title: Large-scale analysis of protein expression changes in human keratinocytes immortalized by human papilloma virus type 16 E6 and E7 oncogenes.
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).
  
  
  CONCLUSIONS
  This large-scale analysis provides a framework for understanding the cooperation between E6 and E7 oncoproteins in HPV-driven carcinogenesis.
authors.affiliation: Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA, USA.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Merkley
authors.affiliation: 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.
authors.forename: Ellen
authors.initials: E
authors.lastname: Hildebrandt
authors.affiliation: Center for Biotechnology and Genomic Medicine, Medical College of Georgia, Augusta, GA, USA.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Podolsky
authors.affiliation: 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.
authors.forename: Hilal
authors.initials: H
authors.lastname: Arnouk
authors.affiliation: Department of Obstetrics and Gynecology, Medical College of Georgia, Augusta, GA, USA.|Department of Family Medicine, Medical College of Georgia, Augusta, GA, USA.
authors.forename: Daron G
authors.initials: DG
authors.lastname: Ferris
authors.affiliation: Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA, USA.
authors.forename: William S
authors.initials: WS
authors.lastname: Dynan
authors.affiliation: 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.
authors.forename: Hubert
authors.initials: H
authors.lastname: Stöppler
references.citation: Oncogene. 1998 Sep 10;17(10):1207-14
references.pmid: 9771963
references.citation: Biochem Biophys Res Commun. 1997 Feb 13;231(2):509-13
references.pmid: 9070310
references.citation: Gynecol Oncol. 2005 Oct;99(1):142-52
references.pmid: 16038965
references.citation: Virchows Arch. 1994;425(2):145-55
references.pmid: 7524976
references.citation: Biochem Biophys Res Commun. 1998 Mar 17;244(2):384-9
references.pmid: 9514931
references.citation: Oncogene. 2007 May 17;26(23):3321-8
references.pmid: 17130828
references.citation: Int J Cancer. 2004 Feb 20;108(5):766-72
references.pmid: 14696105
references.citation: Cell Mol Life Sci. 2006 Mar;63(6):686-97
references.pmid: 16429325
references.citation: Cancer Res. 2007 Feb 15;67(4):1626-35
references.pmid: 17308103
references.citation: Virology. 1997 Dec 8;239(1):97-107
references.pmid: 9426450
references.citation: Am J Pathol. 1992 Mar;140(3):601-12
references.pmid: 1372156
references.citation: Hum Pathol. 2001 Dec;32(12):1351-5
references.pmid: 11774168
references.citation: Nat Med. 2004 Feb;10(2):182-6
references.pmid: 14704791
references.citation: Clin Cancer Res. 2001 Nov;7(11):3328-35
references.pmid: 11705844
references.citation: Genes Dev. 1998 Jul 1;12(13):2061-72
references.pmid: 9649509
references.citation: Mod Pathol. 2000 Sep;13(9):962-72
references.pmid: 11007036
references.citation: J Clin Oncol. 2006 Feb 10;24(5):736-47
references.pmid: 16401683
references.citation: Gynecol Oncol. 2005 Oct;99(1):26-35
references.pmid: 16051329
references.citation: Br J Dermatol. 2002 Jul;147(1):13-9
references.pmid: 12100179
references.citation: Am J Surg Pathol. 2005 Aug;29(8):1062-6
references.pmid: 16006801
references.citation: Oncogene. 2004 Apr 15;23(17):2988-95
references.pmid: 14968115
references.citation: Clin Cancer Res. 2006 Dec 15;12(24):7279-83
references.pmid: 17189399
references.citation: Clin Sci (Lond). 2006 May;110(5):525-41
references.pmid: 16597322
references.citation: Mol Cell Biol. 1999 Apr;19(4):3103-14
references.pmid: 10082577
references.citation: Cancer Sci. 2007 Aug;98(8):1234-40
references.pmid: 17506794
references.citation: Mol Cell. 2005 Jan 21;17(2):251-64
references.pmid: 15664194
references.citation: J Virol. 2005 Mar;79(6):3737-47
references.pmid: 15731267
references.citation: J Virol. 2006 Jun;80(11):5301-7
references.pmid: 16699010
references.citation: J Biol Chem. 2002 Feb 22;277(8):6131-6
references.pmid: 11711542
references.citation: J Proteome Res. 2007 Nov;6(11):4256-68
references.pmid: 17902640
references.citation: J Biol Chem. 2004 Oct 1;279(40):41975-84
references.pmid: 15280382
references.citation: J Natl Cancer Inst. 2003 Dec 3;95(23):1772-83
references.pmid: 14652239
references.citation: Virology. 1989 Nov;173(1):302-10
references.pmid: 2479170
references.citation: Int J Oncol. 2006 Apr;28(4):873-81
references.pmid: 16525636
references.citation: Proteomics. 2004 Mar;4(3):839-48
references.pmid: 14997504
references.citation: Hum Pathol. 2008 Jan;39(1):87-95
references.pmid: 17949781
references.citation: Clin Cancer Res. 2003 Aug 15;9(9):3418-24
references.pmid: 12960131
references.citation: Cancer Cell. 2005 Mar;7(3):263-73
references.pmid: 15766664
references.citation: J Virol. 1996 May;70(5):3269-79
references.pmid: 8627810
references.citation: Electrophoresis. 1997 Oct;18(11):2071-7
references.pmid: 9420172
references.citation: Virology. 2004 Jun 20;324(1):17-27
references.pmid: 15183049
references.citation: Virology. 2008 Aug 1;377(2):408-18
references.pmid: 18519144
references.citation: Mod Pathol. 2005 Aug;18(8):1102-6
references.pmid: 15731774
references.citation: Nat Med. 2004 Feb;10(2):175-81
references.pmid: 14704789
references.citation: Anticancer Res. 2001 May-Jun;21(3B):2085-90
references.pmid: 11497302
references.citation: Semin Cancer Biol. 2006 Jun;16(3):214-24
references.pmid: 16698281
references.citation: J Virol. 1995 Oct;69(10):6389-99
references.pmid: 7666540
references.citation: J Biol Chem. 2005 Aug 19;280(33):29885-98
references.pmid: 15944157
references.citation: BMC Cancer. 2006 Sep 12;6:223
references.pmid: 16968546
references.citation: J Virol. 1989 Oct;63(10):4417-21
references.pmid: 2476573
references.citation: J Pathol. 2002 Mar;196(3):316-23
references.pmid: 11857495
references.citation: Cancer Res. 2007 Oct 15;67(20):9643-8
references.pmid: 17942893
references.citation: J Clin Virol. 2000 Oct;19(1-2):1-5
references.pmid: 11091143
references.citation: Anticancer Res. 2002 Nov-Dec;22(6A):3331-5
references.pmid: 12530083
references.citation: J Virol. 2002 Jul;76(14):7040-8
references.pmid: 12072504
references.citation: Nature. 1997 Sep 18;389(6648):300-5
references.pmid: 9305847
references.citation: Proteomics Clin Appl. 2009 May 5;3(5):516-527
references.pmid: 19834583
references.citation: J Cell Biol. 1988 Aug;107(2):427-46
references.pmid: 2458356
references.citation: J Biol Chem. 1992 Jan 15;267(2):794-803
references.pmid: 1730670
references.citation: Virology. 1994 Jan;198(1):169-74
references.pmid: 8259651
references.citation: Cell Death Differ. 2007 Nov;14(11):1936-47
references.pmid: 17673920
references.citation: Pathol Res Pract. 2007;203(6):445-9
references.pmid: 17543474
references.citation: Cancer Epidemiol Biomarkers Prev. 1996 Jul;5(7):567-75
references.pmid: 8827362
references.citation: EMBO J. 1988 Oct;7(10):3181-7
references.pmid: 2460337
references.citation: Proc Natl Acad Sci U S A. 2001 Apr 24;98(9):5116-21
references.pmid: 11309499
references.citation: Obstet Gynecol. 1993 Sep;82(3):465
references.pmid: 7689193
references.citation: Nat Rev Cancer. 2002 May;2(5):342-50
references.pmid: 12044010
references.citation: J Natl Cancer Inst. 1998 Nov 4;90(21):1626-36
references.pmid: 9811312
references.citation: Oncogene. 2004 Apr 29;23(20):3561-71
references.pmid: 15077181
references.citation: J Dent Res. 2007 Feb;86(2):104-14
references.pmid: 17251508
references.citation: Clin Chem. 2007 Apr;53(4):629-35
references.pmid: 17303689
references.citation: DNA Cell Biol. 2004 Dec;23(12):826-35
references.pmid: 15684709
references.citation: Cancer Lett. 2002 May 28;179(2):205-12
references.pmid: 11888675
references.citation: Prog Cell Cycle Res. 2003;5:383-91
references.pmid: 14593733
references.citation: Proteomics. 2006 Jan;6(2):547-58
references.pmid: 16342241
references.citation: J Virol. 2004 Nov;78(21):11451-60
references.pmid: 15479788