Large-scale analysis of protein expression changes in human keratinocytes immortalized by human papilloma virus type 16 E6 and E7 oncogenes

Mark A. Merkley, Ellen Hildebrandt, Robert H. Podolsky, Hilal Arnouk, Daron Gale Ferris, William S. Dynan, Hubert Stöppler

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Background: Infection with high-risk type human papilloma viruses (HPVs) is associated with cervical carcinomas and with a subset of head and neck squamous cell carcinomas. Viral E6 and E7 oncogenes cooperate to achieve cell immortalization by a mechanism that is not yet fully understood. Here, human keratinocytes were immortalized by long-term expression of HPV type 16 E6 or E7 oncoproteins, or both. Proteomic profiling was used to compare expression levels for 741 discrete protein features. Results: Six replicate measurements were performed for each group using two-dimensional difference gel electrophoresis (2D-DIGE). The median within-group coefficient of variation was 19-21%. Significance of between-group differences was tested based on Significance Analysis of Microarray and fold change. Expression of 170 (23%) of the protein features changed significantly in immortalized cells compared to primary keratinocytes. Most of these changes were qualitatively similar in cells immortalized by E6, E7, or E6/7 expression, indicating convergence on a common phenotype, but fifteen proteins (~2%) were outliers in this regulatory pattern. Ten demonstrated opposite regulation in E6- and E7-expressing cells, including the cell cycle regulator p16INK4a; the carbohydrate binding protein Galectin-7; two differentially migrating forms of the intermediate filament protein Cytokeratin-7; HSPA1A (Hsp70-1); and five unidentified proteins. Five others had a pattern of expression that suggested cooperativity between the co-expressed oncoproteins. Two of these were identified as forms of the small heat shock protein HSPB1 (Hsp27). Conclusion: This large-scale analysis provides a framework for understanding the cooperation between E6 and E7 oncoproteins in HPV-driven carcinogenesis.

Original languageEnglish (US)
Article number1477
Number of pages1
JournalProteome Science
Volume7
DOIs
StatePublished - Aug 23 2009

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Papillomaviridae
Keratinocytes
Viruses
Oncogenes
Oncogene Proteins
Two-Dimensional Difference Gel Electrophoresis
Proteins
Small Heat-Shock Proteins
Keratin-7
Galectins
Intermediate Filament Proteins
Microarray Analysis
Microarrays
Electrophoresis
Proteomics
Cell Cycle
Carcinogenesis
Gels
Cells
Carcinoma

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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Large-scale analysis of protein expression changes in human keratinocytes immortalized by human papilloma virus type 16 E6 and E7 oncogenes. / Merkley, Mark A.; Hildebrandt, Ellen; Podolsky, Robert H.; Arnouk, Hilal; Ferris, Daron Gale; Dynan, William S.; Stöppler, Hubert.

In: Proteome Science, Vol. 7, 1477, 23.08.2009.

Research output: Contribution to journalArticle

Merkley, Mark A. ; Hildebrandt, Ellen ; Podolsky, Robert H. ; Arnouk, Hilal ; Ferris, Daron Gale ; Dynan, William S. ; Stöppler, Hubert. / Large-scale analysis of protein expression changes in human keratinocytes immortalized by human papilloma virus type 16 E6 and E7 oncogenes. In: Proteome Science. 2009 ; Vol. 7.
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AB - Background: Infection with high-risk type human papilloma viruses (HPVs) is associated with cervical carcinomas and with a subset of head and neck squamous cell carcinomas. Viral E6 and E7 oncogenes cooperate to achieve cell immortalization by a mechanism that is not yet fully understood. Here, human keratinocytes were immortalized by long-term expression of HPV type 16 E6 or E7 oncoproteins, or both. Proteomic profiling was used to compare expression levels for 741 discrete protein features. Results: Six replicate measurements were performed for each group using two-dimensional difference gel electrophoresis (2D-DIGE). The median within-group coefficient of variation was 19-21%. Significance of between-group differences was tested based on Significance Analysis of Microarray and fold change. Expression of 170 (23%) of the protein features changed significantly in immortalized cells compared to primary keratinocytes. Most of these changes were qualitatively similar in cells immortalized by E6, E7, or E6/7 expression, indicating convergence on a common phenotype, but fifteen proteins (~2%) were outliers in this regulatory pattern. Ten demonstrated opposite regulation in E6- and E7-expressing cells, including the cell cycle regulator p16INK4a; the carbohydrate binding protein Galectin-7; two differentially migrating forms of the intermediate filament protein Cytokeratin-7; HSPA1A (Hsp70-1); and five unidentified proteins. Five others had a pattern of expression that suggested cooperativity between the co-expressed oncoproteins. Two of these were identified as forms of the small heat shock protein HSPB1 (Hsp27). Conclusion: This large-scale analysis provides a framework for understanding the cooperation between E6 and E7 oncoproteins in HPV-driven carcinogenesis.

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