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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 4, 2004 as doi:10.1096/fj.03-0946fje. |
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Departments of Medicine and Stomatology, University of California, San Francisco, California, USA
1Correspondence: Box 0216, UCSF, San Francisco, CA 94143, USA. E-mail: joelp{at}medicine.ucsf.edu
SPECIFIC AIMS
The goals of this study were to 1) develop a fast, specific, quantitative real-time nested PCR method to perform functional genomics at the level of laser microdissected homogeneous cell populations from heterogeneous neoplastic lesions and 2) establish that lesions developing in the K14-HPV16 transgenic mouse model are similar to human lesions and express human papillomavirus (HPV) 16 early genes, including the oncogenes E6, E7 and the correctly spliced E2 gene in the basal and the more superficial layers.
PRINCIPAL FINDINGS
1. Laser microdissection of specific epithelial layers from neoplastic lesions
In the K14-HPV16 transgenic mouse model of 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 design and test new HPV-specific therapeutic strategies. We examined HPV gene expression in homogeneous populations of cells microdissected from the stratum basale, stratum spinosum, and stratum corneum of lesions from transgenic mice using PALM microlaser technology.
We characterized HPV16 gene expression at different ages and performed functional genomics at three distinct layers of the epithelium-stratum basale, stratum spinosum, and stratum corneum. Figure 1
shows a schematic diagram of the experimental design and results. To ensure specificity, 
20–25 cells were excised from a particular layer in a single microdissection. Comparable sections from the same biopsy were examined at each time point. The cells were microdissected and pooled to yield 100 cells per cap/tube. This ensured that RNA extracted from each tube resulted from an approximately equal number of homogeneous cell populations.
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Studies have established that lesions in the K14-HPV16 transgenic mice show characteristic neoplastic progression in the squamous epithelium with advancing age. We examined biopsies from the ears of transgenic mice, since the ear consistently develops severe progressive histopathology from hyperplasia to papillomatosis to dysplasia. Figure 2
a shows the ear epithelium of an adult wild-type FVB/N mouse that consists of one to three cell layers shown microdissected in (Fig. 2b, c
). Transgenic mice by 1–2 months developed mild to moderate hyperplasia, seen as mild thickness of the ear and snout skin with an increase in number of cell layers to 8–10 (Fig. 2d
). The differentiation pattern of the epithelium was normal at this stage. The ear from a 5.5- to 6-month-old mouse exhibited regions of hyperplasia and mild dysplasia (Fig. 2g
). Hyperplastic regions showed an increase in cell size and the thickness of the epithelium was increased by 3- to 4-fold; the ear and the snout appeared thickened and rough in texture due to hyperkeratosis and acanthosis. By 9 months of age, the ear and snout showed extreme hyperkeratosis, acanthosis, and papillomatosis. At this stage, small tumors and highly keratotic lesions were seen on the upper body. Histologically, these lesions showed high-grade dysplasia (Fig. 2j
) or well-differentiated squamous cell carcinomas.
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Figure 2e
shows a microdissection of basal layers from a hyperplastic epithelium. Figure 2h, k
shows a microdissection from low- and high-grade dysplastic lesions. Figure 2n
shows a microdissection of the stratum basale, stratum spinosum, and stratum corneum from the same site, demonstrating our ability to isolate homogeneous population of cell layers.
2. Quantitation of HPV16 gene expression using real-time analysis
Real-time PCR offers a sensitive, efficient, and reliable approach to cDNA quantitation. We used a reverse-transcription, comparative real-time PCR method where we quantified the transcripts of an endogenous gene (ß-actin) as an RNA internal control. To control for variation in the quantity and quality of RNA, we used mouse ß-actin levels to calculate a relative Ct (
Ct) for target genes: E2, E1/E2 spliced form, E7, E6, and the E6* and E6** spliced forms.
Real-time PCR data are represented as Ct values, where Ct defines the threshold cycle of PCR at which the amplified product was first detected. Ct represents the difference in Ct values derived from that of a HPV16 gene and the control reference mouse ß-actin for different epithelial layers, stratum basale, stratum spinosum, and stratum corneum (Table 1
). Various genes were examined in the different layers at ages 2, 6, and 9 months. Ct represents the difference between the paired tissue samples as calculated by the formula: Ct= Ct of HPV16 gene in stratum basale at 2 months – Ct in different layers at various ages, where Ct of an HPV16 gene at 2 months in the stratum basale was selected as an arbitrary reference value and the Ct for the same gene at various stages in different layers was subtracted from it. The N-fold differential expression was calculated as 2Ct (Table 1)
. The expression of each transcript examined at 2 months in the stratum basale was expressed as 1; expression at different ages in the different layers for that gene was expressed relative to that.
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Comparative calculations (Table 1)
demonstrate differential expression of the E1/E2 and E7 transcripts. Table 1
shows that a relatively large amount of the correctly spliced E1/E2 transcripts was expressed at 2 and 6 months in the stratum basale. The stratum spinosum at the same age expressed approximately half the stratum basale level of E1/E2 transcripts. At 9 months, expression of E1/E2 in the stratum basale was reduced to one-fifth of that observed in the stratum basale at 2 months. E1/E2 expression in the stratum spinosum was substantially reduced by 9 months. Very little expression was observed in the stratum corneum at all stages.
E7 transcripts increased by 7-fold in the stratum spinosum with increasing age (Table 1)
. An increase of 3.4-fold was observed in the stratum basale by 9 months age compared with the stratum basale of a hyperplastic lesion at 2 months. A much reduced expression was found in stratum corneum at all ages. Similar analysis of the relative expression of the full-length E6 gene and the two spliced forms, E6* and E6**, showed that the full-length E6 increased slightly in the stratum spinosum with age. E6* transcripts were the most prominent and increased by 7.5- and 6-fold by 6 and 9 months, respectively, in the stratum spinosum compared with expression in the stratum basale at 2 months. There was also an increase in E6* transcripts in the stratum basale of a dysplastic lesion at 6 months. The E6** transcript was expressed at low amounts at all ages.
All the HPV genes examined were expressed at very low or insignificant amounts in the stratum corneum. None of the genes were expressed or detected in the stroma or dermal layers and no HPV16 gene expression was detected in control biopsies from wild-type FVB/N mice.
CONCLUSIONS AND SIGNIFICANCE
HPVs are the most common sexually transmitted agents. Anogenital HPV infection contributes to the development of anogenital squamous cell cancers and their precursor lesions, high-grade squamous intraepithelial lesions. The most common HPV type in men and women is HPV16. We have shown in vitro evidence for the feasibility of an HPV-specific gene therapy approach that does not require an active immune response. This approach consists of transfection of a cytotoxic gene under transcriptional regulation of the HPV E2 protein and is able to kill HPV-infected cells (N. Sethi and J. Palefsky, Human Gene Therapy, 2003). Testing in animal models is an essential next step in preclinical development.
One transgenic mouse model of HPV16-associated neoplasia is the K14-HPV16 transgenic model. K14-HPV16 transgenic animals develop hyperplastic and/or dysplastic lesions in several inbred backgrounds. Malignant squamous cell carcinomas are typically observed on the epidermis of the ear, chest, and truncal skin.
The ability to investigate HPV gene expression profiles at different stages of precancerous disease and cancer progression is usually limited by the cellular heterogeneity of the lesions. In this study, we developed a specific, fast, and accurate method to characterize HPV gene expression in specific cell populations. We microdissected specific cell layers of the lesional epithelium using the PALM system based on laser microdissection and pressure catapulting technology. LCM was immediately followed by a 2-step, gene-specific RT-PCR and quantitation of cDNA using nested real-time PCR, methods specifically designed for quantification of low-abundance transcripts from as few as 50–100 cells. Using this fast method of gene-specific RT-PCR coupled with real-time nested PCR, we can determine the transcriptional profile of a number of viral and host genes simultaneously.
In this study we performed functional genomics in three distinct layers of the epithelium-stratum basale, stratum spinosum, and stratum corneum. Large amounts of E2 gene expression were detected in the basal, suprabasal, and stratum spinosum layers of hyperplastic and dysplastic lesions. E6 and E7 oncogenes were expressed at higher levels than E2 in all layers of the epithelium. Different spliced variants of E6 were detected, with E6* being the predominant form at all stages and E6** transcript detected at low levels. We noted that the E2 gene was expressed consistently at all ages. A higher level of expression was observed in basal layers of the early lesions compared with the stratum spinosum or older lesions. Levels of E6* and the E7 transcripts increased progressively with age, especially in the stratum spinosum. This led to an increase in the ratio of E6/E7 to E2 transcript levels with advancing age. This is similar to what is observed in human disease, although we did not observe a complete absence of E2 expression in advanced stages. The E6/E7 transcripts were constantly expressed by the K14 promoter in the basal layer, which is constantly dividing, leading to an accumulation of the oncogenes in the stratum spinosum.
In summary, we showed that K14-HPV16 transgenic mice expressed correctly spliced E2 transcripts and are suitable as a preclinical model to test our E2-regulated gene therapy strategy. If shown to be safe and efficacious in mice, this therapeutic approach may be valuable in treating HPV infection in the anogenital tract.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0946fje; doi: 10.1096/fj.03-0946fje
This article has been cited by other articles:
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