skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Molecular Dissection of Induced Platinum Resistance through Functional and Gene Expression Analysis in a Cell Culture Model of Bladder Cancer

Abstract

Here, we report 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 bymore » 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.« less

Authors:
 [1];  [1];  [1];  [2];  [3];  [1];  [1];  [1];  [4];  [3];  [3];  [3];  [3];  [5];  [5];  [1];  [6];  [2];  [7]
  1. Univ. of California, Davis, CA (United States)
  2. Univ. of California, Davis, CA (United States); Accelerated Medical Diagnostics Inc., Dublin, CA (United States)
  3. Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
  4. Accelerated Medical Diagnostics Inc., Dublin, CA (United States)
  5. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
  6. Univ. of California, Davis, CA (United States); VA Northern California Health Care System, Mather, CA (United States)
  7. Wayne State Univ., Detroit, MI (United States)
Publication Date:
Research Org.:
Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1259484
Alternate Identifier(s):
OSTI ID: 1438769
Report Number(s):
LLNL-JRNL-737014
Journal ID: ISSN 1932-6203
Grant/Contract Number:  
AC52-07NA27344
Resource Type:
Accepted Manuscript
Journal Name:
PLoS ONE
Additional Journal Information:
Journal Volume: 11; Journal Issue: 1; Journal ID: ISSN 1932-6203
Publisher:
Public Library of Science
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Wang, Sisi, Zhang, Hongyong, Scharadin, Tiffany M., Zimmermann, Maike, Hu, Bin, Pan, Amy Wang, Vinall, Ruth, Lin, Tzu-yin, Cimino, George, Chain, Patrick, Vuyisich, Momchilo, Gleasner, Cheryl, Mcmurry, Kim, Malfatti, Michael, Turteltaub, Kenneth, de Vere White, Ralph, Pan, Chong-xian, Henderson, Paul T., and Ahmad, Aamir. Molecular Dissection of Induced Platinum Resistance through Functional and Gene Expression Analysis in a Cell Culture Model of Bladder Cancer. United States: N. p., 2016. Web. doi:10.1371/journal.pone.0146256.
Wang, Sisi, Zhang, Hongyong, Scharadin, Tiffany M., Zimmermann, Maike, Hu, Bin, Pan, Amy Wang, Vinall, Ruth, Lin, Tzu-yin, Cimino, George, Chain, Patrick, Vuyisich, Momchilo, Gleasner, Cheryl, Mcmurry, Kim, Malfatti, Michael, Turteltaub, Kenneth, de Vere White, Ralph, Pan, Chong-xian, Henderson, Paul T., & Ahmad, Aamir. Molecular Dissection of Induced Platinum Resistance through Functional and Gene Expression Analysis in a Cell Culture Model of Bladder Cancer. United States. doi:10.1371/journal.pone.0146256.
Wang, Sisi, Zhang, Hongyong, Scharadin, Tiffany M., Zimmermann, Maike, Hu, Bin, Pan, Amy Wang, Vinall, Ruth, Lin, Tzu-yin, Cimino, George, Chain, Patrick, Vuyisich, Momchilo, Gleasner, Cheryl, Mcmurry, Kim, Malfatti, Michael, Turteltaub, Kenneth, de Vere White, Ralph, Pan, Chong-xian, Henderson, Paul T., and Ahmad, Aamir. Fri . "Molecular Dissection of Induced Platinum Resistance through Functional and Gene Expression Analysis in a Cell Culture Model of Bladder Cancer". United States. doi:10.1371/journal.pone.0146256. https://www.osti.gov/servlets/purl/1259484.
@article{osti_1259484,
title = {Molecular Dissection of Induced Platinum Resistance through Functional and Gene Expression Analysis in a Cell Culture Model of Bladder Cancer},
author = {Wang, Sisi and Zhang, Hongyong and Scharadin, Tiffany M. and Zimmermann, Maike and Hu, Bin and Pan, Amy Wang and Vinall, Ruth and Lin, Tzu-yin and Cimino, George and Chain, Patrick and Vuyisich, Momchilo and Gleasner, Cheryl and Mcmurry, Kim and Malfatti, Michael and Turteltaub, Kenneth and de Vere White, Ralph and Pan, Chong-xian and Henderson, Paul T. and Ahmad, Aamir},
abstractNote = {Here, we report 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.},
doi = {10.1371/journal.pone.0146256},
journal = {PLoS ONE},
number = 1,
volume = 11,
place = {United States},
year = {2016},
month = {1}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 5 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

HtrA1 Downregulation Induces Cisplatin Resistance in Lung Adenocarcinoma by Promoting Cancer Stem Cell-Like Properties: HtrA1 DOWNREGULATION INDUCES CISPLATIN RESISTANCE
journal, April 2014

  • Xu, Yongqing; Jiang, Zhiming; Zhang, Zhonghui
  • Journal of Cellular Biochemistry, Vol. 115, Issue 6
  • DOI: 10.1002/jcb.24751

Modulation of the Cellular Pharmacology of Cisplatin and Its Analogs by the Copper Exporters ATP7A and ATP7B
journal, June 2004

  • Samimi, Goli; Katano, Kuniyuki; Holzer, Alison K.
  • Molecular Pharmacology, Vol. 66, Issue 1
  • DOI: 10.1124/mol.66.1.25

Enhanced DNA repair and resistance to cisplatin in human ovarian cancer
journal, December 1988


The platelet-sparing effect of paclitaxel is not related to changes in the pharmacokinetics of carboplatin
journal, November 2000

  • Fujiwara, Keiichi; Yamauchi, Hideaki; Suzuki, Sachiko
  • Cancer Chemotherapy and Pharmacology, Vol. 47, Issue 1
  • DOI: 10.1007/s002800000212

Superior cytotoxicity and DNA cross-link induction by oxaliplatin versus cisplatin at lower cellular uptake in colorectal cancer cell lines
journal, January 2012


Phase II Study of Pemetrexed for Second-Line Treatment of Transitional Cell Cancer of the Urothelium
journal, July 2006

  • Sweeney, Christopher J.; Roth, Bruce J.; Kabbinavar, Fairooz F.
  • Journal of Clinical Oncology, Vol. 24, Issue 21
  • DOI: 10.1200/JCO.2005.03.6699

Overexpression of dihydrodiol dehydrogenase is associated with cisplatin-based chemotherapy resistance in ovarian cancer patients
journal, April 2005


Long noncoding RNAs and the genetics of cancer
journal, May 2013

  • Cheetham, S. W.; Gruhl, F.; Mattick, J. S.
  • British Journal of Cancer, Vol. 108, Issue 12
  • DOI: 10.1038/bjc.2013.233

In vitro formation of DNA adducts by cisplatin, lobaplatin and oxaliplatin in calf thymus DNA in solution and in cultured human cells
journal, January 1996


Protein interactions with platinum–DNA adducts: from structure to function
journal, October 2004


Induction of aldo-keto reductases (AKR1C1 and AKR1C3) abolishes the efficacy of daunorubicin chemotherapy for leukemic U937 cells
journal, January 2014


The angiogenic factor CYR61 in breast cancer: molecular pathology and therapeutic perspectives.
journal, June 2003


Structure, Recognition, and Processing of Cisplatin−DNA Adducts
journal, September 1999

  • Jamieson, Elizabeth R.; Lippard, Stephen J.
  • Chemical Reviews, Vol. 99, Issue 9
  • DOI: 10.1021/cr980421n

Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks
journal, March 2012


Cisplatin resistance by induction of aldo-keto reductase family 1 member C2 in human bladder cancer cells
journal, December 2013

  • Shirato, Akitomi; Kikugawa, Tadahiko; Miura, Noriyoshi
  • Oncology Letters, Vol. 7, Issue 3
  • DOI: 10.3892/ol.2013.1768

ATM and ATR Substrate Analysis Reveals Extensive Protein Networks Responsive to DNA Damage
journal, May 2007


Differential analysis of gene regulation at transcript resolution with RNA-seq
journal, December 2012

  • Trapnell, Cole; Hendrickson, David G.; Sauvageau, Martin
  • Nature Biotechnology, Vol. 31, Issue 1
  • DOI: 10.1038/nbt.2450

Mechanism of cisplatin resistance in human urothelial carcinoma cells
journal, May 2012


Increased nucleotide excision repair in cisplatin-resistant ovarian cancer cells
journal, November 2000


Characterization of a clonal isolate of an oxaliplatin resistant ovarian carcinoma cell line A2780/C10
journal, January 2007


Serine protease HtrA1 modulates chemotherapy-induced cytotoxicity
journal, July 2006

  • Chien, J.
  • Journal of Clinical Investigation, Vol. 116, Issue 7
  • DOI: 10.1172/JCI27698

New platinum antitumor complexes
journal, December 1993


Calcium-dependent FAK/CREB/TNNC1 signalling mediates the effect of stromal MFAP5 on ovarian cancer metastatic potential
journal, October 2014

  • Leung, Cecilia S.; Yeung, Tsz-Lun; Yip, Kay-Pong
  • Nature Communications, Vol. 5, Issue 1
  • DOI: 10.1038/ncomms6092

Assessment of cytotoxicity, apoptosis and DNA damages in Colo320 colorectal cancer cells selected for oxaliplatin resistance: CYTOTOXICITY AND DNA DAMAGE IN COLO320 CELLS
journal, April 2011

  • Virag, Piroska; Brie, Ioana; Fischer-Fodor, Eva
  • Cell Biochemistry and Function, Vol. 29, Issue 5
  • DOI: 10.1002/cbf.1754

CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer
journal, June 2006

  • Weisenberger, Daniel J.; Siegmund, Kimberly D.; Campan, Mihaela
  • Nature Genetics, Vol. 38, Issue 7
  • DOI: 10.1038/ng1834

Cellular processing of platinum anticancer drugs
journal, March 2005

  • Wang, Dong; Lippard, Stephen J.
  • Nature Reviews Drug Discovery, Vol. 4, Issue 4
  • DOI: 10.1038/nrd1691

Nkx1-2 is a transcriptional repressor and is essential for the activation of Brachyury in P19 mouse embryonal carcinoma cell
journal, June 2012

  • Tamashiro, Dana Ann A.; Alarcon, Vernadeth B.; Marikawa, Yusuke
  • Differentiation, Vol. 83, Issue 5
  • DOI: 10.1016/j.diff.2012.02.010

High BAALC expression predicts chemoresistance in adult B-precursor acute lymphoblastic leukemia
journal, May 2010


The CpG Island Methylator Phenotype and Chromosomal Instability Are Inversely Correlated in Sporadic Colorectal Cancer
journal, January 2007


Oxaliplatin induces different cellular and molecular chemoresistance patterns in colorectal cancer cell lines of identical origins
journal, January 2013

  • Virag, Piroska; Fischer-Fodor, Eva; Perde-Schrepler, Maria
  • BMC Genomics, Vol. 14, Issue 1
  • DOI: 10.1186/1471-2164-14-480

Aquaporin 3 (AQP3) participates in the cytotoxic response to nucleoside-derived drugs
journal, September 2012

  • Trigueros-Motos, Laia; Pérez-Torras, Sandra; Casado, F. Javier
  • BMC Cancer, Vol. 12, Issue 1
  • DOI: 10.1186/1471-2407-12-434

Neoadjuvant Chemotherapy plus Cystectomy Compared with Cystectomy Alone for Locally Advanced Bladder Cancer
journal, August 2003

  • Grossman, H. Barton; Natale, Ronald B.; Tangen, Catherine M.
  • New England Journal of Medicine, Vol. 349, Issue 9
  • DOI: 10.1056/NEJMoa022148

A microdosing approach for characterizing formation and repair of carboplatin-DNA monoadducts and chemoresistance
journal, March 2011

  • Henderson, Paul T.; Li, Tao; He, Miaoling
  • International Journal of Cancer, Vol. 129, Issue 6
  • DOI: 10.1002/ijc.25814

The Copper Transporter CTR1 Regulates Cisplatin Uptake in Saccharomyces cerevisiae
journal, November 2002

  • Lin, Xinjian; Okuda, Tsuyoshi; Holzer, Alison
  • Molecular Pharmacology, Vol. 62, Issue 5
  • DOI: 10.1124/mol.62.5.1154

Phase II Trial of Weekly Paclitaxel in Patients With Previously Treated Advanced Urothelial Cancer
journal, February 2002


Enhanced dna repair and tolerance of DNA damage associated with resistance to cis-diammine-dichloroplatinum (II) after in vitro exposure of a human teratoma cell line to fractionated X-irradiation
journal, July 1990

  • Hill, Bridget T.; Shellard, Sharon A.; Hosking, Louise K.
  • International Journal of Radiation Oncology*Biology*Physics, Vol. 19, Issue 1
  • DOI: 10.1016/0360-3016(90)90137-9

MutS Preferentially Recognizes Cisplatin- over Oxaliplatin-modified DNA
journal, November 2001

  • Zdraveski, Zoran Z.; Mello, Jill A.; Farinelli, Christine K.
  • Journal of Biological Chemistry, Vol. 277, Issue 2
  • DOI: 10.1074/jbc.M105382200

Personalized medicine for targeted and platinum-based chemotherapy of lung and bladder cancer
journal, February 2013

  • Cimino, George D.; Pan, Chong-xian; Henderson, Paul T.
  • Bioanalysis, Vol. 5, Issue 3
  • DOI: 10.4155/bio.12.325

HtrA1 sensitizes ovarian cancer cells to cisplatin-induced cytotoxicity by targeting XIAP for degradation
journal, April 2011

  • He, Xiaoping; Khurana, Ashwani; Maguire, Jacie L.
  • International Journal of Cancer, Vol. 130, Issue 5
  • DOI: 10.1002/ijc.26044

Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays
journal, December 1983


Comparative analysis of the mutagenic activity of oxaliplatin and cisplatin in theHprt gene of CHO cells
journal, January 2005

  • Silva, Maria J.; Costa, Paula; Dias, Anabela
  • Environmental and Molecular Mutagenesis, Vol. 46, Issue 2
  • DOI: 10.1002/em.20138

Regulation of Androgen-Responsive Transcription by the Chromatin Remodeling Factor CHD8
journal, June 2010

  • Menon, Tushar; Yates, Joel A.; Bochar, Daniel A.
  • Molecular Endocrinology, Vol. 24, Issue 6
  • DOI: 10.1210/me.2009-0421

Pathophysiological roles of aldo–keto reductases (AKR1C1 and AKR1C3) in development of cisplatin resistance in human colon cancers
journal, February 2013

  • Matsunaga, Toshiyuki; Hojo, Aki; Yamane, Yumi
  • Chemico-Biological Interactions, Vol. 202, Issue 1-3
  • DOI: 10.1016/j.cbi.2012.09.024

Reversal of inflammation-associated dihydrodiol dehydrogenases (AKR1C1 and AKR1C2) overexpression and drug resistance in nonsmall cell lung cancer cells by wogonin and chrysin
journal, January 2007

  • Wang, Hao-Wei; Lin, Chin-Ping; Chiu, Jen-Hwey
  • International Journal of Cancer, Vol. 120, Issue 9
  • DOI: 10.1002/ijc.22402

Cisplatin and Oxaliplatin, but Not Carboplatin and Nedaplatin, Are Substrates for Human Organic Cation Transporters (SLC22A1–3 and Multidrug and Toxin Extrusion Family)
journal, August 2006

  • Yonezawa, Atsushi; Masuda, Satohiro; Yokoo, Sachiko
  • Journal of Pharmacology and Experimental Therapeutics, Vol. 319, Issue 2
  • DOI: 10.1124/jpet.106.110346

Cellular and molecular aspects of drugs of the future: oxaliplatin
journal, November 2002

  • Di Francesco, A. M.; Ruggiero, A.; Riccardi, R.
  • Cellular and Molecular Life Sciences, Vol. 59, Issue 11
  • DOI: 10.1007/PL00012514

Pharmacokinetics of Oxaliplatin in Humans
journal, January 2002

  • Ehrsson, H.; Wallin, I.; Yachnin, J.
  • Medical Oncology, Vol. 19, Issue 4
  • DOI: 10.1385/MO:19:4:261

Blood clearance of radioactively labelled cis-diammine 1,1-cyclobutane dicarboxylate platinum(II) (CBDCA) in cancer patients
journal, August 1983

  • Sharma, H.; Thatcher, N.; Baer, J.
  • Cancer Chemotherapy and Pharmacology, Vol. 11, Issue 1
  • DOI: 10.1007/BF00257407

Oxaliplatin-Induced Damage of Cellular DNA
journal, November 2000

  • Woynarowski, Jan M.; Faivre, Sandrine; Herzig, Maryanne C. S.
  • Molecular Pharmacology, Vol. 58, Issue 5
  • DOI: 10.1124/mol.58.5.920

    Works referencing / citing this record:

    Radiocarbon Tracers in Toxicology and Medicine: Recent Advances in Technology and Science
    journal, May 2019

    • Malfatti, Michael A.; Buchholz, Bruce A.; Enright, Heather A.
    • Toxics, Vol. 7, Issue 2
    • DOI: 10.3390/toxics7020027

    Radiocarbon Tracers in Toxicology and Medicine: Recent Advances in Technology and Science
    journal, May 2019

    • Malfatti, Michael A.; Buchholz, Bruce A.; Enright, Heather A.
    • Toxics, Vol. 7, Issue 2
    • DOI: 10.3390/toxics7020027