans; Melanoma; Membrane Glycoproteins; Mitogen-Activated Protein Kinase Kinases; Nitriles; Platelet-Derived Growth Factor; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-raf; ras Proteins; RNA Interference; RNA, Messenger; RNA, Small Interfering; Signal Transduction

Tenascin-C (TNC), overexpressed in invasive growths, has been implicated in progression of melanoma, but the source and function of this molecule are not well defined. We found TNC expression at the front of invading melanoma cells, and that adding TNC to matrices enhances individual melanoma cell migration. As TNC is a multidomain protein, we examined the role of the TNC EGF-like (EGFL) repeats as these activate motogenic signaling cascades. We overexpressed a TNC fragment containing the assembly and EGFL domains of TNC (TNCEGFL). TNCEGFL-expressing melanoma cells had lower speed and persistence in 2D migration assays due to a shift in the adhesion-contractility balance, as expression of TNCEGFL delayed melanoma cell attachment and spreading. The less adhesive phenotype was due, in part, to increased Rho-associated kinase (ROCK) signaling concomitant with myosin light chain 2 and MYPT phosphorylation. Inhibition of ROCK activity, which drives transcellular contractility, restored adhesion of TNCEGFL-expressing melanoma cells and increased their migration in 2D. In contrast to the diminished migration in 2D, TNCEGFL-expressing melanoma cells had higher invasive potential in Matrigel invasion assays, with cells expressing TNCEGFL having amoeboid morphology. Our findings suggest that melanoma-derived TNCEGFL exert a role in melanoma invasion by modulating ROCK signaling and cell migration.

Topics: Cardiac Myosins; Cell Adhesion; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Humans; Melanoma; Myosin Light Chains; Neoplasm Invasiveness; Peptide Fragments; Phosphorylation; rho-Associated Kinases; Signal Transduction; Skin Neoplasms; Tenascin

Alterations in epidermal growth factor (EGF) expression are known to be of prognostic relevance in human melanoma, but EGF-mediated effects on melanoma have not been extensively studied. As lymph node metastasis usually represents the first major step in melanoma progression, we were trying to identify a potential role of primary tumor-derived EGF in the mediation of melanoma lymph node metastases. Stable EGF knockdown (EGFkd) in EGF-high (M24met) and EGF-low (A375) expressing melanoma cells was generated. Only in EGF-high melanoma cells, EGFkd led to a significant reduction of lymph node metastasis and primary tumor lymphangiogenesis in vivo, as well as impairment of tumor cell migration in vitro. Moreover, EGF-induced sprouting of lymphatic but not of blood endothelial cells was abolished using supernatants of M24met EGFkd cells. In addition, M24met EGFkd tumors showed reduced vascular endothelial growth factor-C (VEGF-C) expression levels. Similarly, in human primary melanomas, a direct correlation between EGF/VEGF-C and EGF/Prox-1 expression levels was found. Finally, melanoma patients with lymph node micrometastases undergoing sentinel node biopsy were found to have significantly elevated EGF serum levels as compared with sentinel lymph node-negative patients. Our data indicate that tumor-derived EGF is important in mediating melanoma lymph node metastasis.

Topics: Animals; Biomarkers, Tumor; Cell Movement; Endothelial Cells; Epidermal Growth Factor; Female; Gene Knockdown Techniques; Homeodomain Proteins; Humans; Lymphangiogenesis; Lymphatic Metastasis; Melanoma; Mice; Mice, SCID; Neoplasm Micrometastasis; Sentinel Lymph Node Biopsy; Skin Neoplasms; Tumor Cells, Cultured; Tumor Suppressor Proteins; Vascular Endothelial Growth Factor C

Discerning lymphatics from blood vessels using lineage-specific markers has advanced our understanding of tumor lymphangiogenesis. Many studies have demonstrated that the newly formed lymphatics are the result of a dynamic and complex interplay between lymphatic endothelium, tumor cells, secreted growth factors, and inhibitors in the tumor microenvironment. Results provide evidence that lymphatics play an active role in cancer metastasis rather than once thought to be passively invaded by infiltrating tumor cells. The latest discovery of Bracher et al. (2012) further supports a dynamic role for lymphatics-mediated melanoma metastasis to sentinel lymph node prompted by tumor-derived epidermal growth factor.

Topics: Animals; Epidermal Growth Factor; Female; Humans; Lymphangiogenesis; Melanoma; Skin Neoplasms; Vascular Endothelial Growth Factor C

BRAF(V600E) drives tumors by dysregulating ERK signaling. In these tumors, we show that high levels of ERK-dependent negative feedback potently suppress ligand-dependent mitogenic signaling and Ras function. BRAF(V600E) activation is Ras independent and it signals as a RAF-inhibitor-sensitive monomer. RAF inhibitors potently inhibit RAF monomers and ERK signaling, causing relief of ERK-dependent feedback, reactivation of ligand-dependent signal transduction, increased Ras-GTP, and generation of RAF-inhibitor-resistant RAF dimers. This results in a rebound in ERK activity and culminates in a new steady state, wherein ERK signaling is elevated compared to its initial nadir after RAF inhibition. In this state, ERK signaling is RAF inhibitor resistant, and MEK inhibitor sensitive, and combined inhibition results in enhancement of ERK pathway inhibition and antitumor activity.

Topics: Cell Line, Tumor; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Fibroblast Growth Factors; Gene Expression Regulation, Neoplastic; Hepatocyte Growth Factor; Humans; Indoles; Intracellular Signaling Peptides and Proteins; Ligands; MAP Kinase Signaling System; Melanoma; Membrane Proteins; Neuregulins; Proto-Oncogene Proteins B-raf; ras Proteins; Receptors, Growth Factor; Sulfonamides; Vemurafenib

The oncoprotein c-Jun is one of the components of the activator protein-1 (AP-1) transcription factor complex. AP-1 regulates the expression of many genes and is involved in a variety of biological functions such as cell transformation, proliferation, differentiation and apoptosis. AP-1 activates a variety of tumor-related genes and therefore promotes tumorigenesis and malignant transformation. Here, we found that epidermal growth factor (EGF) induces phosphorylation of c-Jun by P21-activated kinase (PAK) 2. Our data showed that PAK2 binds and phosphorylates c-Jun at five threonine sites (Thr2, Thr8, Thr89, Thr93 and Thr286) in vitro and ex vivo. Knockdown of PAK2 in JB6 Cl41 (P+) cells had no effect on c-Jun phosphorylation at Ser63 or Ser73 but resulted in decreases in EGF-induced anchorage-independent cell transformation, proliferation and AP-1 activity. Mutation at all five c-Jun threonine sites phosphorylated by PAK2 decreased the transforming ability of JB6 cells. Knockdown of PAK2 in SK-MEL-5 melanoma cells also decreased colony formation, proliferation and AP-1 activity. These results indicated that PAK2/c-Jun signaling plays an important role in EGF-induced cell proliferation and transformation.

Topics: Animals; Blotting, Western; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Epidermal Cells; Epidermal Growth Factor; Epidermis; Humans; Immunoenzyme Techniques; Immunoprecipitation; Melanoma; Mice; Mutation; p21-Activated Kinases; Phosphorylation; Proto-Oncogene Proteins c-jun; RNA, Small Interfering; Skin; Skin Neoplasms; Threonine; Tissue Array Analysis; Transcription Factor AP-1

RasGRP3, an activator for H-Ras, R-Ras and Ras-associated protein-1/2, has emerged as an important mediator of signaling downstream from receptor coupled phosphoinositide turnover in B and T cells. Here, we report that RasGRP3 showed a high level of expression in multiple human melanoma cell lines as well as in a subset of human melanoma tissue samples. Suppression of endogenous RasGRP3 expression in these melanoma cell lines reduced Ras-GTP formation as well as c-Met expression and Akt phosphorylation downstream from hepatocyte growth factor (HGF) or epidermal growth factor (EGF) stimulation. RasGRP3 suppression also inhibited cell proliferation and reduced both colony formation in soft agar and xenograft tumor growth in immunodeficient mice, demonstrating the importance of RasGRP3 for the transformed phenotype of the melanoma cells. Reciprocally, overexpression of RasGRP3 in human primary melanocytes altered cellular morphology, markedly enhanced cell proliferation and rendered the cells tumorigenic in a mouse xenograft model. Suppression of RasGRP3 expression in these cells inhibited downstream RasGRP3 responses and suppressed cell growth, confirming the functional role of RasGRP3 in the altered behavior of these cells. The identification of the role of RasGRP3 in melanoma highlights its importance, as a Ras activator, in the phosphoinositide signaling pathway in human melanoma and provides a new potential therapeutic target.

Topics: Animals; Cell Proliferation; Enzyme Inhibitors; Epidermal Growth Factor; Gene Knockdown Techniques; Guanine Nucleotide Exchange Factors; Hepatocyte Growth Factor; Humans; Male; Melanoma; Mice; Mice, Nude; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-met; ras Guanine Nucleotide Exchange Factors; Signal Transduction; Skin Neoplasms; Xenograft Model Antitumor Assays

This study was conducted to explore the relationship between different treatment-modulated EGFR expression and gefitinib sensitivity.. Gefitinib-sensitive (A431) and -resistant (A375, MALME-3M, and SK-MEL 5) tumour cell lines were treated with epidermal growth factor (EGF), gefitinib or radiation in vitro, and EGFR expression levels were measured by using ELISA.. EGF, and gefitinib treatment resulted in significantly higher levels of total and/or phosphorylated EGFR in sensitive than in resistant tumours and this was associated with gefitinib IC(50). In contrast, radiation-modulated EGFR expression, both total and phosphorylated, did not correlate with the efficacy of gefitinib. Stimulation of proliferation by EGF was significantly stronger in A431 than in the other three lines, indicating sensitive tumours were more EGFR-dependent than resistant tumours for cell proliferation.. These findings imply a potential role of EGF- and gefitinib-modulated EGFR expression in predicting gefitinib sensitivity.

Topics: Antineoplastic Agents; Cell Growth Processes; Cell Line, Tumor; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Melanoma; Neoplasms; Protein Kinase Inhibitors; Quinazolines

A single nucleotide polymorphism (61A>G) in the epidermal growth factor (EGF) gene has been implicated in both melanoma pathogenesis and increased melanoma risk. To further evaluate this association, we conducted a case-control study in a clinic-based Italian population.. Individuals with less than 10 (N = 127) or more than 100 (N = 128) benign nevi, and patients with cutaneous melanoma (N = 418) were investigated for the EGF +61A>G polymorphism, using an automated sequencing approach.. Overall, no difference in EGF genotype frequencies was observed among subjects with different number of nevi as well as when non-melanoma healthy controls were compared with the melanoma patients. However, a heterogeneous distribution of the frequencies of the G/G genotype was detected among cases and controls originating from North Italy (21.1 and 18.3%, respectively) vs. those from South Italy (12.6 and 17.1%, respectively).. Our findings further suggest that EGF +61A>G polymorphism may have a limited impact on predisposition and/or pathogenesis of melanoma and its prevalence may vary in different populations.

Topics: Adult; Epidermal Growth Factor; Female; Genetic Predisposition to Disease; Genotype; Humans; Italy; Male; Melanoma; Middle Aged; Polymorphism, Single Nucleotide; Prevalence; Risk Factors; Skin Neoplasms; Young Adult

The diagnosis of melanoma is becoming ever more frequent. Although surgical excision of early lesions is associated with relatively significant high cure rates, treatment modalities are largely unsuccessful for advanced disease. Characteristics such as cellular heterogeneity and plasticity, expression of certain molecules such as the multidrug resistance protein-1 (MDR1) or the aberrant expression of embryonic signaling molecules and morphogens like Nodal, important for self renewal and pluripotency, suggest that a stem cell-like population may reside in aggressive melanomas. This perspective focuses on preliminary findings obtained in our laboratory which indicate that the expression of the Nodal coreceptor, Cripto-1, in a subset of malignant melanoma cells may be exploited to identify possible melanoma stem cells (MSC). In fact, the use of anti-Cripto-1 antibodies to cell sort Cripto-1-positive cells in the metastatic melanoma cell line C8161 has identified a slow growing, sphere forming subpopulation that expresses increased levels of Oct4, Nanog and MDR1. If current in vivo studies confirm the self renewal and tumorigenic characteristics of these cells, the expression of Cripto-1 may represent a useful marker to identify cancer stem cells in melanoma, and possibly other aggressive tumors as well.

Topics: ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biomarkers, Tumor; Cell Line, Tumor; Epidermal Growth Factor; Epigenesis, Genetic; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Neoplastic Stem Cells; Nodal Protein; Octamer Transcription Factor-3; Protein Kinases; Signal Transduction; Skin Neoplasms; Transforming Growth Factor beta

Topics: Adenine; Epidermal Growth Factor; Guanine; Humans; Melanoma; Osmolar Concentration; Polymorphism, Genetic; Prognosis; Skin Neoplasms

The Src homology and collagen (Src) family of adaptor proteins comprises six Shc-like proteins encoded by three loci in mammals (Shc, Rai, and Sli). Shc-like proteins are tyrosine kinase substrates, which regulate diverse signaling pathways and cellular functions, including Ras and proliferation (p52/p46Shc), phosphatidylinositol 3-kinase and survival (p54Rai), and mitochondrial permeability transition and apoptosis (p66Shc). Here, we report the identification, cloning, and sequence characterization of a new member of the Shc family that we termed RaLP. RaLP encodes a 69-kDa protein characterized by the CH2-PTB-CH1-SH2 modularity, typical of the Shc protein family, and expressed, among adult tissues, only in melanomas. Analysis of RaLP expression during the melanoma progression revealed low expression in normal melanocytes and benign nevi, whereas high levels of RaLP protein were found at the transition from radial growth phase to vertical growth phase and metastatic melanomas, when tumor cells acquire migratory competence and invasive potential. Notably, silencing of RaLP expression in metastatic melanomas by RNA interference reduced tumorigenesis in vivo. Analysis of RaLP in melanoma signal transduction pathways revealed that (a) when ectopically expressed in RaLP-negative melanocytes and nonmetastatic melanoma cells, it functions as a substrate of activated insulin-like growth factor-1 and epidermal growth factor receptors and increases Ras/mitogen-activated protein kinase (MAPK) signaling and cell migration, whereas (b) its silencing in RaLP-positive melanoma cells abrogates cell migration in vitro, without affecting MAPK signaling, suggesting that RaLP activates both Ras-dependent and Ras-independent migratory pathways in melanomas. These findings indicate that RaLP is a specific marker of metastatic melanomas, a critical determinant in the acquisition of the migratory phenotype by melanoma cells, and a potential target for novel anti-melanoma therapeutic strategies.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Cell Growth Processes; Cell Line, Tumor; Cell Movement; Cloning, Molecular; Down-Regulation; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Humans; Insulin-Like Growth Factor I; MAP Kinase Signaling System; Melanoma; Mitogen-Activated Protein Kinases; Molecular Sequence Data; Receptor, IGF Type 1; RNA, Messenger; Shc Signaling Adaptor Proteins; src-Family Kinases; Transfection

Human Cripto, the founder member of the epidermal growth factor-Cripto-FRL1-Cryptic (EGF-CFC) family, plays an important role during early embryonic development and in particular in carcinogenesis and the development of cancer metastases. Cripto-1 is over-expressed in most cancers, but is absent or only weakly expressed in normal cells. For this reason, Cripto-1 could be of potential value in the targeted treatment. There is no information on the expression of Cripto-1 in human uveal melanoma. Cripto-1 reactivity was evaluated by immunohistochemistry on 36 archival uveal melanomas using the polyclonal antibody to Cripto-1. The tumors were divided in to 2 groups. There were 18 uveal melanomas with no intrascleral or extrascleral extension and 18 uveal melanomas with intrascleral/extrascleral extension/liver metastasis. Cripto-1 reactivity was correlated with tumor aggressiveness and cell type. Furthermore, we studied the immunolocalization of Cripto-1 in 4 uveal melanoma cell lines OCM-1, OCM-8, and 92-1, and OMM-1 and in 2 primary uveal melanocyte cultures. Cripto-1 was expressed in both the non-invasive and aggressive uveal melanomas. Cripto-1 was positive in the 4 uveal melanoma cell lines and absent in the primary uveal melanocyte cultures. Retinal tissue did not express Cripto-1. The results suggest that Cripto-1 is expressed in uveal melanoma, negative in the non-neoplastic ocular tissue and point to its use as a target for therapy.

Topics: Adult; Aged; Biomarkers, Tumor; Epidermal Growth Factor; Eye; Female; GPI-Linked Proteins; Humans; Immunoenzyme Techniques; Intercellular Signaling Peptides and Proteins; Liver Neoplasms; Male; Melanocytes; Melanoma; Membrane Glycoproteins; Middle Aged; Neoplasm Invasiveness; Neoplasm Proteins; Sclera; Tumor Cells, Cultured; Uveal Neoplasms

The immunoreactivity of epidermal growth factor receptor (EGFR) ezrin, hepatocyte growth factor receptor (HGF), and c-Met was studied in 60 uveal melanomas and was correlated with clinicopathologic parameters. Metastases were diagnosed in the patients with uveal melanoma between 5 years and 8 years (median, 6.5 years) after enucleation. Using Kaplan-Meier statistical analysis, we found a significant association between high c-Met expression and death due to uveal melanoma (p < 0.03). EGFR was expressed in 18 of 60 (30%) tumors; ezrin was expressed in 30 of 60 (50%) tumors. Tumors with liver metastasis (n = 6) showed higher expression of c-Met (p = 0.0009) compared with the tumors with no extension/extrascleral extension without liver metastasis (groups A-45 and B-9). HGF was negative in all the six tumors that had liver metastasis. Further studies are required to understand the possible mechanism of ligand-independent c-Met activation in patients with uveal melanoma.

Topics: Adolescent; Adult; Aged; Biomarkers, Tumor; Child; Cytoskeletal Proteins; Epidermal Growth Factor; Female; Hepatocyte Growth Factor; Humans; Immunoenzyme Techniques; Liver Neoplasms; Lymphocytes, Tumor-Infiltrating; Male; Melanoma; Middle Aged; Proto-Oncogene Proteins c-met; Survival Rate; Uveal Neoplasms

Like phosphorylation, protein sumoylation likely represents a dynamic PTM to alter protein function in support of cell regulatory systems. The broad-spectrum impact of transient or chronic engagement of signal transduction cascades on protein sumoylation has not been explored. Here, we find that epidermal growth factor (EGF) stimulation evokes a rapid alteration in small ubiquitin modifier (SUMO) target selection, while oncogene expression alters steady-state SUMO-protein profiles. A proteomic SUMO target analysis in melanoma cells identified proteins involved in cellular signaling, growth control, and neural differentiation.

Topics: Cell Line; Cell Line, Tumor; Chromatography, High Pressure Liquid; Epidermal Growth Factor; Gene Expression; Humans; Intracellular Signaling Peptides and Proteins; Melanoma; Nerve Tissue Proteins; NF-kappa B p50 Subunit; Nucleoproteins; Proteins; Proteome; Proto-Oncogene Proteins B-raf; RNA, Small Interfering; Signal Transduction; SUMO-1 Protein; Tandem Mass Spectrometry; Transcription Factors; Transfection

Epidermal growth factor (EGF) receptor family members are expressed by tumor cells and contribute to tumor progression. The expression and activity of EGF receptors in endothelial cells are less well characterized. Analysis of tumor-derived endothelial cells showed that they express EGFR, ErbB2, and ErbB4, whereas their normal counterparts express ErbB2, ErbB3, and ErbB4. The gain in expression of EGFR and the loss of ErbB3 expression in tumor vasculature was also observed in vivo. As a consequence of their expressing EGFR, tumor endothelial cells responded to EGF and other EGF family members by activating both EGFR and ErbB2, by activating the downstream mitogen-activated protein kinase pathway, and by enhanced proliferation. On the other hand, normal endothelial cells did not respond to EGF but instead were responsive to neuregulin (NRG), a ligand for ErbB3 and ErbB4. NRG activated ErbB3 in normal endothelial cells and inhibited growth of these cells. In contrast, tumor endothelial cells, which do not express ErbB3, were not growth inhibited by NRG. Furthermore, due to their expression of EGFR, tumor endothelial cells, unlike normal endothelial cells, are direct targets for EGFR kinase inhibitors. These low-molecular-weight compounds block EGF-induced EGFR activation and proliferation of tumor endothelial cells. These results suggest that a gain of EGF-induced endothelial cell proliferation, and loss of NRG-induced growth inhibition in tumor endothelial cells constitutes a switch that promotes tumor angiogenesis. In addition, these results suggest that EGFR kinase inhibitors may be effective for antiangiogenesis therapy by specifically targeting the tumor, but not the normal, vasculature.

Topics: Animals; Cell Growth Processes; Cell Line, Tumor; Endothelial Cells; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Melanoma; Mice; Mice, Nude; Neovascularization, Pathologic; Protein Kinase Inhibitors; Receptor, ErbB-3

An earlier study reported that a common polymorphism in the 5' untranslated region of the epidermal growth factor (EGF) gene is associated with increased risk for cutaneous malignant melanoma (MM) and Breslow thickness. Since then, several independent studies have reported conflicting results that have challenged this hypothesis. However, none of the previous studies examined survival as the primary outcome. We therefore sought to study the association between this polymorphism and survival. One hundred and thirty patients diagnosed with MM with a Breslow thickness of >1.5 mm were included in this study. In our collective, the G/G genotype represented a significant risk factor for both shorter disease-free period (hazard ratio of 2.246, 95% CI: 1.06-4.78, P=0.036) and overall MM-specific survival (hazard ratio of 3.8, 95% CI: 1.5-9.5, P=0.004) compared with the A/A genotype, while the heterozygous A/G genotype showed an intermediate risk. In the present study, we demonstrate for the first time that the EGF A61G polymorphism is associated with survival. Our data suggest that this polymorphism is a potential marker for disease severity that predicts earlier progression of MM.

Topics: 5' Untranslated Regions; Adult; Aged; Epidermal Growth Factor; Female; Genotype; Humans; Male; Melanoma; Middle Aged; Polymorphism, Genetic; Skin Neoplasms

Altered signaling pathways are key regulators of cellular functions in tumor cells. Constitutive activation of signal transducer and activator of transcription (STAT)3 and -5 may be involved in tumor formation and progression. We have investigated the role of STAT5 in cutaneous melanoma metastases using various RNA and protein techniques. In melanoma specimens, Stat5b transcripts were upregulated approximately 3.8-fold. In 13 of 21 (62%) human melanoma metastases, STAT5 was phosphorylated in comparison to normal human melanocytes and benign nevi. The STAT5 target gene Bcl-2 was frequently upregulated. The investigation of the underlying mechanism revealed specific STAT5 activation by recombinant human epidermal growth factor (rEGF). rEGF-induced activation of STAT5 occurred in vitro through the non-receptor tyrosine kinases transforming gene (src) of Rous Sarcoma virus and Janus kinase 1. Inhibition of Stat5b expression by small interfering RNA strongly reduced the expression of Bcl-2 and led to decreased cell viability and increased apoptosis in the melanoma cell lines A375 and BLM. Transfection with dominant-negative Stat5b caused enhanced cell death and G1 arrest in A375 cells. Our study identifies phosphorylated STAT5 in melanoma and shows regulation through rEGF; STAT5 may thus act as a survival factor for growth of human melanoma and may represent a potential target for molecular therapy.

Topics: Aged; Aged, 80 and over; Apoptosis; Cell Line, Tumor; DNA; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Janus Kinase 1; Male; Melanoma; Middle Aged; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; src-Family Kinases; STAT5 Transcription Factor

The epidermal growth factor receptor (EGFR) plays a central role in cell life by controlling processes such as growth or proliferation. This receptor is commonly overexpressed in a number of epithelial malignancies and its upregulation is often associated with an aggressive phenotype of the tumor. Thus, targeting of EGFR represents a very promising challenge in oncology, and antibodies raised against this receptor have been investigated as potential antitumor agents. Various putative mechanisms of action were proposed for such antibodies, including decreased proliferation, induction of apoptosis, stimulation of the immunological response against targeted cancer cells or combinations thereof. We report here the development of an alternative high affinity molecule that is directed against EGFR. Production of this pentameric protein, named peptabody-EGF, includes expression in a bacterial expression system and subsequent refolding and multimerization of peptabody monomers. The protein complex contains 5 human EGF ligand domains, which confer specific binding towards the extracellular portion of EGFR. Receptor binding of the peptabody-EGF had a strong antiproliferative effect on different cancer cell lines overexpressing EGFR. However, cells expressing constitutive levels of the target receptor were barely affected. Peptabody-EGF treated cancer cells exhibited typical characteristics of apoptosis, which was found to be induced within 30 min after the addition of the peptabody-EGF. In vitro experiments demonstrated a significantly higher binding activity for peptabody-EGF than for the therapeutic monoclonal EGFR antibody Mab-425. Furthermore, the antitumor action provoked by the peptabody-EGF was greatly superior than antibody mediated effects when tested on EGFR overexpressing cancer cell lines. These findings suggest a potential application of this high affinity molecule as a novel tool for anti-EGFR therapy.

Topics: Amino Acid Sequence; Annexin A5; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Molecular Sequence Data; Up-Regulation

Betulinic acid (BA), a pentacyclic triterpene first identified less than a decade ago, has served as a melanoma-specific cytotoxic agent, and yet its specificity is being challenged. Recently, we found that human melanoma cells exhibited less sensitivity to betulinic acid than human skin keratinocytes. This study was designed to investigate the cell signaling pathway leading human melanoma cells to increased resistance to betulinic acid treatment. In vitro experiments using cultured human melanoma cells indicated that betulinic acid transiently induced survivin expression. The expression of survivin started 30 min post-betulinic acid treatment, peaked at 2 h, remained elevated for 8 h and returned to basal level within 24 h. Similarly, epithelial growth factor (EGF) treatment induced expression of survivin in a time-dependent manner. Since epithelial growth factor receptor (EGFR) activation leads to the activation of cell signaling components that are important to cell survival, we next examined whether BA-induced survivin expression is mediated by the EGFR pathway. The results showed that BA induced EGFR tyrosine phosphorylation in a time-dependent manner. Further, BA strongly induced AKT phosphorylation in a similar pattern. AKT activation started 15 min post-treatment, peaked at approximately 1 h, remained elevated for 4 h and returned to basal level within 8 h. BA also induced ERK activation and, in contrast, weakly induced JNK and p38 activation. Pretreatment of EGFR inhibitor PD153035 blocked BA-induced EGFR phosphorylation, ERK and AKT activation, and survivin expression. Results of the MTT dye assay showed that a combination of PD153035 and BA enhanced melanoma cell death. Collectively, we conclude that betulinic acid transiently activated the EGFR/AKT cell survival pathway and induced survivin expression, contributing to less sensitivity in human melanoma cells. The data suggest that a combination of the EGFR inhibitor and betulinic acid may be a better clinical option to treat human melanoma.

Topics: Animals; Antineoplastic Agents, Phytogenic; Betulinic Acid; Blotting, Western; Cell Line; Cell Line, Tumor; Cell Survival; Cells, Cultured; Drug Resistance, Neoplasm; Drug Synergism; Embryo, Mammalian; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Humans; Inhibitor of Apoptosis Proteins; Keratinocytes; Melanoma; Mice; Mice, Knockout; Microtubule-Associated Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Proteins; Pentacyclic Triterpenes; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Quinazolines; Survivin; Time Factors; Triterpenes

We previously reported that fibroblasts induce human microvascular endothelial cells (HMVECs) to differentiate from monolayer to capillarylike morphology. We now test the hypothesis that fibroblasts modulate angiogenesis in melanoma cells.. We tested 12 human melanoma lines (2 radial growth phase (RGP), 3 vertical growth phase (VGP), and 7 metastatic (MM)) for ability to induce HMVECs to invade/migrate into collagen and form capillarylike networks. HMVEC monolayers were overlaid with 3-dimensional collagen gels embedded with melanoma cells alone (M), fibroblasts alone (F), or a 1:1 mixture of the 2 cells (M+F). After 5 days, gels were removed, fixed, and HMVEC networks were quantified by von Willebrand's factor (vWF) immunofluorescence. The influence of soluble factors on HMVEC invasion/migration into collagen was assessed with the use of acellular 3-D collagen gels overlaid on HMVEC monolayers, cultured with conditioned media (CM) derived from monolayers of M, F, or M+F. Angiogenic growth factors involved in the observed invasion/migration were identified with the use of a RayBio Cytokine Antibody Array (RayBiotech, Norcross, Ga).. Cell line-specific variability in melanoma-supported angiogenesis was observed only when in combination with fibroblasts (analysis of variance [ANOVA], P < .01). Melanoma plus fibroblasts uniformly resulted in a significantly higher angiogenic response than melanoma alone (P < .05). One vertical growth phase and one metastatic melanoma line, while weakly angiogenic alone, induced significantly higher angiogenesis than either fibroblast or melanoma alone (P < .05) when combined with fibroblasts. CM from M or M+F induced significantly less HMVEC invasion/migration into collagen than CM from fibroblasts alone. Interleukin 8, monocyte chemotactic protein-1, and tissue inhibitor of metalloproteinase-2 were identified as significantly elevated in the media derived from M+F cultures, compared with either cell type alone.. To our knowledge, this is the first report demonstrating that melanoma-supported angiogenesis in collagen is more significantly influenced by normal skin-derived fibroblasts than by the intrinsic biology of the melanoma cell type. Interleukin 8, monocyte chemotactic protein-1, and tissue inhibitor of metalloproteinase-2 are implicated as potential paracrine factors regulating this observed effect.

Topics: Angiogenic Proteins; Becaplermin; Cell Line, Tumor; Cells, Cultured; Collagen; Culture Media, Conditioned; Endothelium, Vascular; Epidermal Growth Factor; Extracellular Matrix; Fibroblasts; Humans; Melanoma; Neovascularization, Pathologic; Neovascularization, Physiologic; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Transforming Growth Factor beta; Umbilical Veins

Melanomas are heterogeneous tumours, and differentiation from other melanocytic lesions may cause problems. It may be possible that the distribution and/or colocalization pattern of different markers in the lesions can enable a more accurate diagnosis of melanocytic tumours.. To test this hypothesis, melanocytic naevi, primary melanomas and metastases were investigated.. The distribution and colocalization of markers for proliferation, invasion, angiogenesis and motility of the tumour cells were investigated using antibodies directed against actin, cathepsin B (CatB), transforming growth factor-beta, vascular endothelial growth factor (VEGF), proliferating cell nuclear antigen/Ki-67 and basic fibroblast growth factor (FGF-2). In addition, melanoma markers (HMB-45 and Melan-A) and proteins unrelated to melanoma progression [epidermal growth factor (EGF) and cathepsin H] were investigated.. Malignant melanomas tended to express more markers of malignancy compared with melanocytic naevi, and the differences were statistically significant for EGF and actin immunoreactivity: melanocytic naevi displayed clear EGF labelling more often (60% vs. 5%) and melanomas showed more intense actin labelling (70% vs. 0%). HMB-45+ cells to a large extent also stained with antibodies to CatB but not to EGF or actin; EGF-, FGF-2- and VEGF-immunoreactive cells were predominantly HMB-45-. Similar combinations were observed in melanocytic naevi and in melanomas.. Labelling with EGF may improve the differential diagnosis of melanocytic neoplasias. However, we did not detect a clear-cut increase of markers of malignancy in melanoma. Cells expressing multiple malignancy markers were also found in some melanocytic naevi; this may confirm the dormant potential of melanocytic naevi for melanoma development.

Topics: Biomarkers, Tumor; Cell Movement; Cell Proliferation; Diagnosis, Differential; Epidermal Growth Factor; Humans; Melanoma; Neoplasm Invasiveness; Neoplasm Proteins; Neovascularization, Pathologic; Nevus, Pigmented; Skin Neoplasms

The Ras-mitogen-activated protein (Ras-MAP) kinase pathway regulates various cellular processes, including gene expression, cell proliferation, and survival. Ribosomal S6 kinase (RSK), a key player in this pathway, modulates the activities of several cytoplasmic and nuclear proteins via phosphorylation. Here we report the characterization of the cytoskeletal protein filamin A (FLNa) as a membrane-associated RSK target. We show that the N-terminal kinase domain of RSK phosphorylates FLNa on Ser(2152) in response to mitogens. Inhibition of MAP kinase signaling with UO126 or mutation of Ser(2152) to Ala on FLNa prevents epidermal growth factor (EGF)-stimulated phosphorylation of FLNa in vivo. Furthermore, phosphorylation of FLNa on Ser(2152) is significantly enhanced by the expression of wild-type RSK and antagonized by kinase-inactive RSK or specific reduction of endogenous RSK. Strikingly, EGF-induced, FLNa-dependent migration of human melanoma cells is significantly reduced by UO126 treatment. Together, these data provide substantial evidence that RSK phosphorylates FLNa on Ser(2152) in vivo. Given that phosphorylation of FLNa on Ser(2152) is required for Pak1-mediated membrane ruffling, our results suggest a novel role for RSK in the regulation of the actin cytoskeleton.

Topics: Amino Acid Sequence; Animals; Butadienes; Cell Line, Tumor; Cell Movement; Colforsin; Contractile Proteins; Enzyme Inhibitors; Epidermal Growth Factor; Filamins; Humans; MAP Kinase Signaling System; Melanoma; Microfilament Proteins; Molecular Sequence Data; Nitriles; Phosphorylation; Protein Isoforms; Protein Structure, Tertiary; Ribosomal Protein S6 Kinases; Sequence Alignment; Serine

Overexpression of the epidermal growth factor (EGF) pathway has been implicated in melanoma pathogenesis, and a recent case-control study identified a single nucleotide polymorphism (G to A) in the EGF gene where the G allele was associated with increased EGF expression and an increased risk of melanoma. To further evaluate this association, we conducted a case-control analysis from the Genes, Environment, and Melanoma study at the University of Michigan site using two different study designs. Incident cases of histopathologically confirmed first primary melanoma that were diagnosed between January 1, 2000 and December 31, 2000 from the University of Michigan Melanoma Clinic (n = 330) were compared with the following two different sources of nonmelanoma controls: spouse/friend controls (n = 84) and healthy volunteer controls from a case-control study of psoriasis (n = 148). Using a second analytic design, comparisons between multiple primary melanoma cases (n = 62) and single primary melanoma cases (n = 330) were also evaluated to estimate odds ratios (ORs). Genotyping for the single nucleotide substitution (G to A) at position 61 in the 5' untranslated region of the EGF gene was performed from genomic DNA, and epidemiological risk factors were assessed through a telephone interview. When EGF genotypes were compared between incident primary melanoma cases and the nonmelanoma controls, the risk associated with the homozygous G/G genotype was not statistically significantly associated with an increased risk for incident primary melanoma compared with the homozygous A/A genotype [OR, 1.09; 95% confidence interval (CI); 0.65-1.85]. No strong associations with EGF G/G genotype were observed in comparisons of multiple primary and single primary melanoma cases (OR, 0.66; 95% CI; 0.25-1.73). Case subjects with tumors >/=3.5 mm compared with those <3.5 mm were not significantly associated with the G/G genotype (OR, 0.54; 95% CI; 0.12-2.35). Our data do not support a significant association between melanoma and the EGF 61*G allele or the homozygous G/G genotype. The EGF polymorphism is not a reproducible risk factor for melanoma or thick melanoma in our data. The two analytic approaches used in the study provide evidence against a strong association between EGF 61*G and melanoma and demonstrate the potential utility of case-case designs for evaluating the role of single nucleotide polymorphisms and cancer. Additional independent studies will be required to elucidat

Topics: Alleles; Case-Control Studies; Epidermal Growth Factor; Female; Genetic Predisposition to Disease; Genotype; Humans; Male; Melanoma; Middle Aged; Neoplasm Invasiveness; Polymorphism, Genetic; Sex Factors

The inheritance of a G allele in position 61 in the 5'UTR of the epidermal growth factor (EGF) gene has been reported to increase melanoma susceptibility, a finding we have investigated in this study. The most potent phenotypic risk factor for melanoma is the atypical mole syndrome (AMS) phenotype. Our hypothesis is that the AMS is genetically determined and that nevus genes are also low penetrance melanoma susceptibility genes. We report that the G allele frequencies were the same in 697 healthy women and 380 melanoma cases (OR 0.97, 95% CI 0.8-1.2 p=0.76). We therefore found no evidence that this polymorphism is a melanoma susceptibility gene. Furthermore, we found no evidence that the polymorphism controls the nevus phenotype (nevus number, number atypical nevi or AMS phenotype). We did find some evidence that the G allele may be associated with decreased tumor Breslow thickness (OR 0.5, 95% CI 0.3-0.9) for the A/A genotype versus A/G and G/G combined in tumors of thickness >3.5 vs < or =3.5 mm and may therefore act as a predictor of survival, although this finding is not in accord with the original report. This is the second study to find no association between EGF +61 and melanoma susceptibility.

Topics: 5' Untranslated Regions; Adult; Case-Control Studies; Epidermal Growth Factor; Female; Gene Frequency; Genetic Predisposition to Disease; Genotype; Humans; Male; Melanoma; Middle Aged; Nevus; Penetrance; Polymorphism, Genetic; Skin Neoplasms

A common single nucleotide polymorphism (SNP) in the 5' untranslated region (5'UTR) of the epidermal growth factor (EGF) gene modulates the level of transcription of this gene and hence is associated with serum levels of EGF. This variant may be associated with melanoma risk, but conflicting findings have been reported. An Australian melanoma case-control sample was typed for the EGF+61A>G transversion (rs4444903). The sample comprised 753 melanoma cases from 738 families stratified by family history of melanoma and 2387 controls from 645 unselected twin families. Ancestry of the cases and controls was recorded, and the twins had undergone skin examination to assess total body nevus count, degree of freckling and pigmentation phenotype. SNP genotyping was carried out via primer extension followed by matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectroscopy. The EGF+61 SNP was not found to be significantly associated with melanoma status or with development of nevi or freckles. Among melanoma cases, however, G homozygotes had thicker tumors (p=0.05), in keeping with two previous studies. The EGF polymorphism does not appear to predispose to melanoma or nevus development, but its significant association with tumor thickness implies that it may be a useful marker of prognosis.

Topics: Case-Control Studies; Epidermal Growth Factor; Gene Frequency; Genetic Predisposition to Disease; Genotype; Humans; Melanoma; Melanosis; Nevus; Polymorphism, Genetic; Prognosis; Risk Factors; Skin Neoplasms

Since Unna's Abtropfung hypothesis, the process of migration of nevus cells in the dermis remains unknown. To investigate its mechanisms, we studied the role of gelatinases in dermal nevus cells obtained from congenital pigmented nevi, which are major actors in the remodeling of basement membrane proteins. Our previous studies have shown that dermal nevus cells express pro-matrix metalloproteinase (MMP)-2 exclusively and cannot return to the dermis when seeded together with keratinocytes on top of the dermis in a skin reconstruction model. To examine why MMP-2 was not in its active form, we used Western blot to study the expression of members of the MMP-2 activation pathway (membrane type 1-MMP and tissue inhibitor of metalloproteinase-2), which proved to be normally expressed. To induce the dermal passage of nevus cells artificially, we also tried to activate gelatinases with phorbol-12-myristate-13-acetate and epidermal growth factor, using epidermis reconstructed with nevus cells. No migration in the dermis could be triggered. We conclude that the absence of active MMP-2 is due to a functional blockade of its activation pathway and may prevent dermal nevus cells from reaching the dermal compartment in skin reconstructs. Furthermore, our findings reinforce the concept that dermal nevus cells originating from congenital nevi are in a quiescent status.

Topics: Adolescent; Cell Line, Tumor; Cell Movement; Cells, Cultured; Child; Child, Preschool; Dermis; Enzyme Activation; Epidermal Growth Factor; Gelatinases; Humans; Infant; Infant, Newborn; Isoenzymes; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinases; Melanoma; Nevus; Nevus, Pigmented; Skin Neoplasms; Tetradecanoylphorbol Acetate; Tissue Inhibitor of Metalloproteinase-2

CMM is the most serious cutaneous malignancy and is increasing in frequency among most Caucasian populations, where the most important risk factor is exposure to UV light. Relatively little is known of the genetic factors that mediate susceptibility to and prognosis in sporadic CMM, although a number of genes have been implicated. A striking association between EGF polymorphism and Breslow thickness of invasive CMM has been reported. We have sought confirmation of this finding in an independent study of 159 patients and 310 controls using TaqMan fluorescence-based genotyping for EGF +61. In our study group, there were no significant differences in EGF genotype frequencies between patients and controls nor was EGF genotype associated with tumour growth phase, stage or mitotic count. However, correlation between EGF genotype and Breslow thickness showed a modestly significant increase in frequency of the EGF (G/G) genotype among tumours >3.5 mm thick (30.0% vs. 9.8%, p = 0.03). In summary, in our group, the EGF +61 polymorphism was not a risk factor for CMM susceptibility, but this polymorphism may play a role in disease progression.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Case-Control Studies; DNA; Epidermal Growth Factor; Female; Gene Frequency; Genetic Predisposition to Disease; Genotype; Humans; Male; Melanoma; Middle Aged; Polymorphism, Single Nucleotide; Prognosis; Skin Neoplasms; United Kingdom

Topics: Epidermal Growth Factor; Genetic Predisposition to Disease; Humans; Melanoma; Polymorphism, Genetic

Malignant melanoma, the most serious cutaneous malignancy, has attracted substantial attention because of its rapidly increasing incidence and the poor prognosis of some tumours. Little is known of the genetic factors that mediate susceptibility to, and outcome of, sporadic malignant melanoma. Because of its role in mitogenesis, which is especially relevant to wound healing, tumorigenesis, and proliferation of epidermal tissues, epidermal growth factor (EGF) is an attractive candidate in which to look for genetic polymorphisms.. We enrolled 135 white European patients with malignant melanoma and 99 healthy white European controls, and screened a selection of DNA samples for polymorphisms in the promoter and 5' untranslated region of the EGF gene by analysis. We then screened DNA samples from all participants for the identified polymorphism by restriction-fragment-length polymorphism (RFLP) analysis. In-vitro EGF production was measured in peripheral-blood mononuclear cells from 34 controls, and the results were compared with the individuals' EGF genotypes.. We identified a single nucleotide substitution (G to A) at position 61 of the EGF gene. Allele frequencies in the controls were 56% EGF 61*A and 44% EGF 61*G. Cells from individuals homozygous for the 61*A allele produced significantly less EGF than cells from 61*G homozygotes (p=0.0004) or heterozygous A/G individuals (p=0.001). Compared with the A/A genotype, G/G was significantly associated with Breslow thickness (p=0.045) and with risk of malignant melanoma (odds ratio 4.9 [95% CI 2.3-10.2], p<0.0001).. This study suggests that high EGF production might be important in the development of malignant melanoma.

Topics: Adult; Alleles; Case-Control Studies; Epidermal Growth Factor; Europe; Female; Genotype; Humans; Male; Melanoma; Middle Aged; Polymorphism, Genetic; Polymorphism, Restriction Fragment Length

Intrinsic expression of the multidrug resistance (MDR) transporter P-glycoprotein (Pgp) may be regulated by reactive oxygen species (ROS). A transient expression of Pgp was observed during the growth of multicellular tumor spheroids. Maximum Pgp expression occurred in tumor spheroids with a high percentage of quiescent, Ki-67-negative cells, elevated glutathione levels, increased expression of the cyclin-dependent kinase inhibitors p27Kip1 and p21WAF-1 as well as reduced ROS levels and minor activity of the mitogen-activated kinase (MAPK) members c-Jun amino-terminal kinase (JNK), extracellular signal-regulated kinase ERK1,2, and p38 MAPK. Raising intracellular ROS by depletion of glutathione with buthionine sulfoximine (BSO) or glutamine starvation resulted in down-regulation of Pgp and p27Kip1, whereas ERK1,2 and JNK were activated. Down-regulation of Pgp was furthermore observed with low concentrations of hydrogen peroxide and epidermal growth factor, indicating that ROS may regulate Pgp expression. The down-regulation of Pgp following BSO treatment was abolished by agents interfering with receptor tyrosine kinase signaling pathways, i.e. the protein kinase C inhibitors bisindolylmaleimide I (BIM-1) and Ro-31-8220, the p21ras farnesyl protein transferase inhibitor III, the c-Raf inhibitor ZM 336372 and PD98059, which inhibits ERK1,2 activation. ROS involved as second messengers in receptor tyrosine kinase signaling pathways may act as negative regulators of Pgp expression.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Buthionine Sulfoximine; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Drug Resistance, Multiple; Enzyme Inhibitors; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Glutathione; Humans; Hydrogen Peroxide; JNK Mitogen-Activated Protein Kinases; Kinetics; Liver Neoplasms; Male; MAP Kinase Signaling System; Melanoma; Microtubule-Associated Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Prostatic Neoplasms; Protein-Tyrosine Kinases; Reactive Oxygen Species; Tumor Cells, Cultured; Tumor Suppressor Proteins

The hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs, has been implicated in intracellular trafficking and signal transduction. Hrs contains a phosphatidylinositol 3-phosphate-binding FYVE domain that contributes to its endosomal targeting. Here we show that Hrs and EEA1, a FYVE domain protein involved in endocytic membrane fusion, are localized to different regions of early endosomes. We demonstrate that Hrs co-localizes with clathrin, and that the C-terminus of Hrs contains a functional clathrin box motif that interacts directly with the terminal beta-propeller domain of clathrin heavy chain. A massive recruitment of clathrin to early endosomes was observed in cells transfected with Hrs, but not with Hrs lacking the C-terminus. Furthermore, the phosphatidylinositol 3-kinase inhibitor wortmannin caused the dissociation of both Hrs and clathrin from endosomes. While overexpression of Hrs did not affect endocytosis and recycling of transferrin, endocytosed epidermal growth factor and dextran were retained in early endosomes. These results provide a molecular mechanism for the recruitment of clathrin onto early endosomes and suggest a function for Hrs in trafficking from early to late endosomes.

Topics: Amino Acid Sequence; Animals; Binding Sites; Cell Line; Clathrin; Cricetinae; Dextrans; Endocytosis; Endosomal Sorting Complexes Required for Transport; Endosomes; Epidermal Growth Factor; Macromolecular Substances; Melanoma; Phosphatidylinositol Phosphates; Phosphoproteins; Protein Conformation; Protein Subunits; Protein Transport; Sequence Alignment; Sequence Homology, Amino Acid; Transferrin; Tumor Cells, Cultured

SHIP-2 is a phosphoinositidylinositol 3,4,5 trisphosphate (PtdIns[3,4,5]P3) 5-phosphatase that contains an NH2-terminal SH2 domain, a central 5-phosphatase domain, and a COOH-terminal proline-rich domain. SHIP-2 negatively regulates insulin signaling. In unstimulated cells, SHIP-2 localized in a perinuclear cytosolic distribution and at the leading edge of the cell. Endogenous and recombinant SHIP-2 localized to membrane ruffles, which were mediated by the COOH-terminal proline-rich domain. To identify proteins that bind to the SHIP-2 proline-rich domain, yeast two-hybrid screening was performed, which isolated actin-binding protein filamin C. In addition, both filamin A and B specifically interacted with SHIP-2 in this assay. SHIP-2 coimmunoprecipitated with filamin from COS-7 cells, and association between these species did not change after epidermal growth factor stimulation. SHIP-2 colocalized with filamin at Z-lines and the sarcolemma in striated muscle sections and at membrane ruffles in COS-7 cells, although the membrane ruffling response was reduced in cells overexpressing SHIP-2. SHIP-2 membrane ruffle localization was dependent on filamin binding, as SHIP-2 was expressed exclusively in the cytosol of filamin-deficient cells. Recombinant SHIP-2 regulated PtdIns(3,4,5)P3 levels and submembraneous actin at membrane ruffles after growth factor stimulation, dependent on SHIP-2 catalytic activity. Collectively these studies demonstrate that filamin-dependent SHIP-2 localization critically regulates phosphatidylinositol 3 kinase signaling to the actin cytoskeleton.

Topics: Actins; Amino Acid Sequence; Animals; Cell Membrane; Contractile Proteins; COS Cells; Epidermal Growth Factor; Filamins; Humans; Inositol Polyphosphate 5-Phosphatases; Melanoma; Mice; Microfilament Proteins; Molecular Sequence Data; Muscle Fibers, Skeletal; Muscle, Skeletal; Myocardium; Oligonucleotide Probes; Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Tumor Cells, Cultured; Two-Hybrid System Techniques; Yeasts

Current adenoviral (Ad) vectors cannot be targeted to specific cell types due to the widespread distribution of the Ad receptor (CAR). Moreover, CAR and/or internalization receptors (alphav integrins) are absent or present at low levels on some cell types, rendering them resistant to Ad-mediated gene delivery. To address these problems, we have developed a novel vector targeting approach that takes advantage of the common cell signaling pathways initiated by ligation of alphav integrins and growth factor receptors. Recombinant growth factor/cytokines (TNF-alpha, IGF-1, EGF) which trigger phosphatidylinositol-3-OH kinase (PI3K) activation, a signaling molecule involved in adenovirus internalization, were fused to a monoclonal antibody specific for the viral penton base. Ad vectors complexed with these bifunctional mAbs increased gene delivery 10 to 50-fold to human melanoma cells lacking alphav integrins. The bifunctional mAbs also enhanced gene delivery by fiberless adenovirus particles which cannot bind to CAR. Improved gene delivery correlated with increased virus internalization and attachment as well as PI3K activity. The use of bifunctional mAbs to trigger specific cell signaling pathways offers a widely applicable method for bypassing the normal Ad receptors in gene delivery and potentially increasing the selectivity of gene transfer.

Topics: Adenoviridae; Androstadienes; Animals; Antigens, CD; beta-Galactosidase; Colorectal Neoplasms; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; Gene Expression; Genetic Engineering; Genetic Therapy; Genetic Vectors; Humans; Insecta; Insulin-Like Growth Factor I; Integrin alphaV; Melanoma; Neoplasms; Phosphoinositide-3 Kinase Inhibitors; Receptors, Virus; Recombinant Fusion Proteins; Signal Transduction; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Wortmannin

We have identified a novel 3845 bp cDNA differentially expressed in a human melanoma metastasis model. Northern blot analysis showed expression in the poorly and intermediately metastasizing cell lines and a marked downregulation in the highly metastatic cell lines. Using RT-PCR expression was also seen in several other tumor cell lines and normal cell types of human origin. cDNA sequence analysis revealed an ORF of 687 amino acids containing seven putative transmembrane domains C-terminally and a long N-terminus. The gene was mapped to 16q13. Highest homology was observed with members of the EGF-TM7 subfamily of the secretin/calcitonin receptor family. We propose the delineation of a subfamily of TM7 proteins, LN-TM7, containing seven transmembrane proteins with a long N-terminal extracellular part.

Topics: Amino Acid Sequence; Base Sequence; Chromosome Mapping; Cloning, Molecular; DNA, Complementary; Epidermal Growth Factor; Gene Expression; Humans; Melanoma; Molecular Sequence Data; Neoplasm Metastasis; RNA, Messenger; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Tumor Cells, Cultured

Previous research in animals supports the use of tyrosine and phenylalanine (Tyr-Phe) restriction as an adjuvant to the treatment of cancer. In this regard, dietary restriction of Tyr-Phe specifically inhibits the growth of B16BL6 melanoma tumors, dramatically suppresses spontaneous hematogenous metastasis, and modulates the sensitivity of these tumor cells to growth factors. Two chimeric toxins, HB-TGF alpha-PE4EKDEL and TGF alpha-PE4EKDEL, were examined for their toxicity against the B16BL6 melanoma cell line, and the ability of Tyr-Phe limitation to modulate the potential of these toxins was examined. Tyr-Phe limitation significantly enhanced the cytotoxic effects of HB-TGF alpha-PE4EKDEL approximately 10-fold toward B16BL6 melanoma, and free heparin diminished the cytotoxicity of HB-TGF alpha-PE4EKDEL. Although TGF alpha-PE4EKDEL is cytotoxic to this cell line, Tyr-Phe limitation did not effect the cytotoxicity of this toxin. Tyr-Phe limitation inhibited the synthesis and secretion of heparin-binding proteins but did not alter the expression of surface heparan sulfate proteoglycans. These data suggest that cell surface heparan sulfate proteoglycan is a target for binding and execution of the cytotoxicity of HB-TGF alpha-PE4EKDEL and that augmentation of cytotoxicity by Tyr-Phe limitation is due to the inhibition of heparin-binding protein production.

Topics: Animals; Antineoplastic Agents; Carrier Proteins; Cell Membrane; Culture Media; Epidermal Growth Factor; Heparin; Melanoma; Mice; Phenylalanine; Proteoglycans; Recombinant Fusion Proteins; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tyrosine

SEK-1, a dual specificity protein kinase that serves as one of the immediate upstream activators of the stress-activated protein kinases (SAPKs), associates specifically with the actin-binding protein, ABP-280, in vitro and in situ. SEK-1 binds to the carboxyl-terminal rod segment of ABP-280, upstream of the ABP carboxyl-terminal dimerization domain. Activation of SEK-1 in situ increases the SEK-1 activity bound to ABP-280 without changing the amount of SEK-1 polypeptide bound. The influence of ABP-280 on SAPK regulation was evaluated in human melanoma cells that lack ABP-280 expression, and in stable transformants of these cells expressing wild type ABP, or an actin-binding but dimerization-deficient mutant ABP (ABPDeltaCT109). ABP-280-deficient cells show an activation of SAPK in response to most stimuli that is comparable to that seen in ABP-280-replete cells; ABP-280-deficient cells, however, fail to show the brisk tumor necrosis factor-alpha (TNF-alpha) activation of SAPK seen in ABP-replete cells and have an 80% reduction in SAPK activation by lysophosphatidic acid. Expression of the dimerization-deficient mutant ABP-280 fails to correct the defective SAPK response to lysophosphatidic acid, but essentially normalizes the TNF-alpha activation of SAPK. Thus, a lack of ABP-280 in melanoma cells causes a defect in the regulation of SAPK that is selective for TNF-alpha and is attributable to the lack of ABP-280 polypeptide itself rather than to the disordered actin cytoskeleton that results therefrom. ABP-280 participates in TNF-alpha signal transduction to SAPKs, in part through the binding of SEK-1.

Topics: Actins; Animals; Anisomycin; B-Lymphocytes; Cell Line; Contractile Proteins; Dimerization; Enzyme Activation; Epidermal Growth Factor; Filamins; Glutathione Transferase; Humans; MAP Kinase Kinase 4; Melanoma; Mice; Microfilament Proteins; Mitogen-Activated Protein Kinase Kinases; Mutagenesis, Site-Directed; Phosphorylation; Protein Kinases; Recombinant Fusion Proteins; T-Lymphocytes; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Internalization of the urokinase-type plasminogen activator (uPA) requires two receptors, the uPA receptor (uPAR) and the low density lipoprotein receptor-related protein (LRP)/alpha2-macroglobulin (alpha2M) receptor. Here, we address whether protein kinases are involved in the internalization of uPA by human melanoma cells. Initially, we found that the internalization of uPA was significantly inhibited by the serine/threonine protein kinase inhibitors staurosporine, K-252a and H-89, but not by the tyrosine kinase inhibitors, genistein and lavendustin A. Internalization of uPA was also inhibited by a pseudosubstrate peptide for cAMP-dependent protein kinase (PKA), but not by a pseudosubstrate peptide for protein kinase C. We confirmed a requirement for PKA-activity and implicated a specific isoform by using an antisense oligonucleotide against the regulatory subunit RI alpha of PKA which suppresses PKA-I activity. Exposure of cells to this oligonucleotide led to a specific, dose-dependent decrease in RI alpha protein and to a significant inhibition in the rate of uPA internalization. We further demonstrate that treatment of melanoma cells with either H-89 or PKA RI alpha antisense oligonucleotides also resulted in a decreased internalization of two other ligands of LRP, activated alpha2M and lactoferrin, indicating that PKA activity is associated with LRP. Finally, we demonstrate that PKA activity is also required for the internalization of transferrin, but not for the internalization of the epidermal growth factor or adenovirus 2, suggesting that in melanoma cells, PKA activity is not generally required for clathrin-mediated endocytosis, but is rather associated with specific internalization receptors.

Topics: Adenoviruses, Human; Carbazoles; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Endocytosis; Enzyme Inhibitors; Epidermal Growth Factor; Genistein; Humans; Indole Alkaloids; Indoles; Isoflavones; Isoquinolines; Lactoferrin; Low Density Lipoprotein Receptor-Related Protein-1; Maleimides; Melanoma; Naphthalenes; Phenols; Protein Kinase C; Receptors, Immunologic; Signal Transduction; Staurosporine; Sulfonamides; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Heparanase is an endo-beta-D-glucuronidase whose enzymatic targets are the glycosaminoglycan chains of heparan sulfate proteoglycans (50). Elevated levels of heparanase are associated with the metastatic potential of melanoma cells, and treatment of murine and human melanoma cells with the prototypic neurotrophin nerve growth factor (NGF) increases the production of heparanase by melanoma cells. We previously reported that physiological concentrations of NGF increased invasion of early passage human brain-metastatic 70W melanoma cells but not melanoma cells metastatic to other sites or nonmetastatic melanoma cells as measured in Matrigel invasion assays. Here we found that treatment of 70W melanoma cells with neurotrophin-3 (NT-3) increased Matrigel invasion, whereas treatment with neurotrophins other than NGF or NT-3 did not influence invasion. Mutants of NGF that do not bind to the neurotrophin receptor p75NTR or other nonneuronal growth factors were not able to enhance the invasion of 70W melanoma cells. When 70W cells were exposed to antisense oligonucleotides directed against p75NTR mRNA, there was a reduction in NGF and NT-3 binding, and the neurotrophins failed to enhance Matrigel invasion. To study the properties of heparanase in neurotrophin-regulated malignant melanoma invasive processes, we developed a sensitive heparanase assay consisting of purified [35S]HS subpopulations separated by agarose gel electrophoresis. Incubation of 70W cells with NGF or NT-3 but not brain-derived neurotrophic factor, neurotrophin-4/5 or mutant NGF resulted in increased release of heparanase activity that was capable of degrading a subpopulation of heparan sulfate molecules.

Topics: Electrophoresis, Agar Gel; Enzyme Activation; Epidermal Growth Factor; Extracellular Matrix; Glucuronidase; Glycoside Hydrolases; Heparitin Sulfate; Humans; Melanoma; Neoplasm Invasiveness; Nerve Growth Factors; Neurotrophin 3; Oligonucleotides, Antisense; Protein Binding; Receptor, Nerve Growth Factor; Receptors, Nerve Growth Factor; Substrate Specificity; Tumor Cells, Cultured

An unusual type of glycosylation has been observed for tissue plasminogen activator (t-PA). The monosaccharide fucose is glycosidically linked to threonine-61 in the epidermal growth factor region of t-PA. The presence of O-linked fucose was demonstrated by carbohydrate analysis and mass spectrometry of tryptic and chymotryptic peptides that contain this site. The susceptibility of the fucose residue to alpha-fucosidase indicated that it was in the alpha-anomeric configuration. Fucosylation of threonine-61 was observed in t-PA isolated from the Bowes melanoma cell line and from recombinant expression systems using Chinese hamster ovary or human embryonic kidney cells. Fucosylation of the homologous residue in prourokinase has also been reported recently. Our results indicate that this novel type of glycosylation may be common to the epidermal growth factor domains found in coagulation and fibrinolytic proteins and, therefore, suggest that the modification may have functional significance.

Topics: Amino Acid Sequence; Animals; Cell Line; Chromatography, High Pressure Liquid; Chymotrypsin; Epidermal Growth Factor; Fucose; Glycosylation; Humans; Melanoma; Molecular Sequence Data; Peptide Fragments; Peptide Mapping; Recombinant Proteins; Threonine; Tissue Plasminogen Activator; Trypsin

The epidermal growth factor receptor (EGFR) level in 56 esophageal cancer tissues was measured by 125I-EGF binding assay to elucidate its role in tumor progression. The survival rate of patients with high EGFR level (more than 50 fmol/mg protein) was significantly lower than that of patients with low EGFR level (less than 50 fmol/mg protein, P less than 0.01), although a correlation between EGFR level and the pathologic findings was not observed. The expression of EGF was examined immunohistochemically using anti-EGF monoclonal antibody in 100 esophageal cancer tissues; EGF-positive tumor cells were detected in 92.0%. The immunoreactivity of EGF was classified arbitrarily into four grades according to the number of stained tumor cells. The expression of EGF significantly correlated with the differentiation of esophageal squamous cell carcinoma (P less than 0.01, by chi-square test). The survival rate of patients with high EGF immunoreactivity (Grade 2 or 3) was much lower than in those with lower grade (0 or 1) tumors, (P less than 0.01). Patients with both high EGFR level and EGF immunoreactivity had a much worse prognosis than if both were low. Furthermore, the mitotic index was higher in groups with both high EGFR and EGF than if both were low (16.39 +/- 5.35 versus 6.90 +/- 3.31). These results suggest that EGF and EGFR in the autocrine system may play an important role in tumor progression in esophageal cancer and their expression could be of prognostic significance.

Topics: Antibodies, Monoclonal; Carcinoma; Carcinoma, Squamous Cell; Cell Differentiation; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Follow-Up Studies; Humans; Immunohistochemistry; Lymphatic Metastasis; Melanoma; Mitotic Index; Neoplasm Staging; Survival Rate

The molecular genetics of melanoma is little understood and has concentrated largely on DNA analyses. We have examined mRNA levels of 21 different oncogenes, antioncogenes, growth factors and proteases in cultured melanocytes and 17 melanoma cell lines. C-mel, c-erb-B2, c-myc, c-src-1, p53, platelet derived growth factor A chain, gro, transforming growth factor alpha, epidermal growth factor receptor and tissue plasminogen activator were all expressed in at least some cell lines. Most striking was the finding that there are significant intercorrelations of c-myc, p53 and c-src-1 levels, and between p53 and c-erb-B2, which may be due to common regulatory control of these genes in cells of the melanocytic lineage.

Topics: Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Humans; Melanocytes; Melanoma; Oncogene Proteins; Phosphoproteins; Plasminogen Activators; Platelet-Derived Growth Factor; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; Proto-Oncogene Proteins pp60(c-src); Proto-Oncogenes; RNA, Messenger; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Histamine receptors are present on the surface of various normal and tumor-derived cell types, where their biological function is incompletely understood. Here we report that histamine not only stimulates cell proliferation under serum-free conditions, but also is chemotactic for human carcinoma (Hela and A431) and melanoma (A875) cells expressing H1 type receptors. Histamine was found to be a potent activator of phospholipase C, leading to polyphosphoinositide hydrolysis and subsequent intracellular Ca2+ mobilization. In addition, histamine also causes the protein kinase C-mediated activation of Na+/H+ exchange, as evidenced by an amiloride-sensitive rise in cytoplasmic pH. All histamine-induced responses, including chemotaxis and DNA synthesis, are completely inhibited by the H1 receptor antagonist pyrilamine, but not by cimetidine, an inhibitor of histamine H2 type receptors. Our results suggest that histamine may have a previously unrecognized role in the migration and proliferation of cells expressing H1 receptors.

Topics: Cell Division; Chemotaxis; DNA, Neoplasm; Epidermal Growth Factor; Growth Substances; HeLa Cells; Histamine; Humans; Inositol Phosphates; Insulin; Kinetics; Melanoma; Receptors, Histamine H1; Tumor Cells, Cultured; Type C Phospholipases

We have examined the effects of transforming growth factor beta 1 (TGF-beta 1) on the growth of paired murine melanoma cell clones that differ with respect to their experimental metastatic potential. Neither poorly (clone 16) nor highly (clone M2) metastatic cells were capable of anchorage-independent growth in 0.3% agar/Dulbecco's modified Eagle's medium in the absence of serum. However, both clones were capable of anchorage-independent growth in 0.3% agar/Dulbecco's modified Eagle's medium containing 10% calf serum. Colony formation in the presence of 10% calf serum was enhanced in a dose-dependent manner by TGF-beta 1 (half-maximal dose, 0.1 ng/ml) and was 5- to 10-fold greater than colony formation in the presence of 10% calf serum alone. Under anchorage-dependent (monolayer) conditions, neither clone grew in the absence of serum or in medium containing less than 1% calf serum. The monolayer growth of poorly metastatic cells (clone 16) was enhanced in a dose-dependent manner by TGF-beta 1 in medium supplemented with calf serum. Growth was 3.5-fold and 2.3-fold greater than untreated controls after 5 days in submitogenic (0.5%) and mitogenic (10%) concentrations of calf serum, respectively. In contrast, TGF-beta 1 had no effect on the monolayer growth of highly metastatic cells (clone M2) either in submitogenic (0.5%) or mitogenic (10%) concentrations of serum. TGF-beta 1 did not directly stimulate DNA synthesis by either poorly or highly metastatic cells when measured 24 h after TGF-beta 1 treatment. The ability of TGF-beta 1 to stimulate the anchorage-independent growth of metastatic melanoma cells suggests that this potent growth factor may play a role in the growth of these cells in vivo. In addition, the differential sensitivity of poorly and highly metastatic cells to TGF-beta 1 may be relevant to their metastatic potential in vivo. While the mechanism(s) by which TGF-beta 1 stimulates the growth of these cells remains unknown, these differentially metastatic clones of the K-1735 murine melanoma should provide a useful model in which to study the effects of transforming growth factor beta on the metastatic phenotype.

Topics: Animals; Cell Division; Culture Media; DNA, Neoplasm; Epidermal Growth Factor; Melanoma; Mice; Neoplasm Metastasis; Transforming Growth Factors; Tumor Cells, Cultured

Normal rat kidney fibroblasts (NRK-B F49) treated at transforming doses with a gamma-like TGF, partially purified from human melanoma, showed a 3 to 5 fold increase in DNA type II topoisomerase activity. A similar effect was observed using EGF and a partially purified alfa TGF from rabbit fetuses. DNA type I topoisomerase activity, from the same cells, was not significantly modified by treatment with these growth factors. Topoisomerase II stimulation was dependent on mRNA synthesis. The possible role of topoisomerase II in phenotypical cell transformation induced by TGF is discussed.

Topics: Animals; DNA Topoisomerases, Type I; DNA Topoisomerases, Type II; Epidermal Growth Factor; Fetus; Fibroblasts; Humans; Kinetics; Melanoma; Rabbits; Rats; Transforming Growth Factors

Five out of 6 cell lines derived from metastatic melanoma lesions grew in a chemically defined base medium consisting of a mixture of calcium-supplemented MCDB 153 and L 15 media in the absence of any polypeptide growth factors. In contrast, under these conditions no growth was seen in any of 5 primary melanoma cell lines tested, including 2 cell lines from patients whose metastatic cells proliferated well in base medium. Growth stimulation of all 11 melanoma cell lines by epidermal growth factor (EGF), transferrin, insulin, and insulin-like growth factor (IGF)-1 alone and in various combinations was studied. Insulin represented the strongest single growth factor for primary and metastatic melanoma cell lines. The metastatic cell lines remained growth-responsive to EGF, insulin and transferrin and responded more vigorously to these exogenously provided mitogens than the primary cell lines. No synergistic or additive growth effects of insulin, transferrin, or EGF for primary and metastatic cell lines were observed. Cross-linking studies with 125I-IGF-1 demonstrate surface expression of the type-I IGF receptor on melanoma cells. Growth stimulation by insulin and IGF-1 was inhibited by adding to the culture medium a monoclonal antibody to the type-I IGF receptor. Our studies indicate that IGF-1 and insulin are major growth factors for melanoma cells and act via the type-I IGF receptor.

Topics: Cell Division; Cell Line; Culture Media; Epidermal Growth Factor; Growth Substances; Humans; Insulin; Insulin-Like Growth Factor I; Melanoma; Transferrin; Tumor Cells, Cultured

The effect of epidermal growth factor (EGF) on the in vitro growth of 186 malignant human tumor specimens (45 melanomas, 32 sarcomas, and 56 lung, 16 gynecological, 14 breast, 12 genitourinary, and 11 gastrointestinal carcinomas) was evaluated in the cellular adhesive matrix human tumor culture system supplemented with transferrin, insulin, hydrocortisone, and estradiol. EGF increased tumor growth by at least 50% in 81% of the 186 tumors and by over 100% in 54%. The enhanced growth induced by EGF was related to an accelerated cellular division independent of tumor type and not to an increase in the actual number of clonogenic units. The drug concentrations of cell cycle-independent Adriamycin and cisplatin needed to achieve a 90% tumor cell kill were not altered by the responsiveness of the tumor to EGF.

Topics: Antineoplastic Agents; Breast Neoplasms; Carcinoma; Cell Division; Cell Line; Cell Survival; Cells, Cultured; Culture Media; Epidermal Growth Factor; Extracellular Matrix; Gastrointestinal Neoplasms; Humans; Lung Neoplasms; Melanoma; Neoplasms; Sarcoma; Urogenital Neoplasms

We have studied the effect of laminin on type IV collagenolytic activity elaborated by malignant cells in culture. Laminin (at concentrations of 4-8 micrograms/ml) added to serum-free culture supernatants of subconfluent A2058 human melanoma cells significantly increased the release of the type IV collagenolytic activity (200-300%). The induction of type IV collagenase was more pronounced (580%) using a fragment of laminin which binds to the cell surface laminin receptor. A monoclonal antibody against the human laminin receptor blocked the effect of laminin on type IV collagenase, suggesting that occupation of the laminin receptor may be necessary for the effect. Increase in the type IV collagenolytic activity mediated by laminin was also demonstrated in two other malignant cell lines, HT fibrosarcoma (168%) and mouse melanoma (B16-F10) (271%). The increase in type IV collagenase was found to be specific for laminin because another cell-binding matrix protein, fibronectin, did not have any effect, and epidermal growth factor and transferrin actually decreased the type IV collagenase in human melanoma culture medium (epidermal growth factor, 50% at 20 ng/ml; and transferrin, 20% at 10 micrograms/ml). These studies suggest that tumor cell binding to laminin, which comprises the first step of basement membrane invasion, will induce the second step, namely the collagenolytic dissolution of the basement membrane.

Topics: Animals; Antibodies, Monoclonal; Cells, Cultured; Epidermal Growth Factor; Fibronectins; Fibrosarcoma; Humans; Laminin; Melanoma; Mice; Microbial Collagenase; Neoplasm Invasiveness; Neoplasm Proteins; Peptide Fragments; Receptors, Immunologic; Receptors, Laminin; Secretory Rate; Transferrin

Epidermal growth factor (EGF)-like factors with EGF competing and cell growth stimulating activity were investigated in malignant and nonmalignant tissues. About 37% of ovarian carcinomas present an increased factor activity between 9.0 and 19.3 ng EGF units/mg protein. In one tumor 175.0 ng EGF units/mg protein were found. In extracts of nonmalignant tissues, the factor concentration was about 1.0-6.4 ng EGF units/mg protein. Isoelectric focusing was performed to characterize these factors. In normal ovaries and ovarian carcinomas, factors with EGF competing activity focus at pH 8.0-9.0, pH 5.7-6.3, and pH 3.6-4.9. In ovarian carcinomas, an additional peak with EGF competing and cell growth stimulating activity was found between pH 6.5 and 7.2. Similar results could be achieved in other malignant tissues investigated. These data indicate the presence of EGF-like factors. EGF itself focuses at pH 4.6 (G. Carpenter and S. Cohen, Annu. Rev. Biochem., 48: 193-216, 1979). Specific EGF binding was determined in 12 ovarian carcinomas. In five of these cases EGF receptors could be detected. In the EGF receptor positive carcinomas, the content of EGF-like growth factors varied between 0 and 9 ng EGF units/mg protein. In EGF receptor negative cases, the content of EGF-like growth factors varied between 0 and 19.3 ng EGF units/mg protein. The clinical data of 19 patients are also demonstrated.

Topics: Animals; Binding, Competitive; Biological Assay; Breast Neoplasms; Carcinoma; Epidermal Growth Factor; ErbB Receptors; Female; Growth Substances; Humans; Isoelectric Point; Melanoma; Mice; Ovarian Neoplasms; Radioligand Assay; Receptors, Cell Surface; Sarcoma

Transforming growth factor type beta (TGF beta) has been purified from serum-free culture fluids of transformed mouse L-929 cells which are capable of continual growth in serum-free medium in the absence of any exogenously added polypeptide growth factors. TGF beta has been purified to homogeneity as judged by NH2-terminal amino acid sequence analysis. Analysis of the purified polypeptide by gel electrophoresis indicates that TGF beta is composed of two polypeptide chains of Mr 12,500 cross-linked by disulfide bonds. TGF beta was characterized by its ability to induce anchorage-dependent normal rat kidney (NRK) cells to grow in soft agar in the presence of epidermal growth factor (EGF). TGF beta was also able to enhance both EGF-induced DNA synthesis and cell proliferation on growth-arrested NRK cells in monolayer cultures under serum-free conditions. We also show that in mouse melanoma B-16 cells under serum-free conditions TGF beta stimulates both DNA synthesis in monolayer cultures and anchorage-independent growth in soft agar. Paradoxically, the anchorage-independent growth in the presence of serum of many human cell lines, including melanomas, and mammary, prostatic, vulvar, and lung carcinomas is inhibited by TGF beta at saturating concentrations similar to those that stimulate colony formation of the rodent cell lines L-929 and B-16 under serum-free conditions. The peculiar action of TGF beta is further revealed by the observations that while EGF and TGF beta synergize to induce inhibition of anchorage-independent growth of A-431 human vulvar carcinoma cells, their effects on the anchorage-independent growth of one human lung carcinoma cell line (A-549) and two human prostatic carcinoma cell lines (PC-3 and DU-145) are antagonistic. Moreover, we show that in the rodent and human cell lines TGF beta interacts with specific cellular receptors which may mediate the actions of TGF beta. We conclude that the expression of both TGF beta and TGF beta receptors by L-929 cells and the stimulation of growth of L-929 cells in serum-free medium by TGF beta suggests that TGF beta may be important for maintaining the transformed state of this tumor cell line, and the way in which a cell responds to TGF beta is dependent on the presence or absence of growth factors contained in the serum.

Topics: Amino Acid Sequence; Breast; Cell Division; Cell Line; Cell Transformation, Neoplastic; DNA; Epidermal Growth Factor; Growth Substances; Humans; Kidney; Melanoma; Molecular Weight; Neoplasm Proteins; Neoplasms; Peptides; Transforming Growth Factors

In this study, we report that conditioned medium (CM) from the human melanoma cell line Hs0294 contains growth-stimulating activity for human and murine hemopoietic progenitor cells of multiple lineages. Crude CM was assayed for hemopoietic progenitor cell growth-stimulating activity (HP-GSA) in plasma clot cultures of 10(5) C57B1/6J mouse bone marrow cells or 10(5) light-density, nonadherent human peripheral blood mononuclear cells. CM concentrations as low as 0.2% had demonstrable HP-GSA for murine and human hemopoietic progenitor cells, although peak activity for all hemopoietic progenitor cell types assayed (murine CFU-Mix, BFU-E, CFU-GM, and CFU-M and human BFU-E) was observed with 5%-10% Hs0294-CM. Growth-stimulating activity for human erythroid progenitors (BFU-E) was comparable to that produced by phytohemagglutinin-stimulated human mononuclear cell CM. Stimulation of murine hemopoietic progenitors by Hs0294-CM was comparable to the activity in pokeweed mitogen-stimulated mouse spleen cell CM. The Hs0294-CM did not contain detectable quantities of erythropoietin or interleukin 3. Human burst-promoting activity was stable through sequential freezing and thawing, heating to 50 degrees C for 1 h or 100 degrees C for 5 min, and incubation at pH 9.8 for 1 h, but lost 70% of its activity when incubated at pH 2.2 for 1 h. More than 95% of the HP-GSA was recovered in the 50%-80% fraction by (NH4)2SO4 precipitation. The HP-GSA had an estimated molecular weight of approximately 21,000. In conclusion, we have shown that a human melanoma cell line, Hs0294, produces multilineage HP-GSA.

Topics: Animals; Cell Line; Chromatography, Gel; Chromatography, Ion Exchange; Colony-Forming Units Assay; Culture Media; Epidermal Growth Factor; Growth Substances; Hematopoiesis; Humans; Melanoma; Mice; Peptides; Transforming Growth Factors

A substance immunochemically cross-reactive with insulin (SICRI) appears in melanoma B16 growing in diabetic and nondiabetic C57BL/6 mice. Progression of tumor size is paralleled by the increase of SICRI levels in the serum of both diabetic and nondiabetic animals; this increase correlates with a decreased concentration of circulating glucose and an elevated concentration of growth hormone in blood. Melanoma B16 grown under serum-free culture conditions secretes SICRI into the medium. Affinity-purified SICRI stimulates glucose uptake by rat epididymal adipocytes and competes with radiolabeled insulin for binding to these cells. Low concentrations of SICRI enhance growth of cultured melanoma B16 cells, whereas high concentrations of this substance have inhibitory growth effects on these cells. Porcine insulin, human insulin-like growth factors I and II, human growth hormone, platelet-derived growth factor, epidermal growth factor, and fibroblast growth factor have negligible influence on growth of melanoma B16.

Topics: Adipose Tissue; Animals; Diabetes Mellitus, Experimental; Epidermal Growth Factor; Fibroblast Growth Factors; Growth Hormone; Homeostasis; Hypoglycemia; Insulin; Melanoma; Mice; Platelet-Derived Growth Factor; Somatomedins

Epidermal growth factor (EGF) receptor is expressed selectively by human melanoma cells which show the presence of an extra copy of chromosome 7. None of the cells of benign pigmented lesions (nevi) or radial growth phase (nonmetastatic) primary melanoma expressed EGF receptor and none of these cells showed an extra copy of chromosome 7. The results indicate that a single extra dose of a gene (for EGF receptor) may provide a selective advantage to cells in the late stages of tumorigenesis.

Topics: Antibodies, Monoclonal; Cell Line; Chromosome Mapping; Chromosomes, Human, 6-12 and X; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation; Genes; Humans; Melanoma; Neoplasm Metastasis; Nevus, Pigmented; Receptors, Cell Surface

A low Mr human transforming growth factor (TGF) present in melanoma patients' urine has been purified approximately 200,000-fold to apparent homogeneity. Initial purification of an acid-soluble fraction of urine was achieved by Bio-Gel P-30 gel filtration chromatography in 1 M acetic acid. TGF activities were demonstrated in the Mr ranges of 30,000 and 6,000-10,000. These competed with epidermal growth factor (EGF) for binding to A431 membrane receptors and induced anchorage-independent growth of untransformed fibroblasts. The low Mr TGF activity obtained from P-30 chromatography was purified to apparent homogeneity by two sequential reverse-phase high performance liquid chromatography steps with a mu Bondapak C18 column first using a linear gradient of acetonitrile going from 0-60% in 120 min and then by rechromatography of the activity over the same column using a shallower gradient of acetonitrile going from 20-40% in 160 min. The isoelectric point of the melanoma patient-derived urinary TGF was determined to be 6.2, which is distinct from that for human EGF. Amino acid composition analysis of the purified urinary TGF (uTGF) revealed that it is composed of at least 42 amino acid residues with a minimum estimated Mr of 4,545. Compositional analysis further revealed distinct similarities and differences between the uTGF, human EGF and TGFs secreted by various transformed human and rodent cell lines.

Topics: Amino Acids; Animals; Binding, Competitive; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Humans; Kidney; Kinetics; Melanoma; Molecular Weight; Neoplasm Proteins; Peptides; Rats; Receptors, Cell Surface; Transforming Growth Factors

Cells derived from a human melanoma strain by low-serum selection in monolayer were found to be capable of growth in semisolid medium, forming colonies ranging from tight to loose or dispersed. These phenotypes were found to be stable after cloning and retesting. Examination of the biological activities of ectopic peptides produced by a clone that gives rise to loose dispersed colonies revealed the presence of a transforming growth factor beta (TGF-beta; apparent Mr 14,700) and a single TGF-alpha species produced at an unusually high concentration (2.2 ng/ml). Coeluted with the TGF-alpha at approximately 22,500 was a mitogenic activity and a previously undescribed activity capable of modulating the phenotype of induced normal rat kidney fibroblast (NRK-49F) colonies in semisolid medium. This activity can modulate the usual tight colony to express a loose or dispersed phenotype characteristic of the producer cells. Both the possible role these ectopic peptides play in the expression of the transformed phenotype by the tumor cells producing them and the possible correlation between the Mr 22,500 epidermal growth factor-like peptide released by this particular tumor line and high molecular weight epidermal growth factor-like peptides found in the urine of cancer patients are discussed.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Humans; Kidney; Melanoma; Molecular Weight; Neoplasm Proteins; Peptides; Radioligand Assay; Rats; Receptors, Cell Surface; Transforming Growth Factors

Specific binding of 125I-labeled epidermal growth factor (EGF) was measured in 62 skin tumors of different severity. Within a group of 28 benign tumors, 11 of 15 condylomata acuminata were receptor positive, whereas the investigated mesenchymal tumors and normal skin as a control were receptor negative. 6 of 18 basal cell epitheliomas bound EGF specifically. In the group of precancerous and malignant skin tumors, 7 of 8 squamous cell carcinomas had the highest number of EGF binding sites and a high affinity state, whereas 5 malignant melanomas were receptor negative. The clinical relevance of these findings is not yet clear due to the short follow-up of the patients.

Topics: Binding Sites; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Condylomata Acuminata; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Melanoma; Receptors, Cell Surface; Skin; Skin Diseases; Skin Neoplasms

Cells derived from a human melanoma strain by low serum selection in monolayer were found to be capable of growth in semi-solid medium, forming colonies ranging from tight, to loose, or dispersed. These phenotypes were found to be stable on cloning and retesting. Examination of the serum-free media conditioned by these clones indicates that the clones release activities capable of inducing agar colonies, in an indicator cell line (NRK, 49F), that express phenotypes similar to those of the melanoma clones used to produce the serum-free conditioned media (SF-CM). These SF-CM contained a transforming growth factor (TGF) beta (apparent Mr 14 700) and a single, apparently high Mr TGF-alpha species. Co-eluting with the TGF-alpha at an Mr of approximately 22 500 was a previously undescribed activity capable of modulating the phenotype of the NRK agar colonies induced by the combination of TFGs alpha and beta. This new activity can modulate the usual tight colony to express a loose or dispersed phenotype characteristic of the producer cells. The possible role these ectopic peptides play in the expression of the transformed phenotype by the tumour cells producing them, and the possible correlation between the 22 500 Mr EGF-like peptide (TGF-alpha) released by this particular tumour line and high Mr EGF-like peptides found in the urine of cancer patients, are both discussed.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Culture Media; Epidermal Growth Factor; ErbB Receptors; Humans; Melanoma; Molecular Weight; Peptides; Phenotype; Radioligand Assay; Rats; Receptors, Cell Surface; Transforming Growth Factors

Primary cell cultures of normal human epidermal keratinocytes and melanocytes and human cell lines established from a primary melanoma (SK-PM-4) and metastatic melanomas (HO#1, SK-MEL21, and SK-MEL37) contain specific and saturable receptors for the tumor promoter phorbol dibutyrate (PDBu). Scatchard analyses of the keratinocytes revealed two classes of binding sites: 1) a high-affinity class (affinity constant = 37 nM; 1.3 X 10(6) sites/cell) and a low-affinity class (affinity constant = 4,880 nM; 7 X 10(7) sites/cell). The melanoma cultures, likewise, showed high- and low-affinity classes of PDBu binding sites. However, the affinity constant values and total numbers of sites in the melanoma cells were lower than the corresponding values in the keratinocytes. The binding of [3H]PDBu to human keratinocytes was inhibited by the tumor promoters 12-O-tetradecanoylphorbol 13-acetate and teleocidin but not by phorbol, which lacks tumor-promoting activity. Human serum also inhibited binding. Specific receptors for epidermal growth factor (EGF) were demonstrated in the keratinocytes and primary melanoma cultures. In contrast, three metastatic melanoma cultures gave negligible levels of EGF binding. Among the various cell types, the extent of [3H]PDBu binding did not correlate with the extent of EGF binding, indicating that these two substances occupy distinctly separate types of receptors.

Topics: Caenorhabditis elegans Proteins; Carrier Proteins; Cell Line; Epidermal Growth Factor; ErbB Receptors; Humans; Melanoma; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Protein Kinase C; Receptors, Cell Surface; Receptors, Drug; Skin Neoplasms; Tetradecanoylphorbol Acetate

Transforming growth factors (TGFs) were purified from serum-free medium conditioned by retrovirus-transformed Fisher rat embryo fibroblasts, mouse 3T3 cells, and two human melanoma cell lines. The purification of each TGF was monitored in a radioreceptor assay based on receptor crossreactivity with mouse submaxillary gland epidermal growth factor (mEGF) and was achieved by gel permeation chromatography of the acid-soluble TGF-containing activity, followed by reverse-phase high-pressure liquid chromatography with sequential use of acetonitrile and 1-propanol in the presence of aqueous trifluoroacetic acid. The amino-terminal sequences of rat, mouse, and human TGFs were determined. Extensive sequence homology was found among TGF polypeptides from different species and cell types. Alignment of the amino acid sequences of rat TGF, mEGF, and human urogastrone (hEGF) reveals statistically significant sequence homology. The reported results suggest that TGFs that compete for binding to the cellular EGF receptor and EGF may have evolved from a common progenitor.

Topics: Amino Acid Sequence; Animals; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Embryo, Mammalian; Epidermal Growth Factor; Fibroblasts; Genes; Humans; Melanoma; Mice; Moloney murine leukemia virus; Neoplasm Metastasis; Peptides; Rats; Rats, Inbred F344; Retroviridae; Sarcoma Viruses, Feline; Transforming Growth Factors

Extracts of conditioned medium (CM) from Hs0294 human malignant melanoma cells stimulate [3H]thymidine incorporation and an increase in cell number in serum-depleted Hs0294 cells. This activity is acid and heat stable, nonproteolytic, protease sensitive, contains disulfide bonds and elutes broadly from a Bio-Gel P-30 column with an approximate molecular weight range of 6,000 to 25,000. Hs0294 CM also stimulates [3H]thymidine incorporation in nonmalignant human nevus cells and normal rat kidney fibroblast cells but not in human fibroblasts. There was only limited competition with 125I-epidermal growth factor in binding assays. Hs0294 CM extracts stimulate anchorage-independent growth in normal rat kidney fibroblast cells in soft agar but not in Hs0294 cells, nevus cells, or human fibroblasts. This second activity elutes from the Bio-Gel P-30 column in two positions with apparent molecular weights of 27,000 and 11,000.

Topics: Agar; Animals; Cell Count; Cell Division; Cell Line; Culture Media; DNA; Epidermal Growth Factor; Growth Substances; Humans; Kidney; Melanoma; Molecular Weight; Nerve Growth Factors; Nevus; Rats

A low molecular weight human transforming growth factor (hTGFs) was isolated from serum-free medium conditioned by a human metastatic melanoma tumor line, A2058. The purification of hTGFs was achieved by gel permeation chromatography of the acid-soluble growth-promoting activity on Bio-Gel P-10 in 1 M acetic acid, followed by reverse phase high pressure liquid chromatography on muBondapak C18 support using a linear gradient of acetonitrile in 0.045% trifluoroacetic acid, and subsequently by rechromatography of the human transforming growth factor-containing fractions with a linear 1-propanol gradient in 0.035% trifluoroacetic acid on the same column. The estimated molecular weight of hTGFs is 7400. It is a single chain polypeptide with three intrachain disulfide bridges and no free sulfhydryl groups. Lacking tyrosine and methionine, but containing three phenylalanine residues, hTGFs is unlike human and mouse epidermal growth factor (EGF). hTGFs competes with 125I-labeled EGF for binding to A431 human carcinoma cells completely and equivalently, and thus is functionally related to EGF. In contrast, hTGFs enabled normal anchorage-dependent rat kidney cells to grow in soft agar, whereas EGF did not stimulate growth of these cells in semisolid medium. The half-maximal growth-stimulating response of hTGFs was reached with one EGF-competing unit or 1.1 ng of hTGFs.

Topics: Amino Acids; Cell Line; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Melanoma; Peptide Biosynthesis; Peptides; Receptors, Cell Surface; Transforming Growth Factors

A partially purified preparation of a transforming growth factor (TGF) obtained from serum-free growth medium conditioned by a human melanoma tumor line was found to stimulate the phosphorylation of a synthetic tyrosine-containing peptide. The sequence of the peptide is related to that of the known site of tyrosine phosphorylation in the Rous sarcoma virus-encoded transforming protein, pp60src. In A431 membranes, the characteristics of TGF- and epidermal growth factor (EGF)-stimulated peptide phosphorylation are nearly identical. The effects of the two growth factors are not additive, suggesting that TGF and EGF stimulate peptide phosphorylation through the same EGF receptor system. This conclusion is supported by the finding that both TGF and EGF stimulate peptide phosphorylation in wild type Swiss 3T3 cell membranes, but neither factor is effective in stimulating peptide phosphorylation in membranes prepared from EGF receptor-deficient NR6 3T3 cells.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Cells, Cultured; Epidermal Growth Factor; Humans; Kinetics; Melanoma; Membrane Proteins; Mice; Peptides; Phosphorylation; Transforming Growth Factors; Tyrosine

Three different human tumor lines in culture, a rhabdomyosarcoma, a bronchogenic carcinoma and a metastatic melanoma, release proteins (transforming growth factors, TGFs) into the medium that confer the transformed phenotype on untransformed fibroblasts. These proteins are acid and heat-stable; produce profound morphologic changes in rat and human fibroblasts; and enable normal anchorage-dependent cells to grow in agar. Removal of the transforming protein results in a reversion of cell phenotype. The major activity interacts with epidermal growth factor (EGF) cell membrane receptors. The peptides from these tumor cells are similar in their action to the sarcoma growth factor (SGF) released by murine sarcoma virus-transformed rodent cells. The most anchorage-independent tumor cells released the most TGFs. EGF-related TGFs were not detectable in fluids from cultures of cells with high numbers of free EGF membrane receptors (normal human fibroblasts and human carcinomas).

Topics: Adult; Aged; Animals; Binding, Competitive; Carcinoma, Bronchogenic; Carcinoma, Squamous Cell; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; DNA; Epidermal Growth Factor; ErbB Receptors; Female; Fibroblasts; Growth Substances; Humans; Lung Neoplasms; Male; Melanoma; Peptides; Rats; Receptors, Cell Surface; Rhabdomyosarcoma