Expression microarray analysis reveals genes associated with in vitro resistance to cisplatin in a cell line model

Figures & data

Table I.  Primers used successfully for RT-qPCR and RT-PCR. Sequences were selected using a design service (Sigma-Aldrich), RTPrimerDB (http://medgen.ugent.be/rtprimerdb/) or PrimerBank (http://pga.mgh.harvard.edu/primerbank/).

Gene	Sense Primer	Antisense Primer	Amplicon Length (bp)
ABCB1	5′-AGGAAGACATGACCAGGTATGC-3′	5′-CCAACATCGTGCACATCAAAC-3′	184
ABCC1	5′-CGGAAACCATCCACGACCCTAA-3′	5′-TCATGAGGAAGTAGGGCCCAAA-3′	387
ABCG2	5′-GCACTCTGACGGTGAGAGAAAA-3′	5′-CCAGACACACCACGGATAAACT-3′	169
MVP	5′-GGCAGGACAATGAGAGGGTACT-3′	5′-CCGAACTTGCCCTGTGACAT-3′	137
ACTG2	5′-CTGGAGAAGAGCTATGAGCTG-3′	5′-ATCTCCTTCTGCATCCTGTC-3′	245
ARHD	5′-CCACGGTGTTTGAGCGGTA-3′	5′-CAGGCGGTCATAGTCATCTTG-3′	101
CTSL	5′-TGGCCTAATGGATTATGCTTTCC-3′	5′-ATGACCTGCATCAATAGCAACA-3′	220
EHF	5′-CAGCCTGGACAGACAGCTAC-3′	5′-CACTCCCACACCTGGTACTT-3′	109
GAPDH*	5′-CATCTTCCAGGAGCGAGATCC-3′	5′-GTGGTGAAGACGCCAGTGG-3′	87
GSTM3	5′-GCGATACTGGCATTTCCCTA-3′	5′-TCTCCAACTGTGCAATCTCG-3′	104
GSTM4	5′-TTGGAGAACCAGGCTATGGAC-3′	5′-TTCCCCAGGAACTGTGAGAAGT-3′	134

*Reference gene

Figure 1.  Relative resistance of MCF-7CR and MCF-7 cells to cisplatin (CDDP) over the concentration range 0–117 µM (final concentration in media). Each point represents the means of at least 3 individual experiments and error bars* represent the standard error of the mean for each point. The IC₅₀ values of MCF-7 and MCF-7CR were 48 µM and 79 µM respectively, making MCF-7CR approximately 1.6-fold more resistant to cisplatin than the MCF-7 parent line. The MCF-7CR cells were significantly more resistant to cisplatin than MCF-7 cells over the dose range 50–117 µM (p < 0.05 by ANOVA). *Error bars are less than the size of the data point symbols.

Figure 2.  RT-PCR analysis of the expression of four membrane bound drug transporters commonly associated with chemotherapy resistance in breast cancer in the parent and derivative cell lines used in this study. L Ladder; M MCF-7; MC MCF-7CR.

Table II.  Expression values for each of the genes identified as significantly associated with the observed in vitro resistance of MCF-7CR to cisplatin using expression MA analysis. Gene ontology information was derived from the GOA database (http://www.ebi.ac.uk/GOA/). *Target selected for RT-qPCR confirmation.

			Fold Expression Level (MCF-7CR relative to MCF-7)
Gene	Description	Known/Proposed Function (& Direction of Regulation)	Increase	Decrease
PRL*	Prolactin	Cell proliferation (+)	5.27
EHF*	ETS Homologous Factor	Transcription (−)	4.84
THBS1	Thrombospondin 1	Cell adhesion (+)	3.82
SLC7A1	Solute Carrier Family 7 Member 1	AA metabolism (+)	3.47
CHST1	Carbohydrate Sulfotransferase 1	Galactose metabolism (+)	3.22
EDN1	Endothelin 1	Cell proliferation (+)	3.13
TRIP11	Thyroid Hormone Receptor Interactor 11	Protein binding (+)	2.81
DUSP4	Dual Specificity Phosphatase 4	Signal transduction (−)	2.77
ACTG2*	Actin Gamma 2	Nucleotide binding (+)		15.77
CTSL*	Cathepsin L	Protein metabolism (+)		4.95
GSTM3*	Glutathione S Transferase M3	Metabolism (+)		4.84
ARMET	Arginine-Rich, Mutated In Early Stage Tumors	Unknown		4.44
PSMD8	26S Proteasome Non-ATPase Regulatory Subunit 8	Protein Metabolism (+)		4.42
HRMT1L2	Protein Arginine Methyltransferase 1	Signal transduction (+)		3.80
		Transferase activity (+)
CCNB1	Cyclin B1	Cell cycle regulation (+)		3.80
		Apoptosis (+)
PSMB2	Proteasome Subunit, Beta Type, 2	Protein metabolism (+)		3.38
MRPL3	Mitochondrial Ribosomal Protein L3	Protein metabolism (+)		3.30
MDK	Midkine	Cell proliferation (+)		3.27
ITGB5	Integrin Beta 5	Cell adhesion (+)		3.23
MYBL2	V-Myb Myeloblastosis Viral Oncogene Homolog (Avian)-Like 2	Cell cycle regulation (+)		3.16
		Apoptosis (−)
GSTM4*	Glutathione S Transferase M4	Metabolism (+)		3.13
LGALS3	Galectin 3	Galactose metabolism (+)		3.01
ARHD*	Rho-Related Protein D	Signal transduction (+)		2.94
MAD2L1	MAD2 Mitotic Arrest Deficient-Like 1	Cell cycle regulation (+/ − )		2.94
SCO2	SCO Cytochrome Oxidase Deficient Homolog 2 (Yeast)	Metal ion binding		2.75
ATP5J2	ATP Synthase, H+ Transporting, Mitochondrial F0 Complex, Subunit F2	Metal ion binding		2.49
GCN5L2	GCN5 General Control Of Amino-Acid Synthesis 5-Like 2 (Yeast)	Regulation of transcription (+)		2.46
PMS2L5	Postmeiotic Segregation Increased 2-Like 5	DNA mismatch repair (+)		2.34

Figure 3.  Fold change in expression values between MCF-7 and MCF-7CR cells for 6 genes derived using expression MA and RT-qPCR analysis.