Dynamic genomic changes in methotrexate-resistant human cancer cell lines beyond DHFR amplification suggest potential new targets for preventing drug resistance.

BACKGROUND: Although DHFR gene amplification has long been known as a major mechanism for methotrexate (MTX) resistance in cancer, the early changes and detailed development of the resistance are not yet fully understood. METHODS: We performed genomic, transcriptional and proteomic analyses of human colon cancer cells with sequentially increasing levels of MTX-resistance. RESULTS: The genomic amplification evolved in three phases (pre-amplification, homogenously staining region (HSR) and extrachromosomal DNA (ecDNA)). We confirm that genomic amplification and increased expression of DHFR, with formation of HSRs and especially ecDNAs, is the major driver of resistance. However, DHFR did not play a detectable role in the early phase. In the late phase (ecDNA), increase in FAM151B protein level may also have an important role by decreasing sensitivity to MTX. In addition, although MSH3 and ZFYVE16 may be subject to different posttranscriptional regulations and therefore protein expressions are decreased in ecDNA stages compared to HSR stages, they still play important roles in MTX resistance. CONCLUSION: The study provides a detailed evolutionary trajectory of MTX-resistance and identifies new targets, especially ecDNAs, which could help to prevent drug resistance. It also presents a proof-of-principal approach which could be applied to other cancer drug resistance studies.

Reduced expression and novel splice variants of ING4 in human gastric adenocarcinoma.

ING4, a new member of the ING (inhibitor of growth) family of tumour suppressor genes, has been found to be deleted or down-regulated in gliomas, breast tumours, and head and neck squamous cell carcinomas. The goal of the present study was to investigate whether the expression and alternative splicing of ING4 transcripts are involved in the initiation and progression of stomach adenocarcinoma. ING4 mRNA and protein expression was examined in gastric adenocarcinoma tissues and human gastric adenocarcinoma cell lines by RT-PCR, real-time RT-PCR, tissue microarray immunohistochemistry, and western blot analysis. Alterations in ING4 transcripts were determined through sequence analysis of ING4 cDNA. Our data showed that ING4 mRNA and protein were dramatically reduced in stomach adenocarcinoma cell lines and tissues, and significantly less in female than in male patients. We also found that reduced ING4 mRNA expression correlated with the stage of the tumour. Interestingly, by sequence analysis, we discovered five novel aberrantly spliced variant forms of ING4_v1 and ING4_v2. These variants cause a codon frame-shift and, eventually, deletion of the NLS or PHD domain contributing to the mislocalization of p53 and/or HAT/HDAC complexes and, subsequently, altered gene expression in gastric adenocarcinoma. These results suggest that attenuated and aberrant ING4 expression may be involved in the initiation and progression of stomach adenocarcinoma.

Elevated serum level and gene polymorphisms of TGF-beta1 in gastric cancer.

Transforming growth factor (TGF)-beta1, as a candidate tumor marker, is currently of interest. In this study, serum TGF-beta1 levels in gastric cancer (GC) patients and healthy volunteers were measured using enzyme-linked immunosorbent assay (ELISA). In addition, single nucleotide polymorphisms (SNPs) of the TGF-beta1 gene at codon 10 and codon 25 were identified by means of amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and sequence analysis. Our results indicated that serum concentrations of TGF-beta1 in GC patients were significantly higher than those in the control, and positively correlated with tumor mass, invasion, metastasis, and clinical stage. The serum TGF-beta1 levels of patients recovering from radical resection were markedly lower than those before surgery. Meanwhile, no deoxyribonucleic acid (DNA) sequence variation at codon 25 of the TGF-beta1 gene was found and a TGF-beta1 gene polymorphism at codon 10 did not show obvious correlations with either TGF-beta1 expression or clinicopathological parameters of GC. Our evidence suggested that serum concentration of TGF-beta1 might be a novel tumor marker for GC and the polymorphisms of TGF-beta1 gene did not play a role as a determinant of serum TGF-beta1 concentration or as a genetic risk factor in the gastric carcinogenesis and progression.

[Suppression of RASSF1A gene on human esophageal carcinoma cells: experiments in vitro and in vivo].

OBJECTIVE: To investigate the effect of Ras association domain family 1A (RASSF1A) gene, a new tumor suppressor gene (TSG), on tumorigenesis of human esophageal carcinoma cells. METHODS: pcDNA3.1 (+)-RASSF1A, a plasmid containing RASSF1A gene, and the blank plasmid pcDNA3.1 (+) were transfected into human esophageal carcinoma cells of the line EC9706. The expression of RASSF 1A protein was examined by Western blotting. The changes of cell cycle of stably-transfected cells were determined by flow cytometry (FCM), and the cellular proliferation was analyzed by MTT assay. Fifteen nude mice were randomly divided into 3 groups to be inoculated subcutaneously with EC9706 cells transfected with pcDNA3.1 (+)-RASSF1A, EC9706 cells transfected with pcDNA3.1 (+), and untransfected EC9706 cells respectively. Other 5 nude mice were used as controls. Four weeks later, the mice were killed to take out the carcinoma tissues. FCM was used to analyze the cell cycle. RESULTS: Western blotting showed that RASSF1A protein was expressed highly in the stably transfected cells. The cell viability and growing speed were decreased obviously in the cells expressing of RASSF1A (both P < 0.01); FCM showed that the proportion of cells at the G(1) phase of the EC9706 cells expressing RASSF1A was significantly higher than those in the blank plasmid group and untransfected group (both P < 0.01). The size of the EC9706 cells obtained from the nude mice inoculated with the EC9706 cells transfected with pcDNA3.1 (+)-RASSF1A was significantly smaller than those of the pcDNA3.1 (+) group and blank plasmid group (both P < 0.05). CONCLUSION: Expression of exogenous RASSF1A inhibits the progression of human esophageal carcinoma cells in vitro and in vivo. As a tumor suppressor gene, it plays an important role in origination, progression and metastasis of esophageal carcinoma.