Transcriptional profiling of dysplastic lesions in K14‐HPV16 transgenic mice using laser microdissection

Abstract

In the K14-HPV16 transgenic mouse model of human papillomavirus (HPV)-associated squamous cell cancers, HPV16 E6 and E7 oncogenes and E1 and E2 regulatory genes are driven by the K14 keratinocyte-specific promoter. HPV transcription varies within the different layers of the epithelium. The correlation between HPV transcription patterns and disease pathogenesis is not well understood. Understanding these patterns is critical to designing and testing new HPV-specific therapeutic strategies. We examined HPV gene expression in homogenous populations of cells microdissected from the stratum basale, stratum spinosum, and stratum corneum of lesions from the transgenic mice using PALM microlaser technology. RNA extracted from each cell layer was subjected to two-step gene-specific RT-PCR and real-time quantitative nested PCR. To ensure specific amplification of spliced transcripts, the primers used for real-time nested PCR spanned the splice sites. High levels of E2 were detected in the basal and supra-basal layers of hyperplastic and dysplastic lesions. E7 and E6* levels increased significantly over time in stratum basale and stratum spinosum. E6** was expressed at much lower levels. We showed that the transgenic mice express correctly spliced E2 transcripts and are suitable as a preclinical model to test a therapeutic strategy using transcriptional regulation by the E2 protein.

Development of Skin Tumors in Mice Transgenic for Early Genes of Human Papillomavirus Type 8

“…The HPV8 transgenes are under control of the cytokeratin-14 promoter, which is well known to target expression to the epidermis and developing hair follicles of mouse embryos and to basal and spinous layers of the adult skin (29)(30)(31). The early HPV genes are transcribed as multiple polycistronic messages generated by alternative splicing (32,33).…”

Quantitative Reverse Transcriptase Polymerase Chain Reaction

“…67,68 Therefore, a combination of both technologies, in which the screening of the involved genes is performed by microarrays and the precise quantifi cation and high throughput screening is performed by real-time PCR, is the ideal method. Similarly, real-time PCR technology will continue to be combined with advanced microdissection techniques 13,[16][17][18][19] or nucleic acids obtained from paraffi n-fi xed archival samples. 14,15 The detection and analysis of minimal residual disease 51,69 and viral loads will remain an important application.…”

“…Since the fi rst documentation of real-time polymerase chain reaction (PCR), 1 it has been used for an increasing and diverse number of applications, including mRNA expression studies, DNA copy number measurements in genomic or viral DNAs, 2-7 allelic discrimination assays, 8,9 expression analysis of specifi c splice variants of genes [10][11][12][13] and gene expression in paraffi n-embedded tissues, 14,15 and laser captured microdissected cells. 13,[16][17][18][19] Therefore, quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) is now essential in molecular diagnostics to quantitatively assess the level of RNA or DNA in a given specimen.…”

“…13,[16][17][18][19] Therefore, quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) is now essential in molecular diagnostics to quantitatively assess the level of RNA or DNA in a given specimen. Q-RT-PCR enables the detection and quantifi cation of very small amounts of DNA, cDNA, or RNA, even down to a single copy.…”

Expression of the HPV11 E2 gene in transgenic mice does not result in alterations of the phenotypic pattern

“…The use of raft cultures has further allowed an analysis of the E2 functions in the context of a stratified structure, but no animal model is so far available permitting to extrapolate these data to the infection situation (Stubenrauch et al 1998). Despite the existence of transgenic mice models for the complete HPV genome, it has so far been impossible to associate defined functions with expression of the E2 protein (Arbeit et al 1996;Schaper et al 2005;Sethi and Palefsky 2004). Thus, transgenic mice carrying only the E2 sequence of HPV11 were generated and the question was addressed whether expression of the viral protein will affect growth or differentiation of tissues, mainly skin, expressing the viral protein.…”