Figure 1.
19q12 amplification in tumors and ovarian tumor cell lines.
(A) Peak regions of amplification between 30–46 Mb on chromosome 19 in 1non-small cell [8], [13], [40], [41], [42]; 2ovarian [3], 3[13]; 4endometrial [9]; 5breast [13], [43], [44]; and 6esophageal tumors [42]. Frequency of occurrence and genes present within peak boundaries indicated. (B) Affymetrix SNP 6.0 mapping microarray copy number of chromosome 19 in ovarian tumor cell lines and (C) between 34–36 Mb for OVCAR-3 and SK-OV-3 cell lines. Copy number shown is the average moving window of 20 markers mapped to Human March 2006 (hg18) genome assembly (source: Sanger Cancer Genome Project Archive). (D) Gene expression determined by qPCR in OVCAR-3 cells relative to SK-OV-3.
Figure 2.
siRNA mediated knock-down of 19q12 genes in ovarian tumor cell lines.
(A) Experimental schematic for combined siRNA transfection and drug treatment. (B) qPCR heatmap showing log2 gene expression ratio to untransfected OVCAR-3 (top) and SK-OV-3 (bottom) cells for each siRNA (columns) at gene targets (rows) with or without cisplatin treatment 72 hours after transfection. (C) Cell viability normalized to no siRNA control cells after transfection with each siRNA. Statistical significance (t-test) calculated by comparison to non-silencing (NS) siRNA in the same cell line using data from three independent MTS assays performed with triplicate wells per condition. Average normalized absorbance at 490 nm and SEM plotted (n = 3), **p-value <0.01. (D) Proportion of apoptotic cells assessed by TUNEL staining in no siRNA control cells or after transfection with NS or CCNE1 targeted siRNA. Data shown from three independent experiments with duplicate wells analyzed per condition. Average percentage of apoptotic cells and SEM plotted (n = 3). ***p-value <0.0001 (Chi squared) calculated by comparison of total cell counts between OVCAR-3 cells treated with a CCNE1 siRNA and all other conditions. (E) CCNE1 protein expression by western blot to confirm siRNA-mediated CCNE1 knockdown at experimental endpoint. (F) Cell viability in additional cell lines after transfection with CCNE1 or non-silencing siRNAs normalized to each cell line with no siRNA added. Statistical significance (t-test) calculated by comparison to NS siRNA in the same cell line. Average normalized absorbance from MTS assay and SEM plotted (n = 3); *p-value <0.05, **p-value <0.01.
Figure 3.
Combined siRNA knock-down and cisplatin treatment in ovarian tumor cell lines.
(A) Cell viability after transfection with individual siRNAs and cisplatin treatment normalized to cisplatin-treated control cells without siRNA. Cisplatin dose of 3 µM or 6 µM was used for OVCAR-3 and SK-OV-3 cells respectively. Average normalized absorbance (490 nm) from three independent MTS assays (triplicate wells per condition) and SEM plotted (n = 3). (B) Cisplatin dose response after transfection with CCNE1 or non-silencing siRNA in OVCAR-3 and SK-OV-3 cell lines. Arrow indicates drug treatment dose used in initial screen; p-value indicates significance of difference between fitted curves. Average normalized MTS assay absorbance to cells without cisplatin treatment, SEM and four-parameter fitted Hill slope plotted (n = 3 for each drug concentration). (D) Cell viability after transfection with CCNE1 or NS siRNAs and cisplatin treatment normalized to cisplatin-treated no siRNA control cells for each cell line. Statistical significance calculated by comparison to NS siRNA, cisplatin-treated cells in the same cell line. Average normalized absorbance from MTS assay and SEM plotted (n = 3). See Table S4 for cisplatin treatment doses.
Figure 4.
Cell cycle distribution after CCNE1 knockdown and cisplatin treatment.
(A) OVCAR-3 cells and (B) SK-OV-3 cell cycle profile (left) and proportion of cells in G1, S or G2 phase (right) for PI stained cells analyzed by flow cytometry 72 hours after transfection with CCNE1 or NS siRNA with or without cisplatin treatment (3 µM or 6 µM for OVCAR-3 and SK-OV-3 cells respectively).
Figure 5.
Clonogenic survival after CCNE1 knockdown and cisplatin treatment.
(A) Experimental time-course for clonogenic survival assay after siRNA transfection and cisplatin treatment. Representative crystal violet stained (B) SK-OV-3 and (C) OVCAR-3 colonies (top) and average proportion of discrete colonies formed (bottom) compared to control cells without siRNA or 1 hour cisplatin treatment (3 µM or 6 µM for OVCAR-3 and SK-OV-3 cells respectively). Error bars indicate SEM (n = 3), **p-value <0.001.
Figure 6.
CCNE1 copy number and gene expression associated with patient outcome.
(A) Patient age distribution stratified by CCNE1 amplification status. Kruskal-Wallis p-value reported, bars indicate mean and SEM. (B) Correlation between CCNE1 copy number by qPCR and gene expression signal by microarray. (C) Kaplan-meier analysis of CCNE1 unamplified (n = 68), gained (n = 21) and amplified (n = 6) ovarian cancer patients and (D) CCNE1 low (n = 31), medium (n = 28) and high (n = 36) expressing samples for progression-free survival and (E & F) overall survival. Log-rank test p-values reported. Stratification by CCNE1 copy number or expression status described in Materials and Methods.
Table 1.
Patient characteristics by CCNE1 copy number status.
Figure 7.
Regions of copy number change and TPX2 gene expression associated with CCNE1 amplification.
(A) Circos plot showing regions of copy number change significantly correlated to CCNE1 amplification (p-value <1×10−5) (B) Significance of copy number correlation to CCNE1 amplification across chromosome 20. Significance threshold used to define correlation peak boundaries indicated by dotted line. (C) TPX2 expression correlation with locus copy number and CCNE1 expression (source: TCGA). (D) Correlation of CCNE1 and TPX2 gene expression in an independent data set (Tothill et al., 2009).
Table 2.
19q12 co-amplified regions of copy number change.
Figure 8.
Functional analysis of TPX2 and CCNE1 co-amplification in ovarian tumor cell lines.
(A) Affymetrix SNP 6.0 mapping microarray copy number of chromosome 20 in ovarian tumor cell lines indicating location of TPX2. (B) Correlation between TPX2 copy number and gene expression by qPCR in cell lines. (C) Cell viability depicted as normalized MTS absorbance (490 nm) from three independent assays to cells treated with no siRNA in siRNA double knockdown experiments. Average normalized absorbance from MTS assay and SEM plotted (n = 3). (E) TPX2 protein expression by western blot to confirm siRNA-mediated knockdown at experimental endpoint for cell lines and CCNE1 in OAW-28 cells.