Investig Clin Urol.  2016 Jan;57(1):63-72. 10.4111/icu.2016.57.1.63.

Upregulated expression of BCL2, MCM7, and CCNE1 indicate cisplatin-resistance in the set of two human bladder cancer cell lines: T24 cisplatin sensitive and T24R2 cisplatin resistant bladder cancer cell lines

• Kim SH
• Ho JN
• Jin H
• Lee SC
• Lee SE
• Hong SK
• Lee JW
• Lee ES
• Byun SS

• KMID: 2363126
• DOI: http://doi.org/10.4111/icu.2016.57.1.63

Abstract

PURPOSE
The mechanism of resistance to cisplatin during treatment of bladder cancer (BC) has been a subject of intense investigation in clinical research. This study aims to identify candidate genes associated with resistance to cisplatin, in order to understand the resistance mechanism of BC cells to the drug, by combining the use of microarray profiling, quantitative reverse transcription-polymerase chain reaction (RT-PCR), and Western blot analyses.
MATERIALS AND METHODS
The cisplatin sensitive human BC cell line (T24) and the cisplatin resistant BC cell line, T24R2, were used for microarray analysis to determine the differential expression of genes that are significant in cisplatin resistance. Candidate upregulated genes belonging to three well-known cancer-related KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways (p53 tumor suppressor, apoptosis, and cell cycle) were selected from the microarray data. These candidate genes, differentially expressed in T24 and T24R2, were then confirmed by quantitative RT-PCR and western blot. A fold change > or =2 with a p-value <0.05 was considered significant.
RESULTS
A total of 18 significantly upregulated genes were detected in the three selected cancer-related pathways in both microarray and RT-PCR analyses. These genes were PRKAR2A, PRKAR2B, CYCS, BCL2, BIRC3, DFFB, CASP6, CDK6, CCNE1, STEAP3, MCM7, ORC2, ORC5, ANAPC1, and ANAPC7, CDC7, CDC27, and SKP1 . Western blot analyses also confirmed the upregulation of BCL2, MCM7, and CCNE1 at the protein level, indicating their crucial association with cisplatin resistance.
CONCLUSIONS
The BCL2, MCM7, and CCNE1 genes might play distinctive roles in cisplatin resistance in BC.

Keyword

Cell line; Cisplatin; Drug resistance; Microarray analysis; Urinary bladder neoplasms

MeSH Terms

Antineoplastic Agents/*pharmacology
Cell Line, Tumor
Cisplatin/*pharmacology
Cyclin E/*biosynthesis/genetics
Drug Resistance, Neoplasm/genetics
Gene Expression Regulation, Neoplastic
Genes, Neoplasm
Humans
Minichromosome Maintenance Complex Component 7/*biosynthesis/genetics
Oncogene Proteins/*biosynthesis/genetics
Protein Array Analysis/methods
Proto-Oncogene Proteins c-bcl-2/*biosynthesis/genetics
Reverse Transcriptase Polymerase Chain Reaction/methods
Up-Regulation
Urinary Bladder Neoplasms/*drug therapy/genetics/metabolism
Antineoplastic Agents
Cisplatin
Cyclin E
Minichromosome Maintenance Complex Component 7
Oncogene Proteins
Proto-Oncogene Proteins c-bcl-2

Figure

• 

  Fig. 1 List of primers used in quantitative real-time polymerase chain reaction studies of the 18 upregulated genes and two housekeeping controls.

• 

  Fig. 2 Differential gene expression in T24 and T24R2 cell lines. (A) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway classification of differentially regulated genes. (B) Logarithmic scatter plot of microarray results. ECM, extracellular membrane.

• 

  Fig. 3 Gene expression analysis by quantitative real-time polymerase chain reaction.

• 

  Fig. 4 Results of the Western blot analyses of BCL2, CCNE1, and MCM7.

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