Loss of FBXW7 expression can lead to MYC overexpression and has been associated with poor prognosis in GC patients. However, MYC activation by FBXW7 loss triggers activation of p53, which plays a key role in the regulation of cellular responses selleck chem inhibitor to DNA damage and abnormal expression of oncogenes. Induction of cell cycle arrest by p53 allows for DNA repair or apoptosis induction. Thus, concomitant loss of FBXW7 and TP53 is necessary to induce genetic instability and tumorigenesis. In the present study, we investigated MYC, FBXW7, and TP53 gene copy number variation and mRNA and protein expression in GC samples and gastric adenocar cinoma cell lines. Possible associations between our findings and the clinicopathological features and or invasion and migration capability of the cell lines were also evaluated.
Methods Clinical samples Samples were obtained from 33 GC patients who under went surgical treatment at the Jo?o de Barros Barreto University Hospital in Par State, Brazil. Dissected tumor and paired non neoplastic tissue specimens were immediately cut from the stomach and frozen in liquid nitrogen until RNA extraction. The clinicopathological features of the patient samples are shown in Table 1. GC samples were classified according to Lauren. All GC samples showed the presence of Helicobacter pylori, and the cagA virulence factor was determined by PCR analysis of ureA and cagA as described by Clayton et al. and Covacci et al. respectively. All patients had negative histories of exposure to either chemotherapy or radiotherapy before surgery, and there were no other co occurrences of diag nosed cancers.
Informed consent with approval of the ethics committee of the Federal University of Par was obtained. Cells lines Gastric adenocarcinoma cell lines ACP02 and ACP03 were cultured in complete RPMI medium supplemented with 10% fetal bovine serum, 1% penicillin streptomycin, and 1% kanamycin. Copy number variation DNA was extracted using a DNAQiamp mini kit according to the manufacturers instructions. Duplex quantitative real time PCR was performed using the FAM MGB labeled TaqMan probes for MYC, FBXW7, or TP53, and VIC TAMRA labeled TaqMan CNV RNAse P was used for the internal control. All real time qPCR reactions were performed in quadruplicate with gDNA according to the manufacturers protocol using a 7500 Fast Real Time PCR system.
The copy number of each sample was estimated by CNV analysis using Copy Caller Software V1. 0. Known Human Genomic DNA was used for calibration. Dacomitinib Quantitative real time reverse transcriptase PCR Total RNA was extracted with TRI Reagent Solution following the manufacturers instructions. RNA concentration and quality were determined using a NanoDrop spectropho tometer and 1% agarose gels. Complementary DNA was synthesized using a High Capacity cDNA Archive kit according to the manufacturers recommendations.