RESUMEN
The molecular mechanisms involved in tumor progression from a low-grade astrocytoma to the most malignant glioblastoma multiforme (GBM) have been hampered due to lack of suitable experimental models. We have established a model of tumor progression comprising of two cell lines derived from the same astrocytoma tumor with a set of features corresponding to low-grade glioma (as in HNGC-1) and high-grade GBM (as in HNGC-2). The HNGC-1 cell line is slow-growing, contact-inhibited, nontumorigenic, and noninvasive, whereas HNGC-2 is a rapidly proliferating, anchorage-independent, highly tumorigenic, and invasive cell line. The proliferation of cell lines is independent of the addition of exogenous growth factors. Interestingly, the HNGC-2 cell line displays a near-haploid karyotype except for a disomy of chromosome 2. The two cell lines express the neuronal precursor and progenitor markers vimentin, nestin, MAP-2, and NFP160, as well as glial differentiation protein S100beta. The HNGC-1 cell line also expresses markers of mature neurons like Tuj1 and GFAP, an astrocytic differentiation marker, hence contributing toward a more morphologically differentiated phenotype with a propensity for neural differentiation in vitro. Additionally, overexpression of epidermal growth factor receptor and c-erbB2, and loss of fibronectin were observed only in the HNGC-2 cell line, implicating the significance of these pathways in tumor progression. This in vitro model system assumes importance in unraveling the cellular and molecular mechanisms in differentiation, transformation, and gliomagenesis.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral/metabolismo , Transformación Celular Neoplásica , Glioma/metabolismo , Proteínas del Tejido Nervioso , Anciano , Western Blotting , Neoplasias Encefálicas/patología , Línea Celular Tumoral/patología , Receptores ErbB/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Proteína Ácida Fibrilar de la Glía/metabolismo , Glioma/patología , Humanos , Proteínas de Filamentos Intermediarios/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Cariotipificación , Masculino , Microscopía Confocal , Invasividad Neoplásica/patología , Nestina , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor ErbB-2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas S100/metabolismo , Transducción de Señal/fisiología , Tubulina (Proteína)/metabolismo , Vimentina/metabolismoRESUMEN
Multi-cellular spheroids (MCS) generated from tumor cells serve as excellent in vitro models for understanding the mechanisms of tumor progression and micro-metastasis. We have compared the expression of molecular markers with reference to their growth as conventional adherent monolayers (2-D) and anchorage independent cultures (3-D) using two mouse melanoma cell lines, B16F10 and Clone M3. The two cell lines differed in their ability to form spheroids with respect to their aggregation potential, with B16F10 forming large clusters compared to Clone M3. A panel of molecular markers comprising cell adhesion molecules, cyclin dependent kinase inhibitors and members of the cadherin-catenin complex were analyzed by flow cytometry in 2-D and 3-D cultures. There was a distinct difference in the patterns of expression of CD44(S) and variant isoforms v3, v10 in spheroids compared to cells grown as monolayers in both cell lines. Also, there was an increase in cells positive for CDK inhibitor p27 in 3-D cultures from the B16F10 cell line. The expression of alpha and gamma catenin was down regulated in spheroids. As these molecules are implicated in the regulation of cell proliferation, alterations in the expression of these molecules in 3-D cultures compared to their 2-D counterparts suggests the importance of spheroids as experimental model for tumorigenesis.