RESUMEN
HYPOTHESIS: To determine the expression of the tyrosine kinases platelet-derived growth factor receptor (PDGFR) and c-Kit in vestibular schwannoma (VS) and to determine the potential role of imatinib mesylate (Gleevec) in regulating the growth and cell death of this tumor. BACKGROUND: Protein tyrosine kinases are transmembrane tyrosine kinase receptors that transduce signals from inside and outside the cell and function as relay points for signaling pathways. They have a key role in numerous processes that affect cell proliferation, tumorigenesis, cancer invasion, metastasis, and modulation of apoptosis. A few of these kinases have been demonstrated to be overexpressed and dysregulated in many carcinomas, sarcomas, and benign tumors. METHODS: Immunohistochemical staining was used to investigate the expression of PDGFR and c-Kit in archived acoustic neuroma tissue. Clinical data including size of tumors, age, sex, and symptoms were correlated with kinase expression, whereas Western blot analysis and immunofluorescence were performed to demonstrate the expression and localization of PDGFR and c-Kit in HEI193, an immortalized VS cell line. Clonogenic survival assays were performed to assess proliferation inhibition by Gleevec. Gleevec's effect on the cell cycle profile also was investigated via flow cytometry analysis. RESULTS: Expression of PDGFR in the formalin-fixed VS tumor tissue was observed in 23 (67.5%) of the 34 samples. C-kit was expressed in 18 (52.9%) of the 34 samples. Western blot analysis demonstrates positive expression of c-Kit and PDGFR-Q in HEI193 and a primary VS culture. Western blot analysis showed downregulation of phospho-c-kit and phospho-PDGFR-Q with 5 and 10 uM Gleevec. Immunofluorescent staining of this cell line also reveals that PDGFR-ß is localized primarily in the cytoplasm, whereas c-Kit is both nuclear and cytoplasmic. Cell cycle analysis of HEI193 96 hours after incubation with Gleevec indicates a dose-dependent increase in G1 from 61.6% to 70.7% and 74% at 5 and 10 uM of Gleevec, respectively. Colony formation assays demonstrate dose-dependent growth inhibition by Gleevec, in the HEI193 cell line as well as in a VS cell culture derived from a fresh tumor. CONCLUSION: The expression of PDGFR-Q and c-Kit in VS tissue may indicate novel molecular targets involved in the development of this tumor. Direct inhibition of these molecules by Gleevec may have relevant therapeutic applications.
Asunto(s)
Antineoplásicos/farmacología , Muerte Celular/efectos de los fármacos , Neuroma Acústico/metabolismo , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Pirimidinas/farmacología , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Adulto , Anciano , Antineoplásicos/uso terapéutico , Benzamidas , Western Blotting , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Citometría de Flujo , Humanos , Mesilato de Imatinib , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Neuroma Acústico/tratamiento farmacológico , Fosforilación/efectos de los fármacos , Piperazinas/uso terapéutico , Pirimidinas/uso terapéutico , Células Tumorales CultivadasRESUMEN
OBJECTIVES: To determine the expression of the p53 family member p73 in vestibular schwannoma (VS) and to determine the potential role of this tumor suppressor in regulating the proliferation of HEI193, a human papillomavirus E6-E7 immortalized VS cell line. METHODS: Immunohistochemical staining was used to investigate the expression of p73 in 34 cases of archived VS tissue, while Western blot analysis and immunofluorescence were performed to demonstrate the expression and localization of p73 in HEI193. After transfection of a full-length p73 plasmid (TAp73alpha), flow cytometry analysis was performed to determine the effect of p73 expression on cell cycle distribution, while annexin V-FITC (fluorescein isothiocyanate) analysis and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling) assay were used to measure apoptosis. The effect of p73 expression on ionizing radiation-induced cell death was also investigated with annexin V staining, TUNEL assay, and flow cytometry analysis. RESULTS: Of the 34 vestibular schwannoma tissues examined, p73 was expressed in 14 (41%) but was not expressed in HEI193. Transfection of p73 alone resulted in increased apoptosis and necrosis, and G(1) accumulation with concomitant induction of p21. The presence of p73 also significantly increased early apoptosis (P = .046), late apoptosis (P < .001), and necrosis (P = .009) on exposure of the HEI193 cells to ionizing radiation. CONCLUSION: Forced expression of p73, perhaps by gene therapy, to induce apoptosis directly or to sensitize VS tumors to ionizing radiation may have relevant therapeutic applications.