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


CONCLUSIONS
This large-scale analysis provides a framework for understanding the cooperation between E6 and E7 oncoproteins in HPV-driven carcinogenesis.
Merkley
Mark A
MA
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA, USA.
Hildebrandt
Ellen
E
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA, USA.|Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130, USA.
Podolsky
Robert H
RH
Center for Biotechnology and Genomic Medicine, Medical College of Georgia, Augusta, GA, USA.
Arnouk
Hilal
H
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA, USA.|Division of Hematology/Oncology, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Ferris
Daron G
DG
Department of Obstetrics and Gynecology, Medical College of Georgia, Augusta, GA, USA.|Department of Family Medicine, Medical College of Georgia, Augusta, GA, USA.
Dynan
William S
WS
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA, USA.
Stöppler
Hubert
H
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA, USA.|Department of Obstetrics and Gynecology, Medical College of Georgia, Augusta, GA, USA.
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