Transcriptional profiling of dysplastic lesions in K14-HPV16 transgenic mice using laser microdissection.
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.
Sethi
Neerja
N
Department of Medicine, University of California, San Francisco, California, USA.
Palefsky
Joel
J
D000818:Animals; D002294:Carcinoma, Squamous Cell / Q000209:etiology / Q000235:genetics* / Q000648:ultrastructure; D003199:Computer Systems; D004268:DNA-Binding Proteins / Q000235:genetics*; D004195:Disease Models, Animal; D004210:Dissection; D004817:Epidermis / Q000378:metabolism; D005814:Genes, Viral; D006801:Humans; D053547:Keratin-14; D007633:Keratins / Q000235:genetics*; D007834:Lasers; D051379:Mice; D008822:Mice, Transgenic; D008846:Micromanipulation; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D027383:Papillomaviridae / Q000235:genetics* / Q000472:pathogenicity; D050725:Papillomavirus E7 Proteins; D030361:Papillomavirus Infections; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics*; D012878:Skin Neoplasms / Q000209:etiology / Q000235:genetics* / Q000648:ultrastructure; D014158:Transcription, Genetic; D019076:Transgenes
D004268:DNA-Binding Proteins; C093478:E1 protein, Human papillomavirus 16; C054855:E2 protein, Human papillomavirus type 16; C068651:E6 protein, Human papillomavirus type 16; C507540:KRT14 protein, human; D053547:Keratin-14; C507446:Krt14 protein, mouse; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012097:Repressor Proteins; C059731:oncogene protein E7, Human papillomavirus type 16; D007633:Keratins