RESUMEN
PURPOSE: Advanced testicular germ cell tumours (GCT) generally have a good prognosis owing to their unique sensitivity towards cisplatin-based chemotherapies. However, cisplatin-resistant GCT have a poor outcome. Further studies are mandatory to better understand resistance mechanisms and develop therapeutic strategies for refractory GCTs. METHODS: Protein levels in cisplatin-resistant GCT cell lines of NTERA-2, NCCIT and 2102EP were analyzed by quantitative proteomic mass spectrometry (MS) in combination with stable isotope labelling by amino acids in cell culture (SILAC). Differentially abundant protein markers of acquired cisplatin resistance were validated by Western blotting. Comprehensive bioinformatical annotation using gene set enrichment analyses (GSEA) and STRING interaction analysis were performed to identify commonly affected pathways in cisplatin resistance and the data were compared to the GCT cohort of the 'The Cancer Genome Atlas'. RESULTS: A total of 4375 proteins were quantified by MS, 144 of which were found to be differentially abundant between isogenic resistant and sensitive cell line pairs (24 proteins for NTERA-2, 60 proteins for NCCIT, 75 proteins for 2102EP). Western blotting confirmed regulation of key resistance-associated proteins (CBS, ANXA1, LDHA, CTH, FDXR). GSEA revealed a statistically significant enrichment of DNA repair-associated proteins in all three resistant cell lines and specific additional processes for individual cell lines. CONCLUSION: High resolution MS combined with SILAC is a powerful tool and 144 significantly deregulated proteins were found in cisplatin-resistant GCT cell lines. Our study provides the largest proteomic in vitro library for cisplatin resistance in GCT, yet, enabling further studies to develop new treatment options for patients with refractory GCT.
Asunto(s)
Antineoplásicos , Neoplasias de Células Germinales y Embrionarias , Neoplasias Testiculares , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Cisplatino/farmacología , Cisplatino/uso terapéutico , Resistencia a Antineoplásicos , Humanos , Masculino , Espectrometría de Masas , Neoplasias de Células Germinales y Embrionarias/tratamiento farmacológico , Proteómica , Neoplasias Testiculares/tratamiento farmacológico , Neoplasias Testiculares/patologíaRESUMEN
BACKGROUND AND PURPOSE: Adenoid cystic carcinomas (ACC) are characterized by high rate of local recurrence and late distant metastasis. Chromosomal changes in the evolution from primary tumors to metastatic disease of ACC have not been appointed. Here we investigated the chromosomal alterations of 53 primary tumors from ACC patients with different progressive states by shallow whole genome sequencing to identify potential new markers for metastatic spread. METHODS: Illumina paired-end libraries were generated using DNA from the primary tumor of 53 ACC patients. Fragmented DNA was end-repaired, A-tailed and multiplex sequencing adapters were ligated. Sequence data were mapped to HG19 and a copy-number analysis was conducted using the QDNAseq R package (version 1.10.0). Outliers were removed and data was smoothed by applying the circular binary segmentation algorithm implemented in the R package copynumber version 1.22.0. A modified chromosomal instability (CNI) score was used to analyze deletions and amplifications. RESULTS: Cluster analysis of the whole genome sequencing revealed that the frequency of chromosomal aberrations were increased in ACC with local recurrence and distant metastases in comparison to ACC patients with no metastatic spread. Specifically, chromosome 6 and 12 and exclusively the entire chromosome 4 showed an increased frequency of chromosomal alterations with tumor progression. CONCLUSION: Our data show a molecular evolution from primary tumors to local recurrences and distant metastases and pinpoint the critical chromosomal regions involved in this process. These regions should be in the focus of the search for therapeutic targets of progressive ACC.
Asunto(s)
Carcinoma Adenoide Quístico/genética , Neoplasias de las Glándulas Salivales/genética , Secuenciación Completa del Genoma/métodos , Carcinoma Adenoide Quístico/patología , Aberraciones Cromosómicas , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Neoplasias de las Glándulas Salivales/patologíaRESUMEN
Current methods to analyze gene expression measure steady-state levels of mRNA. To specifically analyze mRNA transcription, we have developed a technique that can be applied in vivo in intact cells and animals. Our method makes use of the cellular pyrimidine salvage pathway and is based on affinity-chromatographic isolation of thiolated mRNA. When combined with data on mRNA steady-state levels, this method is able to assess the relative contributions of mRNA synthesis and degradation/stabilization. It overcomes limitations associated with currently available methods such as mechanistic intervention that disrupts cellular physiology, or the inability to apply the techniques in vivo. Our method was first tested in serum response of cultured fibroblast cells and then applied to the study of renal ischemia reperfusion injury, demonstrating its applicability for whole organs in vivo. Combined with data on mRNA steady-state levels, this method provided a detailed analysis of regulatory mechanisms of mRNA expression and the relative contributions of RNA synthesis and turnover within distinct pathways, and identification of genes expressed at low abundance at the transcriptional level.