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Journal of Virology, July 2004, p. 7478-7489, Vol. 78, No. 14
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.14.7478-7489.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Gene Profiling of Cottontail Rabbit Papillomavirus-Induced Carcinomas Identifies Upregulated Genes Directly Involved in Stroma Invasion as Shown by Small Interfering RNA-Mediated Gene Silencing

Evamaria Huber, Daniela Vlasny, Sonja Jeckel, Frank Stubenrauch, and Thomas Iftner*

Sektion Experimentelle Virologie, Universitaetsklinikum Tuebingen, 72076 Tuebingen, Germany

Received 27 November 2003/ Accepted 12 March 2004

To investigate changes in cellular gene expression associated with malignant progression, we identified differentially expressed genes in a cottontail rabbit papillomavirus (CRPV) squamous carcinoma model employing New Zealand White rabbits. The technique of suppression subtractive cDNA hybridization was applied to pairs of mRNA isolates from CRPV-induced benign papillomas and carcinomas, with each pair derived from the same individual rabbit. The differential expression of 23 subtracted cDNAs was further confirmed by quantitative reverse transcription-PCR (RT-PCR) with additional biopsies. Eight papilloma-carcinoma pairs examined showed a constant upregulation of the transcripts for the multifunctional adaptor protein 14-3-3 {zeta} and the Y-box binding transcription factor YB-1, whereas transcripts for m-type calpain 2 and NB thymosin ß, which are involved in cell motility and tissue invasion, as well as casein kinase 1 {alpha}, chaperonin, and annexin I, were found to be upregulated in the majority of the cases. RNA-RNA in situ hybridization and laser capture microdissection in combination with quantitative RT-PCR analysis verified the deregulated expression of the transcripts in the tumor cells. In contrast, CRPV E7 transcript levels remained rather constant indicating no requirement for a further upregulation of E7 expression following tumor induction. Small interfering RNA-mediated interference with expression of genes encoding YB-1, m-type calpain 2, or NB thymosin ß in a CRPV-positive cell line established from New Zealand White rabbit keratinocytes resulted in decreased cell invasion in matrigel chamber assays.

* Corresponding author. Mailing address: Experimentelle Virologie, Universitaetsklinikum Tuebingen, Elfriede-Aulhorn Strasse 6, 72076 Tuebingen, Germany. Phone: 49 7071 2980246. Fax: 49 7071 295419. E-mail: tsiftner{at}med.uni-tuebingen.de.

Journal of Virology, July 2004, p. 7478-7489, Vol. 78, No. 14
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.14.7478-7489.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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