The BRCA/Fanconi anemia (FA) pathway plays a key role in the
The BRCA/Fanconi anemia (FA) pathway plays a key role in the repair of DNA double strand breaks. BRCA/FA components. The BRCA/FA pathway was upregulated by GEM and cisplatin (CDDP) exposure. Inhibition using siRNA and RAD51 inhibitor sensitized GR cells to GEM or CDDP. The CD24+/44+ population was increased in GR and parent BTC cells treated with GEM or CDDP and highly expressed BRCA/FA genes. FANCD2 was related to CD24 expression in resected BTC specimens. Inhibition of the BRCA/FA pathway under GEM reduced the CD24+/44+ population in MzChA1-GR cells. Thus, high expression of the BRCA/FA pathway is one mechanism of chemoresistance against GEM and/or CDDP and is related to the CD24+/44+ population in BTC. and in various cancers, including lung, colon, breast, ovary and stomach cancer.10C16 GEM is a nucleoside analogue that inhibits DNA elongation and ribonucleotide reductase.17 In addition, GEM may contribute to DNA damage18 and cells may be SB 431542 manufacture sensitized by the inhibition of checkpoint kinase?1 (CHK1), which coordinates the DDR,19 suggesting that other DDR proteins are involved in chemoresistance. The DDR generally protects against genomic instability, which enables cancer development.20,21 Among the DDR-related genes, the BRCA/Fanconi anemia (FA) pathway genes play a role in homologous recombination repair (HRR), particularly the repair of fatal DNA double strand breaks,22 and are related to the development of several cancers. BRCA/FA pathway genes are well known tumor suppressors,21C23 but the percentage of mutations is limited.23 However, the downregulation of BRCA2 may cause radio-sensitization20 and chemosensitization.24,25 We hypothesized that upregulation of BRCA/FA pathway components caused chemoresistance in BTC. Our objective was to investigate the SC35 role of the BRCA/FA pathway in BTC, focusing on several key molecules in the BRCA/FA pathway. In addition to is a central gene in this pathway and associated with cell cycle control at the S/G2 checkpoint. RAD51c is a SB 431542 manufacture final factor in this pathway and directly repairs DNA damage in combination with other components. 22 We also investigated the relationship between the DDR and CD24+/44+ population, which was reported as a candidate marker for extracting cancer stem cells (CSC) in BTC.26 Enrichment of CSC is a well-known mechanism of chemoresistance.27 The DDR works in stem cells28 and may contribute to the CSC-like population in several cancers.29,30 Our results demonstrate that inhibition of the BRCA/FA pathway not only sensitizes BTC cells to GEM or CDDP, but also reduces the CD24+/44+ population in BTC. Materials SB 431542 manufacture and Methods Establishment of gemcitabine-resistant biliary tract cancer cells (MzChA1-GR, CCLP1-GR and KMCH1-GR) Human BTC cell lines (MzChA1, CCLP1 and KMCH1) were kindly provided by Dr Gregory J. Gores of the Mayo Clinic, Rochester, MN, USA.31C34 The GEM-resistant MzChA1 cell line (MzChA1-GR) was recently established in our department.35 The primary MzChA1-GR cell line was developed through exposure to increasing concentrations of GEM (0.2C2.0?ng/mL) with repeated subculturing until the cells became fully resistant. Primary MzChA1-GR cells were cultured in GEM-free medium for 3?weeks prior to the next limiting dilution. After the primary MzChA1-GR cells were confirmed to be significantly more resistant to GEM than the parent cells, a single MzChA1-GR cell was seeded in a SB 431542 manufacture 96-well microplate by limiting dilution. Eight MzChA1-GR clones were established from the primary MzChA1-GR cell. To reduce the risk of contamination, we cultured each cell line separately with 6 months interval and each GR cell was also established by different two scientists. The MzChA1-GR cells were cultured under the same conditions as other cell lines, without GEM.35 The concordances of short tandem repeat were 21% in MzChA1 and MzChA1-GR, 81% in CCLP1 and CCLP1-GR, and 90% in KMCH1 and KMCH1-GR (BEX, Tokyo, Japan) like as the previous report included the data of STR changes by DNA damage drug treatment.36 The GEM-resistant CCLP1 cell line (CCLP1-GR) and the GEM-resistant KMCH1 cell line (KMCH1-GR) were developed through exposure to increasing concentrations of GEM (CCLP1, from 5 to?300?ng/mL; KMCH1, from 0.3 to?100?ng/mL) and established using the same method as MzChA1-GR. Microarray analysis DNA microarray analysis was performed using a 3D-Gene Human Oligo chip 25k (Toray SB 431542 manufacture Industries, Tokyo, Japan). We compared the MzChA1-parent and three MzChA1-GR clones. We determined that RRM1 and dCK mRNA levels were generally upregulated in all GR cells. The normalized data were used to identify genes whose expression appeared to be upregulated or downregulated. Immunocytochemistry Immunocytochemistry studies of H2AX were performed using BTC cells. As a positive control, we also assessed BTC cells 4?h after 6 Gray irradiation using a Gamma Cell 40 Exactor (Nordion International, Ottawa, ON, Canada). Briefly, cells were cultured on six-well chamber slides, fixed with 4% paraformaldehyde, and permeabilized. The cells were then incubated with monoclonal mouse anti-H2AX (diluted 1:500 [Millipore, Billerica, MA, USA]), followed by Alexa Fluor anti-mouse IgG conjugated to Alexa Fluor 488 (diluted 1:500 [Cell.