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Quantitative proteomic and interaction network analysis of cisplatin resistance in HeLa cells.

Chavez JD, Hoopmann MR, Weisbrod CR, Takara K, Bruce JE - PLoS ONE (2011)

Bottom Line: Unfortunately mRNA levels do not always correlate with protein expression levels due to post-transcriptional changes in protein abundance.A total of 856 proteins were identified and quantified, with 374 displaying significantly altered expression levels between the cell lines.Several of these proteins have been previously implicated in resistance towards platinum-based and other drugs, while many represent new potential markers or therapeutic targets.

View Article: PubMed Central - PubMed

Affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
Cisplatin along with other platinum based drugs are some of the most widely used chemotherapeutic agents. However drug resistance is a major problem for the successful chemotherapeutic treatment of cancer. Current evidence suggests that drug resistance is a multifactorial problem due to changes in the expression levels and activity of a wide number of proteins. A majority of the studies to date have quantified mRNA levels between drug resistant and drug sensitive cell lines. Unfortunately mRNA levels do not always correlate with protein expression levels due to post-transcriptional changes in protein abundance. Therefore global quantitative proteomics screens are needed to identify the protein targets that are differentially expressed in drug resistant cell lines. Here we employ a quantitative proteomics technique using stable isotope labeling with amino acids in cell culture (SILAC) coupled with mass spectrometry to quantify changes in protein levels between cisplatin resistant (HeLa/CDDP) and sensitive HeLa cells in an unbiased fashion. A total of 856 proteins were identified and quantified, with 374 displaying significantly altered expression levels between the cell lines. Expression level data was then integrated with a network of protein-protein interactions, and biological pathways to obtain a systems level view of proteome changes which occur with cisplatin resistance. Several of these proteins have been previously implicated in resistance towards platinum-based and other drugs, while many represent new potential markers or therapeutic targets.

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Distribution and heat map analysis of quantified proteins.A Histograms illustrating of the distribution of RIA’s observed for the control samples (blue) and the mixed samples (yellow). B Histogram of the distribution of averaged RIA’s with overlaid distributions of the proteins identified by ANOVA with significantly decreased ratios (green) and significantly increased ratios (red). Bars are included indicating a±1.25 fold change cutoff which was determined from cumulative distribution analysis of the control sample at the 99% confidence level. C Heat map generated from SILAC data comparing cisplatin resistant HeLa cells to normal HeLa cells. Lane 1 is light cisplatin resistant and heavy normal cells. Lane 2 is heavy cisplatin resistant and light normal cells. Lane 3 is a one to one mixture of light and heavy cisplatin resistant cells. Lane 4 is a one to one mixture of light and heavy normal cells.
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pone-0019892-g002: Distribution and heat map analysis of quantified proteins.A Histograms illustrating of the distribution of RIA’s observed for the control samples (blue) and the mixed samples (yellow). B Histogram of the distribution of averaged RIA’s with overlaid distributions of the proteins identified by ANOVA with significantly decreased ratios (green) and significantly increased ratios (red). Bars are included indicating a±1.25 fold change cutoff which was determined from cumulative distribution analysis of the control sample at the 99% confidence level. C Heat map generated from SILAC data comparing cisplatin resistant HeLa cells to normal HeLa cells. Lane 1 is light cisplatin resistant and heavy normal cells. Lane 2 is heavy cisplatin resistant and light normal cells. Lane 3 is a one to one mixture of light and heavy cisplatin resistant cells. Lane 4 is a one to one mixture of light and heavy normal cells.

Mentions: Histograms illustrating the distribution of relative isotope abundance (RIA, area of light isotopic distribution divided by the sum of the areas for the light and heavy isotopic distributions) values for the control samples and the mixed samples are shown in Figure 2A. The protein ratios from the mixed samples show a broad distribution compared to those from the control samples indicating that several proteins are differentially expressed in HeLa/CDDP versus the drug sensitive cells. To determine which proteins showed altered expression in HeLa/CDDP compared to HeLa, an analysis of variance (ANOVA) was performed to compare the mean SILAC ratio for each protein from the mixed sample type to the control sample type. The resulting F-statistic and corresponding p-values from the ANOVA are included in Table S1. In total 387 proteins were found to have significantly shifted expression levels (p<0.01), of which 184 proteins were observed with increased expression levels and 203 proteins were observed with decreased expression levels in HeLa/CDDP. The 184 proteins with increased expression levels had measured RIA ratios between 0.516 and 0.819 with an average of 0.595, corresponding to a range from 1.07 to 4.54 fold increase with an average of 1.54 fold increase in expression levels. The 203 proteins with decreased expression levels had measured RIA ratios between 0.490 and 0.228 with an average of 0.422, corresponding to a range from 1.04 to 3.38 fold change with an average of 1.39 fold decrease in expression levels. Table 1 lists the ten proteins with the largest increase in expression levels and the ten proteins with the largest decrease in expression levels. Figure 2B displays the distributions of RIA values for the proteins identified by ANOVA with significantly altered expression levels. To further examine the magnitude of change in protein expression levels that could be expected to result from normal biological variation, cumulative distribution analysis was performed on the control samples, resulting in that a 1.25 fold change or greater (RIA less than 0.444 or RIA greater than 0.556) is not due to biological noise at the 99% confidence level. A total of 142 proteins from the 184 proteins with increased expression and with ANOVA p-values <0.01 also exceed the 1.25 fold change cutoff, while a total of 152 proteins from the 203 proteins with decreased expression and p-values <0.01 exceed the −1.25 fold change cutoff. The magnitude of the observed protein expression level changes is on the same order as the increased relative resistance of HeLa/CDDP vs. HeLa to cisplatin (2.6 fold) [13]. Hierarchical clustering of the 856 proteins based on their SILAC ratios was performed to generate a dendrogram and colored heat map shown in Figure 2C. The heat map contains four columns corresponding to the two mixed SILAC samples and two control SILAC samples (column 1-light HeLa/CDDP:heavy HeLa, column 2-heavy HeLa/CDDP:light HeLa, column 3- light HeLa/CDDP:heavy HeLa/CDDP, column 4- light HeLa:heavy HeLa). The heat map image reveals three general clustered regions which include proteins with increased expression in HeLa/CDDP, proteins with decreased expression in HeLa/CDDP, and proteins with unchanged expression levels. Each lane in the map is an average of three biological replicates each consisting of technical duplicates. In general excellent agreement is observed between the inverse mixed SILAC samples (Figure 2C, columns 1 & 2). A heat map displaying the signals from each of the biological replicates is included as Figure S1.


Quantitative proteomic and interaction network analysis of cisplatin resistance in HeLa cells.

Chavez JD, Hoopmann MR, Weisbrod CR, Takara K, Bruce JE - PLoS ONE (2011)

Distribution and heat map analysis of quantified proteins.A Histograms illustrating of the distribution of RIA’s observed for the control samples (blue) and the mixed samples (yellow). B Histogram of the distribution of averaged RIA’s with overlaid distributions of the proteins identified by ANOVA with significantly decreased ratios (green) and significantly increased ratios (red). Bars are included indicating a±1.25 fold change cutoff which was determined from cumulative distribution analysis of the control sample at the 99% confidence level. C Heat map generated from SILAC data comparing cisplatin resistant HeLa cells to normal HeLa cells. Lane 1 is light cisplatin resistant and heavy normal cells. Lane 2 is heavy cisplatin resistant and light normal cells. Lane 3 is a one to one mixture of light and heavy cisplatin resistant cells. Lane 4 is a one to one mixture of light and heavy normal cells.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102677&req=5

pone-0019892-g002: Distribution and heat map analysis of quantified proteins.A Histograms illustrating of the distribution of RIA’s observed for the control samples (blue) and the mixed samples (yellow). B Histogram of the distribution of averaged RIA’s with overlaid distributions of the proteins identified by ANOVA with significantly decreased ratios (green) and significantly increased ratios (red). Bars are included indicating a±1.25 fold change cutoff which was determined from cumulative distribution analysis of the control sample at the 99% confidence level. C Heat map generated from SILAC data comparing cisplatin resistant HeLa cells to normal HeLa cells. Lane 1 is light cisplatin resistant and heavy normal cells. Lane 2 is heavy cisplatin resistant and light normal cells. Lane 3 is a one to one mixture of light and heavy cisplatin resistant cells. Lane 4 is a one to one mixture of light and heavy normal cells.
Mentions: Histograms illustrating the distribution of relative isotope abundance (RIA, area of light isotopic distribution divided by the sum of the areas for the light and heavy isotopic distributions) values for the control samples and the mixed samples are shown in Figure 2A. The protein ratios from the mixed samples show a broad distribution compared to those from the control samples indicating that several proteins are differentially expressed in HeLa/CDDP versus the drug sensitive cells. To determine which proteins showed altered expression in HeLa/CDDP compared to HeLa, an analysis of variance (ANOVA) was performed to compare the mean SILAC ratio for each protein from the mixed sample type to the control sample type. The resulting F-statistic and corresponding p-values from the ANOVA are included in Table S1. In total 387 proteins were found to have significantly shifted expression levels (p<0.01), of which 184 proteins were observed with increased expression levels and 203 proteins were observed with decreased expression levels in HeLa/CDDP. The 184 proteins with increased expression levels had measured RIA ratios between 0.516 and 0.819 with an average of 0.595, corresponding to a range from 1.07 to 4.54 fold increase with an average of 1.54 fold increase in expression levels. The 203 proteins with decreased expression levels had measured RIA ratios between 0.490 and 0.228 with an average of 0.422, corresponding to a range from 1.04 to 3.38 fold change with an average of 1.39 fold decrease in expression levels. Table 1 lists the ten proteins with the largest increase in expression levels and the ten proteins with the largest decrease in expression levels. Figure 2B displays the distributions of RIA values for the proteins identified by ANOVA with significantly altered expression levels. To further examine the magnitude of change in protein expression levels that could be expected to result from normal biological variation, cumulative distribution analysis was performed on the control samples, resulting in that a 1.25 fold change or greater (RIA less than 0.444 or RIA greater than 0.556) is not due to biological noise at the 99% confidence level. A total of 142 proteins from the 184 proteins with increased expression and with ANOVA p-values <0.01 also exceed the 1.25 fold change cutoff, while a total of 152 proteins from the 203 proteins with decreased expression and p-values <0.01 exceed the −1.25 fold change cutoff. The magnitude of the observed protein expression level changes is on the same order as the increased relative resistance of HeLa/CDDP vs. HeLa to cisplatin (2.6 fold) [13]. Hierarchical clustering of the 856 proteins based on their SILAC ratios was performed to generate a dendrogram and colored heat map shown in Figure 2C. The heat map contains four columns corresponding to the two mixed SILAC samples and two control SILAC samples (column 1-light HeLa/CDDP:heavy HeLa, column 2-heavy HeLa/CDDP:light HeLa, column 3- light HeLa/CDDP:heavy HeLa/CDDP, column 4- light HeLa:heavy HeLa). The heat map image reveals three general clustered regions which include proteins with increased expression in HeLa/CDDP, proteins with decreased expression in HeLa/CDDP, and proteins with unchanged expression levels. Each lane in the map is an average of three biological replicates each consisting of technical duplicates. In general excellent agreement is observed between the inverse mixed SILAC samples (Figure 2C, columns 1 & 2). A heat map displaying the signals from each of the biological replicates is included as Figure S1.

Bottom Line: Unfortunately mRNA levels do not always correlate with protein expression levels due to post-transcriptional changes in protein abundance.A total of 856 proteins were identified and quantified, with 374 displaying significantly altered expression levels between the cell lines.Several of these proteins have been previously implicated in resistance towards platinum-based and other drugs, while many represent new potential markers or therapeutic targets.

View Article: PubMed Central - PubMed

Affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
Cisplatin along with other platinum based drugs are some of the most widely used chemotherapeutic agents. However drug resistance is a major problem for the successful chemotherapeutic treatment of cancer. Current evidence suggests that drug resistance is a multifactorial problem due to changes in the expression levels and activity of a wide number of proteins. A majority of the studies to date have quantified mRNA levels between drug resistant and drug sensitive cell lines. Unfortunately mRNA levels do not always correlate with protein expression levels due to post-transcriptional changes in protein abundance. Therefore global quantitative proteomics screens are needed to identify the protein targets that are differentially expressed in drug resistant cell lines. Here we employ a quantitative proteomics technique using stable isotope labeling with amino acids in cell culture (SILAC) coupled with mass spectrometry to quantify changes in protein levels between cisplatin resistant (HeLa/CDDP) and sensitive HeLa cells in an unbiased fashion. A total of 856 proteins were identified and quantified, with 374 displaying significantly altered expression levels between the cell lines. Expression level data was then integrated with a network of protein-protein interactions, and biological pathways to obtain a systems level view of proteome changes which occur with cisplatin resistance. Several of these proteins have been previously implicated in resistance towards platinum-based and other drugs, while many represent new potential markers or therapeutic targets.

Show MeSH
Related in: MedlinePlus