title: Comprehensive screening of genes resistant to an anticancer drug in esophageal squamous cell carcinoma.
abstract: Drug resistance to chemotherapy is a major issue in esophageal cancer management. Drug resistance may be mediated by genetic changes in the tumor; therefore, the identification of gene mutations may lead to better therapeutic outcomes. We used a novel method involving transposons to screen and identify drug-resistant genes. Transposons are DNA sequences that move from one location on the gene to another. A modified piggyBac transposon was designed as an insertion mutagen, and a cytomegalovirus (CMV) promoter sequence was added to induce strong transcription. When the transposon is inserted to the upstream of a certain gene, the gene will be overexpressed while when intserted down or intragenically, it will be downregulated. After establishing a transposon-tagged cell library, we treated cell lines derived from esophageal squamous cell carcinomas (ESCC) [Tohoku esophagus (TE)] with cisplatin (CDDP). We performed splinkerette PCR and TOPO cloning on the resistant colonies. Bacterial colonies were sequenced, and next-generation sequencing was used to identify the overexpressed/downregulated sequences as candidate genes for CDDP resistance. We established 4 cell lines of transposon-tagged cells, TE4, 5, 9 and 15. We treated the two relatively viable cell lines, TE4 and TE15, with CDDP. We identified 37 candidate genes from 8 resistant colonies. Eight genes were overexpressed whilst 29 were downregulated. Among these genes was Janus kinase 2 (JAK2) that is implicated in the progression of myeloproliferative neoplasms. We identified 37 candidate genes responsible for CDDP resistance in the two cell lines derived from ESCC cells. The method is inexpensive, relatively simple, and capable of introducing activating and de-activating mutations in the genome, allowing for drug-resistant genes to be identified.
authors.affiliation: Department of Surgery, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160-8582, Japan.
authors.forename: Mai
authors.initials: M
authors.lastname: Tsutsui
authors.affiliation: Department of Surgery, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160-8582, Japan.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Kawakubo
authors.affiliation: Department of Surgery, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160-8582, Japan.
authors.forename: Testsu
authors.initials: T
authors.lastname: Hayashida
authors.affiliation: Department of Surgery, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160-8582, Japan.
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Fukuda
authors.affiliation: Department of Surgery, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160-8582, Japan.
authors.forename: Rieko
authors.initials: R
authors.lastname: Nakamura
authors.affiliation: Department of Surgery, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160-8582, Japan.
authors.forename: Tsunehiro
authors.initials: T
authors.lastname: Takahashi
authors.affiliation: Department of Surgery, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160-8582, Japan.
authors.forename: Norihito
authors.initials: N
authors.lastname: Wada
authors.affiliation: Department of Surgery, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160-8582, Japan.
authors.forename: Yoshiro
authors.initials: Y
authors.lastname: Saikawa
authors.affiliation: Department of Surgery, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160-8582, Japan.
authors.forename: Tai
authors.initials: T
authors.lastname: Omori
authors.affiliation: Department of Surgery, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160-8582, Japan.
authors.forename: Hiroya
authors.initials: H
authors.lastname: Takeuchi
authors.affiliation: Department of Surgery, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160-8582, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Kitagawa
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D002294:Carcinoma, Squamous Cell / Q000235:genetics*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology*; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis / Q000379:methods*; D004251:DNA Transposable Elements; D019008:Drug Resistance, Neoplasm; D004938:Esophageal Neoplasms / Q000235:genetics*; D000077277:Esophageal Squamous Cell Carcinoma; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans
chemical_list: D000970:Antineoplastic Agents; D004251:DNA Transposable Elements; D002945:Cisplatin