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Resistance to TFT by decreased TK or overexpression of sPLA2

ABSTRACT: Trifluorothymidine (TFT) is part of the novel oral formulation TAS-102, which is currently evaluated in phase II studies. Drug resistance is an important limitation of cancer therapy. The aim of the present study was to induce resistance in the colon cancer cell line H630 using two different exposure schedules and to analyze the resistance mechanism. Cells were exposed either continuously or intermittently to TFT, resulting in H630-cTFT and H630-4TFT, respectively. Cells were analyzed for cross resistance, cell cycle, protein expression and activity of thymidine phosphorylase (TP), thymidine kinase (TK) and thymidylate synthase (TS), gene expression (microarray) and genomic alterations (array CGH). Both cell lines were cross-resistant to 2’-deoxy-5-fluorouridine (>170-fold). Exposure to IC75-TFT increased the S- and G2/M-phase of H630 cells, while in the resistant variants no change was observed. The two main target enzymes TS and TP remained unchanged in both TFT-resistant variants. In H630-4TFT cells, TK protein expression and activity were decreased, possibly related to an aberrant TK protein, clearly resulting in less activated TFT and most likely the mechanism of TFT resistance. Surprisingly, in H630-cTFT microarray analysis revealed a strong increase in mRNA levels of secretory phospholipase-A2 (sPLA2; 47-fold), which was also found by RT-PCR (211 fold increase). Inhibition of sPLA2 reversed TFT resistance partially. In addition, H630-cTFT had many chromosomal aberrations, but the exact role of sPLA2 in TFT-resistance remains unclear. Altogether, induction of resistance to TFT can lead to different mechanisms of resistance, including decreased TK protein expression and enzyme activity, as well as (phospho)lipid metabolism.

ORGANISM(S): Homo sapiens

PROVIDER: GSE18137 | GEO | 2010/06/15

SECONDARY ACCESSION(S): PRJNA119425

REPOSITORIES: GEO

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Project description:Purpose. How vitamin A contributes to the maintenance of the wet-surfaced phenotype at the ocular surface is not well understood. We sought to identify vitamin A responsive genes in ocular surface epithelia using gene microarray analysis of cultures of a human conjunctival epithelial cell line (HCjE) grown with all-trans-retinoic acid (RA). The analysis showed that the membrane-associated mucin MUC16 was induced by RA and that secretory phospholipase A2 Group IIA (sPLA2-IIA), the gene most upregulated by RA, was induced earlier. Since eicosanoids, metabolites of arachidonic acid, which is produced by sPLA2 catalysis of membrane phospholipids, have been demonstrated to affect mucin production, we sought to determine if the sPLA2 induction in HCjE cells was associated with RA induction of MUC16. Methods. HCjE cells were cultured with or without RA for 3, 6, 24 and 48 hours. Complementary RNA prepared from RNA of the HCjE cells was hybridized to human gene chips (HG-U133A; Affymetrix) and analyzed using Rosetta Resolver software. Microarray data on mucin expression were validated by real-time PCR. To investigate whether sPLA2 is associated with RA-induced MUC16 upregulation, HCjE cells were incubated with RA and the broad spectrum PLA2 inhibitor, aristolochic acid (ArA) or the specific sPLA2-IIA inhibitor LY315920, followed by analysis of MUC16 mRNA and protein by real-time PCR and Western blot analysis. Results. After RA addition, 28 transcripts were upregulated and 6 downregulated by over 2.0-fold (p < 0.01) at both 3 and 6 hours (early phase). Eighty gene transcripts were upregulated and 45 downregulated at both 24 and 48 hours (late phase). Group IIA sPLA2, significantly upregulated by 24 hours, and MUC16 were the most upregulated RNAs by RA at 48 hours. sPLA2 upregulation by RA was confirmed by Western blot analysis. When HCjE cells were incubated with RA plus ArA or specific inhibitor of sPLA2-IIA, LY315920, the RA-induced MUC16 mRNA was significantly reduced (p < 0.01). Conclusion. The retinoic acid-associated upregulation of membrane-associated mucin MUC16 at late phase appears to be through sPLA2-IIA. Upregulation of this hydrophilic membrane-associated mucin may be one of the important mechanisms by which vitamin A facilitates maintenance of the wet-surfaced phenotype on the ocular surface. Experiment Overall Design: Time course of retinoic acid treatment of human conjunctival epithelial cells: 0 (control), 3, 6, 12, 24, and 48 hours. 2 samples per time point.

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Resistance to TFT by decreased TK or overexpression of sPLA2

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