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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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Related in: MedlinePlus

Western blot analysis.Cropped image of Western blot results comparing levels of four proteins (DDB1, Ku80, CD44, DJ-1) between HeLa and HeLa/CDDP. ACTB was also monitored as a quantitative control. * The normalized ratio for each protein was calculated by averaging the signal intensity from three biological replicates from HeLa/CDDP and dividing it by the average signal intensity from 3 biological replicates from HeLa. This ratio was then normalized using the average ratio of signal intensity of ACTB to the respective protein of interest band. In general there is good agreement between the ratios obtained from Western blotting and from SILAC. Full Western blot images are available in Figure S2.
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pone-0019892-g003: Western blot analysis.Cropped image of Western blot results comparing levels of four proteins (DDB1, Ku80, CD44, DJ-1) between HeLa and HeLa/CDDP. ACTB was also monitored as a quantitative control. * The normalized ratio for each protein was calculated by averaging the signal intensity from three biological replicates from HeLa/CDDP and dividing it by the average signal intensity from 3 biological replicates from HeLa. This ratio was then normalized using the average ratio of signal intensity of ACTB to the respective protein of interest band. In general there is good agreement between the ratios obtained from Western blotting and from SILAC. Full Western blot images are available in Figure S2.

Mentions: To confirm the differential expression of a few key target proteins including CD44, DDB-1, DJ-1 and XRCC5, Western blotting was performed. Levels of β-actin were monitored as a quantitative control. A cropped version of the Western blot results with a comparison of the protein ratio calculated from the Western blot and the SILAC results can be seen in Figure 3. In general there is good agreement between the two techniques as each of these proteins was identified with increased expression levels in HeLa/CDDP compared with normal HeLa. Images of the full Western blots for each of the selected proteins are included in Figure S2.


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)

Western blot analysis.Cropped image of Western blot results comparing levels of four proteins (DDB1, Ku80, CD44, DJ-1) between HeLa and HeLa/CDDP. ACTB was also monitored as a quantitative control. * The normalized ratio for each protein was calculated by averaging the signal intensity from three biological replicates from HeLa/CDDP and dividing it by the average signal intensity from 3 biological replicates from HeLa. This ratio was then normalized using the average ratio of signal intensity of ACTB to the respective protein of interest band. In general there is good agreement between the ratios obtained from Western blotting and from SILAC. Full Western blot images are available in Figure S2.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102677&req=5

pone-0019892-g003: Western blot analysis.Cropped image of Western blot results comparing levels of four proteins (DDB1, Ku80, CD44, DJ-1) between HeLa and HeLa/CDDP. ACTB was also monitored as a quantitative control. * The normalized ratio for each protein was calculated by averaging the signal intensity from three biological replicates from HeLa/CDDP and dividing it by the average signal intensity from 3 biological replicates from HeLa. This ratio was then normalized using the average ratio of signal intensity of ACTB to the respective protein of interest band. In general there is good agreement between the ratios obtained from Western blotting and from SILAC. Full Western blot images are available in Figure S2.
Mentions: To confirm the differential expression of a few key target proteins including CD44, DDB-1, DJ-1 and XRCC5, Western blotting was performed. Levels of β-actin were monitored as a quantitative control. A cropped version of the Western blot results with a comparison of the protein ratio calculated from the Western blot and the SILAC results can be seen in Figure 3. In general there is good agreement between the two techniques as each of these proteins was identified with increased expression levels in HeLa/CDDP compared with normal HeLa. Images of the full Western blots for each of the selected proteins are included in Figure S2.

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