The overall goal of this career development proposal is to assist in establishing the independent research career of the candidate in the study of tumor virus-host cell interactions and specifically of the oncogenic transformation caused by high risk human papillomavirus (HPV) infections that lead to cervical and anogenital cancers. The training environments are the laboratory of the mentor, Dr. Denise Galloway, at the Fred Hutchinson Cancer Research Center, and the University of Washington and Children's Hospital and Regional Medical Center.
The specific aims of the proposed research project focus on elucidating the mechanism by which HPV activates telomerase in cellular immortalization through NFX1 and determining the role of NFX1 in normal and high-risk HPV E6-expressing cells. In high-risk HPV infection, the E6 protein upregulates expression of the catalytic subunit of telomerase, hTERT, and activates telomerase. The candidate's preliminary data has shown that a splice variant of the protein Nuclear Factor binds to the X1 box (NFX1-123) interacts with HPV 16E6. The overexpression or knockdown of NFX1-123 in HPV 16E6- expressing cells affects hTERT mRNA levels and telomerase activity, and interaction of NFX1-123 with cytoplasmic poly(A) binding proteins appears critical for this effect.
The first aim of the proposed research project is to determine whether NFX1-123 functions as a classic transcriptional activator of hTERT using gel shift and chromatin immunoprecipitation assays.
The second aim i s to determine whether NFX1-123 acts as a post-transcriptional regulator of hTERT using RNA transfection and RNA binding assays. Both of these mechanisms of regulation may be important and are not mutually exclusive. Finally, the third aim is to determine the global effects of NFX1-123 in epithelial cells with and without HPV 16E6 expression using gene expression microarray studies and protein purification assays. Understanding the biology of high-risk HPV-mediated cellular immortalization will clarify how a tumor virus can affect a host cell's self regulation, and determining how NFX1-123 affects hTERT expression is likely critical to understanding how most cancers activate telomerase.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08CA131171-05
Application #
8288838
Study Section
Subcommittee G - Education (NCI)
Program Officer
Perkins, Susan N
Project Start
2008-07-01
Project End
2013-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
5
Fiscal Year
2012
Total Cost
$134,379
Indirect Cost
$9,954
Name
Seattle Children's Hospital
Department
Type
DUNS #
048682157
City
Seattle
State
WA
Country
United States
Zip Code
98105
Xu, Mei; Katzenellenbogen, Rachel A; Grandori, Carla et al. (2013) An unbiased in vivo screen reveals multiple transcription factors that control HPV E6-regulated hTERT in keratinocytes. Virology 446:17-24
Vliet-Gregg, Portia A; Hamilton, Jennifer R; Katzenellenbogen, Rachel A (2013) NFX1-123 and human papillomavirus 16E6 increase Notch expression in keratinocytes. J Virol 87:13741-50
Katzenellenbogen, Rachel A; Vliet-Gregg, Portia; Xu, Mei et al. (2010) Cytoplasmic poly(A) binding proteins regulate telomerase activity and cell growth in human papillomavirus type 16 E6-expressing keratinocytes. J Virol 84:12934-44
Howie, Heather L; Katzenellenbogen, Rachel A; Galloway, Denise A (2009) Papillomavirus E6 proteins. Virology 384:324-34
Katzenellenbogen, Rachel A; Vliet-Gregg, Portia; Xu, Mei et al. (2009) NFX1-123 increases hTERT expression and telomerase activity posttranscriptionally in human papillomavirus type 16 E6 keratinocytes. J Virol 83:6446-56