RESUMEN
BACKGROUND: Activity of both c-Jun and cyclin D1 is deemed critical for melanoma cell proliferation. This functionality is corroborated by frequently elevated expression and activity of these proteins in human melanomas. Correspondingly, alleviating c-Jun and cyclin D1 function is vital to the success of antimelanoma therapeutics. OBJECTIVES: To understand the role of the c-Jun N-terminal kinase (JNK) signalling pathway in melanoma cell proliferation and survival. METHODS: The effect of JNK inhibitors SP600125 and JNK-IN-8 on the proliferation and survival of genetically highly representative human melanoma cell lines was studied in assays of proliferation and apoptosis. Changes in c-Jun and cyclin D1 protein and mRNA levels in response to JNK and mitogen-activated protein kinase kinase (MEK) inhibition were investigated through immunoblotting and quantitative reverse-transcription polymerase chain reaction. The effects of JNK and MEK inhibitors on cell-cycle distribution were assessed by flow cytometry. RESULTS: We demonstrate the requirement of JNK signalling in melanoma cell proliferation and survival. While JNK inhibition suppressed the expression and activity of c-Jun, it failed to suppress cyclin D1 levels. Consistently with its inability to downregulate cyclin D1, JNK inhibition failed to induce G1 arrest. In contrast, the blockade of MEK-extracellular signal-regulated kinase (ERK) signalling, although unable to suppress c-Jun activity and expression, paradoxically abated cyclin D1 levels and triggered G1 arrest. This previously unreported dual disconnect between JNK-cyclin D1 and ERK-c-Jun levels was confirmed by concomitant JNK and BRAF inhibition, which suppressed both c-Jun and cyclin D1 levels and exhibited a heightened antiproliferative response. CONCLUSIONS: Dual disjunction between JNK-cyclin D1 and ERK-c-Jun signalling forms the basis for further investigation of combined JNK and MAPK signalling blockade as a more effective therapeutic approach in human melanoma.
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Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Melanoma/mortalidad , Neoplasias Cutáneas/mortalidad , Antracenos/farmacología , Proliferación Celular , Ciclina D1/metabolismo , Humanos , Melanoma/patología , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/fisiología , Neoplasias Cutáneas/patología , Células Tumorales CultivadasRESUMEN
Melanoma is a heterogeneous tumor with different subpopulations showing different proliferation rates. Slow-cycling cells were previously identified in melanoma, but not fully biologically characterized. Using the label-retention method, we identified a subpopulation of slow-cycling cells, defined as label-retaining cells (LRC), with strong invasive properties. We demonstrate through live imaging that LRC are leaving the primary tumor mass at a very early stage and disseminate to peripheral organs. Through global proteome analyses, we identified the secreted protein SerpinE2/protease nexin-1 as causative for the highly invasive potential of LRC in melanomas.
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Ciclo Celular , Melanoma/patología , Serpina E2/metabolismo , Neoplasias Cutáneas/patología , Animales , Línea Celular Tumoral , Separación Celular/métodos , Citometría de Flujo/métodos , Humanos , Melanocitos/metabolismo , Melanocitos/patología , Ratones , Invasividad Neoplásica/patología , Proteómica , Piel/citología , Piel/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
MITF (microphthalmia-associated transcription factor) is a frequently amplified lineage-specific oncogene in human melanoma, whose role in intrinsic drug resistance has not been systematically investigated. Utilizing chemical inhibitors for major signaling pathways/cellular processes, we witness MITF as an elicitor of intrinsic drug resistance. To search kinase(s) targets able to bypass MITF-conferred drug resistance, we employed a multi-kinase inhibitor-directed chemical proteomics-based differential affinity screen in human melanocytes carrying ectopic MITF overexpression. A subsequent methodical interrogation informed mitotic Ser/Thr kinase Aurora Kinase A (AURKA) as a crucial regulator of melanoma cell proliferation and migration, independent of the underlying molecular alterations, including TP53 functional status and MITF levels. Crucially, assessing the efficacy of investigational AURKA inhibitor MLN8237, we pre-emptively witness the procurement of a molecular program consistent with acquired drug resistance. This involved induction of multiple MAPK (mitogen-activated protein kinase) signaling pathway components and their downstream proliferation effectors (Cyclin D1 and c-JUN) and apoptotic regulators (MITF and Bcl-2). A concomitant AURKA/BRAF and AURKA/MEK targeting overcame MAPK signaling activation-associated resistance signature in BRAF- and NRAS-mutated melanomas, respectively, and elicited heightened anti-proliferative activity and apoptotic cell death. These findings reveal a previously unreported MAPK signaling-mediated mechanism of immediate resistance to AURKA inhibitors. These findings could bear significant implications for the application and the success of anti-AURKA approaches that have already entered phase-II clinical trials for human melanoma.
Asunto(s)
Apoptosis , Aurora Quinasa A/metabolismo , Resistencia a Antineoplásicos , Melanoma/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Transducción de Señal , Aurora Quinasa A/antagonistas & inhibidores , Aurora Quinasa A/genética , Azepinas/farmacología , Línea Celular Tumoral , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Factor de Transcripción Asociado a Microftalmía/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Pirimidinas/farmacología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
We have recently shown the CD44 variant isoform 10 (CD44v10) to be expressed on reactive as well as malignant cutaneous lymphocytes; however, the functional consequences of CD44v10 expression on lymphocytes are not elucidated. By using appropriately transfected lymphatic cells we analyzed the role of CD44v10 on lymphocytes in cell-matrix adhesion and homotypic and heterotypic cell-cell adhesion assays. Despite a low binding affinity to hyaluronan, CD44v10-expressing lymphocytes exhibited heterotypic cell-cell adhesion to inflamed dermal microvascular endothelium and keratinocytes, as indicated by Stamper-Woodruff assays on tissue sections of delayed type hypersensitivity reactions and adhesion assays with cultured keratinocytes and cytokine-stimulated human dermal microvascular endothelial cells. Antibody-blocking assays excluded interaction of CD44v10 with the principal CD44 ligand hyaluronan as well as involvement of selectins or integrins in these heterotypic cell-cell adhesion assays. In contrast, cellular aggregation assays with fluorescence-labeled CD44v10- and CD44H-expressing lymphocytes revealed homotypic CD44v10/CD44v10 binding as well as binding of CD44v10 with CD44H. Heterotypic cell-cell adhesion assays with ultraviolet-A-irradiated CD44v-negative cytokine-stimulated endothelial cells demonstrated binding kinetics of CD44v10-expressing lymphocytes paralleling those of endothelial CD44H expression. These results imply that a hyaluronan-independent CD44v10/CD44H-mediated pathway is involved in lymphocyte infiltration into the dermis and epidermis of inflamed skin and suggest modulation of CD44H expression on inflamed dermal microvascular endothelium as a mechanism of ultraviolet-A-induced therapeutic effects on the skin.
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Endotelio Vascular/fisiología , Receptores de Hialuranos/análisis , Ácido Hialurónico/fisiología , Queratinocitos/fisiología , Linfocitos/fisiología , Piel/irrigación sanguínea , Adhesión Celular , Células Cultivadas , Endotelio Vascular/citología , Exones , Variación Genética , Humanos , Receptores de Hialuranos/metabolismo , Receptores de Hialuranos/fisiología , Ácido Hialurónico/metabolismo , Integrinas/fisiología , Microcirculación , Selectinas/fisiologíaRESUMEN
Ras gene mutations have been implicated in the pathogenesis of a variety of human tumors. Mutated ras genes have been isolated from human melanoma cell lines, but subsequent studies indicated that ras gene mutations may be a rare event in melanocytic lesions. Recently, a study reported a high frequency of ras mutations correlated with increasing invasion level. To address this inconsistency in the published data, we analyzed 50 primary melanomas to correlate invasion level, tumor thickness, histologic typing, and body localization with point mutations around codons 12/13/61 of the three ras genes. After micro-dissection of paraffin-embedded tumor tissue, ras gene mutations were analyzed by direct sequencing of tumor DNA amplified by polymerase chain reaction. Only two melanomas exhibited ras gene mutations, one sample containing a transition from A to G at position 2 of N-ras codon 61 and the other exhibiting a transversion from C to A at position 1 and a transition from A to G at position 2 of N-ras codon 61. Both tumors were classified as Clark level IV, with a tumor thickness of 2.5 and 1.2 mm, respectively. Both were typed as superficial spreading melanoma and localized to intermittently sun-exposed body sites. The low frequency of ras mutations in malignant melanoma and the lack of ras mutations in melanoma samples from constantly sun-exposed body sites argue against the hypothesis of ras mutations as a marker of progression in malignant melanoma and the suggestion that ras mutations occur predominantly in melanomas from constantly sun-exposed body sites.
Asunto(s)
Genes ras/genética , Melanoma/genética , Secuencia de Bases , ADN de Neoplasias , Amplificación de Genes , Humanos , Adhesión en Parafina , Mutación Puntual , Reacción en Cadena de la PolimerasaRESUMEN
Tumor cell invasion and metastasis are considered to represent a multistep process leading to the degradation of the extracellular matrix by proteolytic enzymes. The functional activity of matrix metalloproteinases (MMPs) is controlled by tissue inhibitor of metalloproteinases-2 (TIMP-2), which has been shown to inhibit tumor cell invasion and metastasis in vitro and in vivo. To assess the role of TIMP-2 in skin-derived epithelial tumors, we have analyzed the expression of TIMP-2 mRNA in primary tissue samples from human cutaneous basal (BCC) and squamous cell carcinoma (SCC) for a correlation with their different invasive and metastatic potential. Comparative quantitative analysis of TIMP-2 mRNA levels by Northern blot hybridization demonstrated significantly higher TIMP-2 tissue levels in BCC than in SCC, indicating an inverse correlation between TIMP-2 expression and the metastatic capacity of these tumors in vivo. By in situ hybridization, tumor stromal cells were identified as the principal source of TIMP-2 mRNA in both BCC and SCC. A comparable distribution has been reported previously for several matrix metalloproteinases in cutaneous BCC and SCC, indicating co-localization of metalloproteinases with their respective inhibitor. These results may suggest that TIMP-2 substantially contributes to the biological behavior of epithelium-derived skin tumors by significantly inhibiting tumor cell metastasis.
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Carcinoma Basocelular/genética , Carcinoma de Células Escamosas/genética , Proteínas/genética , Neoplasias Cutáneas/genética , Carcinoma Basocelular/química , Carcinoma de Células Escamosas/química , Expresión Génica , Humanos , Inhibidores de Proteasas/metabolismo , ARN Mensajero/análisis , Piel/química , Neoplasias Cutáneas/química , Inhibidor Tisular de Metaloproteinasa-2 , Transcripción GenéticaRESUMEN
The remarkable functional diversity of the cell surface receptor CD44 may be due to expression of multiple variant isoforms generated by alternative splicing of variant exons. Functional and correlative data implicate a role of CD44 variant isoforms in adhesion dependent processes such as lymphocyte recirculation and tumor progression and metastasis. We have analyzed 25 primary cutaneous lymphomas and 35 reactive lymphoid cell skin infiltrates or T cell-mediated skin diseases for the expression of CD44 variant isoforms. Irrespective of histologic typing, staging, and grading, cutaneous lymphomas as well as nonmalignant skin-infiltrating CD3+ CD4+ and CD8+ T and CD19+ B lymphocytes exhibited a strong expression of CD44v10 and a moderate expression of CD44v3 as determined by immunohistochemistry, immunofluorescence microscopy, and mRNA analysis. Expression of v5, v6, v7, and v9-containing CD44 variant isoforms was not detected. Furthermore, flow cytometry revealed expression of CD44v10 on a significant proportion of peripheral blood lymphocytes from Sézary's syndrome patients and a remarkable co-expression with cutaneous lymphocyte antigen. These results indicate a distinct CD44 variant isoform expression pattern associated with skin-homing lymphocytes different to lymphatic cells at noncutaneous sites. This differential expression pattern of CD44 variant isoforms may contribute to the development of lymphocyte skin infiltrates and/or the unique biologic behavior of cutaneous lymphomas.
Asunto(s)
Empalme Alternativo , Variación Genética , Receptores de Hialuranos/genética , Linfocitos/inmunología , Linfoma/inmunología , Neoplasias Cutáneas/inmunología , Formación de Anticuerpos , Progresión de la Enfermedad , Exones , Humanos , Metástasis Linfática , Linfoma/genética , Linfoma/patología , Enfermedades de la Piel/inmunología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Regulación hacia ArribaRESUMEN
Using the differentiation antigen Pmel17/gp100 to genetically immunize C57BL/6 mice (H-2(b)), we and colleagues noticed that only mice that had received the human homolog but not animals injected with the murine counterpart were protected against the growth of syngeneic B16 melanoma cells. The goal of this study was to determine whether the state of nonresponsiveness to the autoantigen Pmel17/gp100 can be broken by immunization with a plasmid DNA construct encoding the autologous form of the molecule. A construct containing the murine form of Pmel17 was administered intradermally to DBA/2 mice (H-2(d)), which were then investigated for the presence of Pmel17/gp100-specific immunity. We show that administration of plasmid DNA coding for the autologous melanoma-associated antigen Pmel17/gp100 protects DBA/2 mice against the growth of Pmel17-positive M3 melanoma cells but not against Pmel17-negative M3 melanoma cells or unrelated P815 mastocytoma cells. Cell depletion experiments demonstrated that this protective effect is mediated by T lymphocytes. The notion that Pmel17/gp100 represents the biologically relevant target in this system was supported by the observations (i) that recipients of Pmel17/gp100 DNA mount an antigen-specific cytotoxic T lymphocyte response and (ii) that M3 tumors growing in mice immunized with autologous Pmel17/gp100 had lost expression of this melanoma-associated antigen whereas M3 melanomas appearing in control-vector-treated animals were still Pmel17/gp100-positive. These results indicate that intracutaneous genetic immunization with autologous melanoma-associated antigen Pmel17/gp100 encoding plasmid DNA can lead to protection against melanoma cells as a result of the induction of a melanoma-associated antigen-specific and protective T-cell-mediated immune response. J Invest Dermatol 115:1082-1087 2000
Asunto(s)
Proteínas/inmunología , Animales , Antígenos de Neoplasias , Epítopos , Femenino , Humanos , Inmunidad Celular , Inmunización , Inyecciones Intradérmicas , Antígenos Específicos del Melanoma , Glicoproteínas de Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Neoplasias/prevención & control , Proteínas/genética , Linfocitos T/inmunología , Vacunación , Antígeno gp100 del MelanomaRESUMEN
In basal cell carcinoma, release of proteolytic activity is implicated in extracellular matrix degradation and tumor infiltration. The stromelysin metalloproteinase family is a major candidate for the matrix proteolytic activity in infiltrative tumors. However, in murine models of basal cell carcinoma, neither stromelysin 1 nor 2 appears to play a role in tumor infiltration. We have analyzed the expression of the newly described stromelysin 3 in human basal cell carcinoma using Northern blot analysis and in situ hybridization. In 12 of 14 cases, levels of stromelysin 3 expression were more than tenfold above those observed in normal skin. In one of five cases of squamous cell carcinoma, stromelysin 3 expression was tenfold above levels seen in normal skin. Stromelysin 3 expression was either undetectable or extremely weak in all five cases of infiltrative malignant melanoma. In basal cell carcinoma, stromelysin 3 transcripts were localized by in situ hybridization to the stromal tissue immediately adjacent to basal cell carcinoma, the tumor cells themselves being negative. Therefore, expression of stromelysin 3 in stromal cells may be expected to play a significant role in destruction of the basal membrane zone and extracellular matrix in basal cell carcinoma invasion.
Asunto(s)
Carcinoma Basocelular/enzimología , Metaloendopeptidasas/metabolismo , Neoplasias Cutáneas/enzimología , Northern Blotting , Carcinoma Basocelular/genética , Carcinoma Basocelular/patología , Sondas de ADN , Expresión Génica , Humanos , Hibridación in Situ , Metaloproteinasa 11 de la Matriz , Metaloendopeptidasas/genética , Sondas ARN , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patologíaRESUMEN
The influence of the immunosuppressants, cyclosporin A (CsA) and FK506, on cAMP formation was studied in T cells from healthy controls and patients with psoriasis. While basal cAMP levels were not affected, CsA (1 microM) and FK506 (2 nM) prevented the isoprenaline (0.1 microM)-induced increase in cAMP formation. Half-maximal inhibition by FK506 and CsA was observed at about 0.2 nM and 20 nM, respectively. In addition, both agents significantly reduced (by about 50%) the forskolin (8 microM)-stimulated cAMP formation. No differences were noted in cAMP responses (basal, stimulation by isoprenaline, inhibition by CsA and FK506) of T cells from healthy controls and psoriatic patients. We conclude that CsA and FK506 potently and efficiently interfere with the stimulatory adenylyl cyclase pathway in T cells and that regulation of T cell cAMP formation is apparently not altered in psoriasis.
Asunto(s)
AMP Cíclico/biosíntesis , Ciclosporina/farmacología , Inmunosupresores/farmacología , Linfocitos T/efectos de los fármacos , Tacrolimus/farmacología , Adulto , Anciano , Colforsina/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Isoproterenol/farmacología , Masculino , Persona de Mediana Edad , Psoriasis/metabolismo , Linfocitos T/metabolismoRESUMEN
The predominant cutaneous side effect of lithium is the exacerbation or aggravation of psoriasis, but the pathogenesis is still unclear. The hyperproliferation of keratinocytes and a dense lesional infiltrate of mononuclear cells are the hallmarks of psoriatic skin lesions. Interactions between keratinocytes and T cells are thought to be one reason for an increased secretion of proinflammatory cytokines and growth factors. To investigate whether lithium influences cytokines of the "psoriatic cytokine network', we established a coculture model with keratinocytes from psoriatic patients and from healthy controls cultured with HUT 78 lymphocytes and measured the cytokine levels of Il-2, Il-6, Il-8, IFN gamma and TGF alpha in the culture supernatants after treatment with lithium. Il-6 levels were slightly elevated in the supernatants obtained from psoriatic and control keratinocyte cultures after lithium treatment, but IFN gamma and Il-2 levels were elevated only in the lithium-treated cocultures with psoriatic keratinocytes. In contrast, these two cytokines were not affected by lithium in HUT 78 monocultures or in cocultures with normal epidermal cells. We also found slightly elevated TGF alpha levels in lithium-treated psoriatic cocultures but not in control cultures. We therefore demonstrated that lithium influences the cell communication of psoriatic keratinocytes with HUT 78 lymphocytes by triggering the secretion of TGF alpha, Il-2 and, massively, IFN gamma. It seems possible that lithium also influences similar parts of the psoriatic cytokine network in vivo.
Asunto(s)
Citocinas/fisiología , Queratinocitos/efectos de los fármacos , Litio/efectos adversos , Linfocitos/efectos de los fármacos , Psoriasis/metabolismo , Adulto , Anciano , Células Cultivadas , Técnicas de Cocultivo , Medios de Cultivo/metabolismo , Citocinas/metabolismo , Femenino , Humanos , Queratinocitos/metabolismo , Linfocitos/metabolismo , Masculino , Persona de Mediana Edad , Psoriasis/patología , Psoriasis/fisiopatologíaRESUMEN
Tumor cells with alterations in the MHC class I peptide-loading complex are unable to load peptide antigens onto MHC class I molecules. These alterations result in destabilization of MHC class I expression on the tumor cell surface and thus play a critical role in escape from immunological recognition by the acquired cellular immune system. By forming physical links between class I heavy chains and TAP molecules, a component of the class I peptide-loading complex, tapasin, plays an important role in the assembly of MHC class I molecules with peptides in the endoplasmic reticulum. In the present study, we compared 104 human melanoma lesions representing different stages of tumor progression for their expression of tapasin in tumor cells by immunohistochemistry. Tapasin downregulation was significantly associated with tumor progression. Whereas 100% of melanomata in situ and 96.2% of primary melanomas with a Breslow index of
Asunto(s)
Antiportadores/metabolismo , Inmunoglobulinas/metabolismo , Melanoma/metabolismo , Neoplasias Cutáneas/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Progresión de la Enfermedad , Regulación hacia Abajo , Femenino , Humanos , Inmunohistoquímica/métodos , Masculino , Proteínas de Transporte de Membrana , Persona de Mediana Edad , Coloración y EtiquetadoRESUMEN
The microphthalmia-associated transcription factor (MITF) is indispensable for the viability of melanocytic cells, is an oncogene in melanoma and has a cell type-specific expression pattern. As the modulation of MITF activity by direct chemical targeting remains a challenge, we assessed a panel of drugs for their ability to downregulate MITF expression or activity by targeting its upstream modulators. We found that the multi-kinase inhibitors midostaurin and sunitinib downregulate MITF protein levels. To identify the target molecules shared by both the drugs in melanocytic cells, a chemical proteomic approach was applied and AMP-activated kinase (AMPK) was identified as the relevant target for the observed phenotype. RNA interference and chemical inhibition of AMPK led to a decrease in MITF protein levels. Reduction of MITF protein levels was the result of proteasomal degradation, which was preceded by enhanced phosphorylation of MITF mediated by ERK. As expected, downregulation of MITF protein levels by AMPK inhibition was associated with decreased viability. Together, these results identify AMPK as an important regulator for the maintenance of MITF protein levels in melanocytic cells.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Melanocitos/metabolismo , Melanoma/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Western Blotting , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cromatografía Liquida , Inhibidores Enzimáticos/farmacología , Técnica del Anticuerpo Fluorescente , Humanos , Indoles/farmacología , Espectrometría de Masas , Melanocitos/efectos de los fármacos , Melanoma/genética , Factor de Transcripción Asociado a Microftalmía/genética , Oncogenes , Pirroles/farmacología , Interferencia de ARN , ARN Interferente Pequeño , Estaurosporina/análogos & derivados , Estaurosporina/farmacología , Sunitinib , TransfecciónRESUMEN
METHOD: The data from our patients of the last 10 years were evaluated in relation to survival and recurrence. RESULTS: Very different surgical therapy-in some cases due to refusal of the therapy suggested-was performed in eight patients (including wedge shaped excision as well as ablation of the auricle). The mean survival ranged from 40 months up to the present. One patient died due to a local recurrence and regional and systemic metastases. One patient experienced local recurrence but is still well 16 months after he underwent surgical revision. Two patients died independently of the tumor. DISCUSSION: Comparison of our results with those of other studies confirms that the extent of the applied resection has of minor effect on recurrence and survival for this disease. Tumor thickness is the most important independent prognostic factor.
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Neoplasias del Oído/cirugía , Oído Externo/cirugía , Melanoma/cirugía , Neoplasias Cutáneas/cirugía , Adulto , Anciano , Neoplasias del Oído/mortalidad , Neoplasias del Oído/patología , Oído Externo/patología , Femenino , Estudios de Seguimiento , Humanos , Escisión del Ganglio Linfático , Masculino , Melanoma/mortalidad , Melanoma/patología , Persona de Mediana Edad , Recurrencia Local de Neoplasia/mortalidad , Recurrencia Local de Neoplasia/patología , Recurrencia Local de Neoplasia/cirugía , Estadificación de Neoplasias , Pronóstico , Estudios Retrospectivos , Biopsia del Ganglio Linfático Centinela , Neoplasias Cutáneas/mortalidad , Neoplasias Cutáneas/patología , Tasa de SupervivenciaRESUMEN
Thus far, the use of classical anti-cancer treatment modalities had only rarely a beneficial impact on the prognosis of patients with metastatic melanoma. We as physicians have therefore the obligation as well as the chance to develop and test new therapeutic strategies. Our growing knowledge about the genetic basis of melanoma provides one platform to fulfil this task. Another one comes from our increasing understanding of the molecular and cellular mechanisms involved in the induction/modulation of immune responses, as well as the progress made in the field of identification of melanoma antigens, and allows for the development of a new generation of vaccines. The aim of this article is to discuss several of these new concepts towards the use of immune and gene therapy of melanoma.
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Vacunas contra el Cáncer/uso terapéutico , Terapia Genética/métodos , Melanoma/terapia , Antígenos de Neoplasias/uso terapéutico , Humanos , Melanoma/inmunología , Melanoma/secundarioRESUMEN
Pmel17/gp100-encoded tumor-associated antigens are recognized by cytotoxic T lymphocytes in melanoma patients and may represent attractive target antigens for immuno- and gene-therapeutic strategies. An important prerequisite for identification and monitoring of melanoma patients that could potentially benefit from Pmel17/gp100-based immuno- and gene-therapies is the detailed knowledge of Pmel17/gp100 expression in vivo. Immunophenotyping is considerably hampered by the different immunoreactivities of Pmel17/gp100-reactive antibodies. Therefore, we analyzed an extended series of different primary normal and malignant human tumor specimens for Pmel17/gp100 expression at the mRNA level. Transcripts were detectable in all malignant melanoma tissue specimens representing all stages of tumor progression, with significant levels even in early and amelanotic melanoma lesions. In contrast, normal melanocytes exhibited significantly less Pmel17/gp100 mRNA in vivo, as determined by comparative in situ hybridization. Tissue specimens from the retina and substantia nigra also contained Pmel17/gp100 mRNA, whereas other normal and malignant human tissues were negative. As determined by comparative in situ hybridisation and HMB-45 immunostaining, even tumor tissue lacking Pmel17/gp100 immunoreactivity contained Pmel17/gp100 transcripts. Our results indicate a melanocytic-cell-lineage-restricted expression of Pmel17/gp100 with significant transcript levels in all stages of melanoma progression, including early and amelanotic melanoma lesions, and a significantly differential expression between melanoma cells and normal melanocytes in vivo. Owing to its higher sensitivity, phenotyping of individual tumor specimens by mRNA expression analysis seems to be more valuable than phenotyping by immunostaining.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/análisis , Terapia Genética/métodos , Inmunoterapia/métodos , Melanoma/terapia , Proteínas de Neoplasias/análisis , Neoplasias Cutáneas/terapia , Linfocitos T Citotóxicos/patología , Anticuerpos Antineoplásicos/análisis , Anticuerpos Antineoplásicos/inmunología , Anticuerpos Antineoplásicos/metabolismo , Antígenos de Neoplasias , Antígenos de Carbohidratos Asociados a Tumores/genética , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Northern Blotting , Regulación Neoplásica de la Expresión Génica , Terapia Genética/normas , Humanos , Inmunohistoquímica , Inmunofenotipificación , Inmunoterapia/normas , Hibridación in Situ , Melanoma/inmunología , Melanoma/patología , Antígenos Específicos del Melanoma , Glicoproteínas de Membrana , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Nevo/inmunología , Nevo/patología , Nevo/terapia , Proteínas , ARN Mensajero/análisis , ARN Mensajero/genética , Sensibilidad y Especificidad , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Linfocitos T Citotóxicos/inmunología , Transcripción Genética , Células Tumorales Cultivadas , Neoplasias de la Úvea/inmunología , Neoplasias de la Úvea/patología , Neoplasias de la Úvea/terapia , Antígeno gp100 del MelanomaRESUMEN
Chromosome 9p21 is frequently deleted in malignant melanoma, and one familial malignant melanoma gene has been linked to 9p21-22. Recently, the cyclin D-dependent kinase inhibitors (CDKIs) p16INK4a and p15INK4b have been localized within chromosome 9p21, and the presence of p16INK4a point mutations has been demonstrated in familial melanoma and melanoma cell lines in vitro. To analyse the role of these CDKIs in sporadic human cutaneous non-metastatic malignant melanoma, we examined 36 primary tumour specimens representing different stages of melanoma progression and their corresponding normal skin samples for the three mechanisms of CDKI inactivation described so far. Homozygous codeletion of the p16INK4a and the p15INK4b gene was detected by Southern blot analysis in two tumour samples. By direct sequencing of polymerase chain reaction (PCR)-amplified microdissected genomic DNA; no somatic or germline p16INK4a point mutations or small deletions were detected in the remaining 34 tumour samples; one individual exhibited the previously described germline codon 148 (Ala-->Thr) polymorphism. In these tumour specimens, comparative semiquantitative reverse PCR analysis of p16INK4a transcript levels revealed no evidence for repression of p16INK4a gene transcription and thus for p16INK4a promoter inactivation by DNA methylation. These results indicate homozygous p16INK4a and p15INK4b loss to occur in a subset of cutaneous melanomas and suggest, in view of the frequent loss of heterozygosity on chromosome 9p21, the presence of another tumour suppressor gene within this chromosomal region.
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Genes Supresores de Tumor/genética , Genes p16/genética , Melanoma/genética , Neoplasias Cutáneas/genética , Southern Blotting , Eliminación de Gen , Expresión Génica , Humanos , Mutación , Proteínas de Neoplasias/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Neoplásico/genética , Sensibilidad y EspecificidadRESUMEN
Molecular oxygen plays a central role in the pathogenesis and therapy of chronic wounds. When reactive oxygen species are overproduced, oxidative stress results, with detrimental cytotoxic effects causing delayed wound healing. Therefore, elimination of reactive oxygen species could be an important strategy to improve healing of chronic wounds. Currently first therapeutic strategies targeting reactive oxygen species by antioxidants are being introduced into the treatment of chronic wounds.
Asunto(s)
Estrés Oxidativo/fisiología , Cicatrización de Heridas/fisiología , Heridas y Lesiones/fisiopatología , Animales , Antioxidantes/uso terapéutico , Enfermedad Crónica , Humanos , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Resultado del Tratamiento , Cicatrización de Heridas/efectos de los fármacos , Heridas y Lesiones/tratamiento farmacológicoRESUMEN
Epithelioid sarcoma is a rare soft tissue tumor that, due to its clinically unspecific features, is frequently mistaken for other benign and malignant entities. We report on a 63-year-old male presenting with a relapse of an epithelioid sarcoma. At clinical inspection, the tumor presented as an erythematous nodule with ulceration in the center. Despite its slow growth, epithelioid sarcoma should be regarded as an aggressive neoplasm exhibiting frequent local recurrences and subsequent lymphatic and visceral spreading in about half of the patients. Histopathological findings are the presence of large epithelioid and spindle cells with immunoreactivity for cytokeratin and vimentin.