title: Molecular Dissection of Induced Platinum Resistance through Functional and Gene Expression Analysis in a Cell Culture Model of Bladder Cancer.
abstract: We report herein the development, functional and molecular characterization of an isogenic, paired bladder cancer cell culture model system for studying platinum drug resistance. The 5637 human bladder cancer cell line was cultured over ten months with stepwise increases in oxaliplatin concentration to generate a drug resistant 5637R sub cell line. The MTT assay was used to measure the cytotoxicity of several bladder cancer drugs. Liquid scintillation counting allowed quantification of cellular drug uptake and efflux of radiolabeled oxaliplatin and carboplatin. The impact of intracellular drug inactivation was assessed by chemical modulation of glutathione levels. Oxaliplatin- and carboplatin-DNA adduct formation and repair was measured using accelerator mass spectrometry. Resistance factors including apoptosis, growth factor signaling and others were assessed with RNAseq of both cell lines and included confirmation of selected transcripts by RT-PCR. Oxaliplatin, carboplatin, cisplatin and gemcitabine were significantly less cytotoxic to 5637R cells compared to the 5637 cells. In contrast, doxorubicin, methotrexate and vinblastine had no cell line dependent difference in cytotoxicity. Upon exposure to therapeutically relevant doses of oxaliplatin, 5637R cells had lower drug-DNA adduct levels than 5637 cells. This difference was partially accounted for by pre-DNA damage mechanisms such as drug uptake and intracellular inactivation by glutathione, as well as faster oxaliplatin-DNA adduct repair. In contrast, both cell lines had no significant differences in carboplatin cell uptake, efflux and drug-DNA adduct formation and repair, suggesting distinct resistance mechanisms for these two closely related drugs. The functional studies were augmented by RNAseq analysis, which demonstrated a significant change in expression of 83 transcripts, including 50 known genes and 22 novel transcripts. Most of the transcripts were not previously associated with bladder cancer chemoresistance. This model system and the associated phenotypic and genotypic data has the potential to identify some novel details of resistance mechanisms of clinical importance to bladder cancer.
authors.affiliation: Department of Internal Medicine, Division of Hematology and Oncology, University of California Davis, Sacramento, California, United States of America.
authors.forename: Sisi
authors.initials: S
authors.lastname: Wang
authors.affiliation: Department of Internal Medicine, Division of Hematology and Oncology, University of California Davis, Sacramento, California, United States of America.
authors.forename: Hongyong
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Internal Medicine, Division of Hematology and Oncology, University of California Davis, Sacramento, California, United States of America.
authors.forename: Tiffany M
authors.initials: TM
authors.lastname: Scharadin
authors.affiliation: Department of Internal Medicine, Division of Hematology and Oncology, University of California Davis, Sacramento, California, United States of America.|Accelerated Medical Diagnostics Incorporated, Dublin, California, United States of America.
authors.forename: Maike
authors.initials: M
authors.lastname: Zimmermann
authors.affiliation: Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America.
authors.forename: Bin
authors.initials: B
authors.lastname: Hu
authors.affiliation: Department of Internal Medicine, Division of Hematology and Oncology, University of California Davis, Sacramento, California, United States of America.
authors.forename: Amy Wang
authors.initials: AW
authors.lastname: Pan
authors.affiliation: Department of Urology, University of California Davis, Sacramento, California, United States of America.
authors.forename: Ruth
authors.initials: R
authors.lastname: Vinall
authors.affiliation: Department of Internal Medicine, Division of Hematology and Oncology, University of California Davis, Sacramento, California, United States of America.
authors.forename: Tzu-yin
authors.initials: TY
authors.lastname: Lin
authors.affiliation: Accelerated Medical Diagnostics Incorporated, Dublin, California, United States of America.
authors.forename: George
authors.initials: G
authors.lastname: Cimino
authors.affiliation: Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America.
authors.forename: Patrick
authors.initials: P
authors.lastname: Chain
authors.affiliation: Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America.
authors.forename: Momchilo
authors.initials: M
authors.lastname: Vuyisich
authors.affiliation: Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America.
authors.forename: Cheryl
authors.initials: C
authors.lastname: Gleasner
authors.affiliation: Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America.
authors.forename: Kim
authors.initials: K
authors.lastname: Mcmurry
authors.affiliation: Lawrence Livermore National Laboratory, Livermore, California, United States of America.
authors.forename: Michael
authors.initials: M
authors.lastname: Malfatti
authors.affiliation: Lawrence Livermore National Laboratory, Livermore, California, United States of America.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Turteltaub
authors.affiliation: Department of Urology, University of California Davis, Sacramento, California, United States of America.
authors.forename: Ralph
authors.initials: R
authors.lastname: de Vere White
authors.affiliation: Department of Internal Medicine, Division of Hematology and Oncology, University of California Davis, Sacramento, California, United States of America.|Department of Urology, University of California Davis, Sacramento, California, United States of America.|VA Northern California Health Care System, Mather, California, United States of America.
authors.forename: Chong-xian
authors.initials: CX
authors.lastname: Pan
authors.affiliation: Department of Internal Medicine, Division of Hematology and Oncology, University of California Davis, Sacramento, California, United States of America.|Accelerated Medical Diagnostics Incorporated, Dublin, California, United States of America.
authors.forename: Paul T
authors.initials: PT
authors.lastname: Henderson
mesh_terms: D000970:Antineoplastic Agents / Q000493:pharmacokinetics* / Q000494:pharmacology*; D017209:Apoptosis / Q000235:genetics; D001692:Biological Transport / Q000235:genetics; D016190:Carboplatin / Q000378:metabolism; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000493:pharmacokinetics / Q000494:pharmacology; D018736:DNA Adducts / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects; D004260:DNA Repair / Q000235:genetics; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000494:pharmacology; D004317:Doxorubicin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D020869:Gene Expression Profiling; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008727:Methotrexate / Q000494:pharmacology; D009944:Organoplatinum Compounds / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D000077150:Oxaliplatin; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy* / Q000235:genetics; D014747:Vinblastine / Q000494:pharmacology; D000093542:Gemcitabine
chemical_list: D000970:Antineoplastic Agents; D018736:DNA Adducts; D009944:Organoplatinum Compounds; C096276:carboplatin-DNA adduct; D000077150:Oxaliplatin; D003841:Deoxycytidine; D014747:Vinblastine; D004317:Doxorubicin; D016190:Carboplatin; D005978:Glutathione; D002945:Cisplatin; D008727:Methotrexate; D000093542:Gemcitabine