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. 2004 Sep;78(17):9041-50.
doi: 10.1128/JVI.78.17.9041-9050.2004.

Global effects of human papillomavirus type 18 E6/E7 in an organotypic keratinocyte culture system

Peggy A Garner-Hamrick  1 , J M Fostel, Wei-Ming Chien, N Sanjib Banerjee, Louise T Chow, Thomas R Broker, Chris Fisher
Affiliations

Global effects of human papillomavirus type 18 E6/E7 in an organotypic keratinocyte culture system

Peggy A Garner-Hamrick et al. J Virol. 2004 Sep.

Abstract

The effects of human papillomavirus type 18 (HPV-18) E6 and E7 proteins on global patterns of host gene expression in primary human keratinocytes grown in organotypic raft culture system were assessed. Primary human keratinocytes were infected with retroviruses that express the wild-type HPV-18 E6 and E7 genes from the native differentiation-dependent HPV enhancer-promoter. Total RNA was isolated from raft cultures and used to generate probes for querying Affymetrix U95A microarrays, which contain >12,500 human gene sequences. Quadruplicate arrays of each E6/E7-transduced and empty vector-transduced samples were analyzed by 16 pairwise comparisons. Transcripts altered in > or =12 comparisons were selected for further analysis. With this approach, HPV-18 E6/E7 expression significantly altered the expression of 1,381 genes. A large increase in transcripts associated with DNA and RNA metabolism was observed, with major increases noted for transcription factors, splicing factors, and DNA replication elements, among others. Multiple genes associated with protein translation were downregulated. In addition, major alterations were found in transcripts associated with the cell cycle and cell differentiation. Our study provides a systematic description of transcript changes brought about by HPV-18 E6/E7 in a physiologically relevant model and should furnish a solid source of information to guide future studies.

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Figures

FIG. 1.
FIG. 1.
Micrographs showing bromodeoxyuridine incorporation and morphology of pLC and HPV-18 E6/E7 raft cultures. Note stimulation of bromodeoxyuridine incorporation in the differentiating compartment in the E6/E7 culture.
FIG. 2.
FIG. 2.
Scatter plot of a representative graph of the log ratio of experimental (E6/E7-2B)/control(pLC-2B) plotted against the sum of experimental E6/E7-2B and pLC-2B. (Top) Only genes in the increased/decreased categories are shown. (Bottom) Off/on and on/off genes are also shown and appear as arms of the graph. In each graph, every point represents an individual transcript. Each color stripe to the left or right of 0 represents >1, 1, 2, and 3 standard deviations from the mean.
FIG. 3.
FIG. 3.
Histograms depicting numbers of reproducible transcript changes versus total number of comparisons (top). The 1,381 genes that were found altered in ≥12 of 16 comparisons were selected for further analysis. Histograms on the bottom show the distribution of 1,381 transcripts, altered in ≥12 of 16 comparisons, according to fold change.
FIG. 4.
FIG. 4.
Incyte functional hierarchies of transcripts reproduced in ≥12 of 16 comparisons. This and the following figure are meant to provide an overall view of transcript changes. Note that because increased/decreased genes may appear multiple times within these hierarchies, the number of genes provided in this and the following figure is relative, not absolute.
FIG. 5.
FIG. 5.
Incyte pathway hierarchies of transcripts reproduced in at least 12 of 16 comparisons. Note that because increased/decreased genes may appear multiple times within these hierarchies, the number of genes provided in this and the previous figure is relative, not absolute.
FIG. 6.
FIG. 6.
Changes in transcripts associated with the DNA synthesis pathway. A grey bar indicates that gene did not achieve reproducibility in ≥12 of 16 comparisons. The colored bars indicate the fold difference relative to controls (see inset) of transcripts that were reproducibly changed in ≥12 of 16 comparisons.
FIG. 7.
FIG. 7.
Bar graph comparing Taqman PCR data (grey bars) to Affymetrix array (black bars) ADIs.
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