Skin hyperproliferation and susceptibility to chemical carcinogenesis in transgenic mice expressing E6 and E7 of human papillomavirus type 38 - PubMed (original) (raw)
Skin hyperproliferation and susceptibility to chemical carcinogenesis in transgenic mice expressing E6 and E7 of human papillomavirus type 38
Wen Dong et al. J Virol. 2005 Dec.
Abstract
The oncoproteins E6 and E7 of human papillomavirus type 38 (HPV38) display several transforming activities in vitro, including immortalization of primary human keratinocytes. To evaluate the oncogenic activities of the viral proteins in an in vivo model, we generated transgenic mice expressing HPV38 E6 and E7 under the control of the bovine homologue of the human keratin 10 (K10) promoter. Two distinct lines of HPV38 E6/E7-expressing transgenic mice that express the viral genes at different levels were obtained. In both lines, HPV38 E6 and E7 induced cellular proliferation, hyperplasia, and dysplasia in the epidermis. The rate of occurrence of these events was proportional to the levels of HPV38 E6 and E7 expression in the two transgenic lines. Exposure of the epidermis of nontransgenic mice to UV led to p21WAF1 accumulation and cell cycle arrest. In contrast, keratinocytes from transgenic mice continued to proliferate and were not positive for p21WAF1, indicating that cell cycle checkpoints are altered in keratinocytes expressing the viral genes. Although the HPV38 E6/E7-expressing transgenic mice did not develop spontaneous tumors during their life span, two-stage carcinogen treatment led to a high incidence of papillomas, keratoacanthomas, and squamous-cell carcinomas in HPV38 mice compared with nontransgenic animals. Together, these data show that HPV38 E6 and E7 display transforming properties in vivo, providing further support for the role of HPV38 in carcinogenesis.
Figures
FIG. 1.
HPV38 E6 and E7 expression in Tg mice. (A) Schematic representation of K10-HPV38 E6/E7 construct. (B) RT-PCR. Total RNA was extracted from skin of the dorsal region of 5- to 6-week-old mice. Semiquantitative PCR was performed on 1/5 serial dilutions of cDNA for HPV38 E7 by use of specific primers located in the 5′ and 3′ regions of the E7 sequence (lines 1 through 3). As a positive control, GAPDH was amplified. For a negative control, PCR was performed for each sample without reverse transcription (lane 4 of each panel). (C) HPV38 E6 and E7 are expressed in the epidermis of HPV38 E6/E7-Tg mice. Skin of the ear region of 5- to 6-week-old mice was snap frozen. In situ hybridization of sections of HPV38 E6/E7-Tg mice was performed using HPV38 E6/E7 antisense (upper panels) and sense (lower panels) probes. Magnifications of a representative dark field and the corresponding bright-field pictures of the ear section of an HPV38 E6/E7-Tg animal are shown (original magnifications were ×20 for dark field and ×40 for bright field).
FIG. 2.
Histopathological analysis of skin specimens of non-Tg and Tg mice. Paraffin-embedded sections of ear skin of non-Tg and Tg mice were prepared and stained with hematoxylin and eosin (HE). (A) Representative morphologically normal skin sections of non-Tg (FVB/N) and HPV38 E6/E7-Tg mice. (B) Representative hyperplastic (middle panel) and dysplastic (right panel) skin sections of HPV38 E6/E7-Tg mice are shown (original magnification, ×40). A section from a HPV16 E6/E7-Tg mouse was also included for comparison.
FIG. 3.
Analysis of skin from HPV38 E6/E7-Tg mice. (A) Immunostaining for proliferative markers in skin from non-Tg and Tg animals. Sections of ear skin from different mice as indicated in the figure were stained for BrdU (top panel [original magnification, ×20]) and Ki-67 (bottom panel [original magnification, ×40]). (B) Quantification of BrdU- and Ki-67-positive cells in non-Tg and Tg mouse epidermis. The percentages of BrdU- and Ki-67-positive cells in the epidermis were determined by counting 400 to 500 HE-stained cells under magnification (×40) in five to six different fields of epidermis. Differences between the BrdU- or Ki-67-positive cells in the HPV38 E6/E7-Tg mice (lines 2 and 6) and the FVB/N non-Tg mice were statistically significant (P < 0.001) as determined by Student's t test. (C) Expression of HPV38 E6 and E7 in epidermis. In situ hybridization was performed using HPV38 E6/E7 as a probe in antisense (upper panels) and sense (lower panels) orientation. Representative dark-field and bright-field pictures of a hyperproliferative patch of the ear of an HPV38 E6/E7-Tg mouse are shown (original magnifications were ×20 for dark field and ×40 for bright field). (D) BrdU- and Ki-67-positive cells are detected in the suprabasal layers of epidermis with increased thickness (original magnification, ×40).
FIG. 4.
Cellular proliferation in the epidermis of non-Tg and Tg mice after UVB irradiation. (A) BrdU and Ki-67 immunostaining of skin of mice exposed or non-exposed to UVB. Non-Tg and Tg animals were irradiated with 0.45 J/cm2 UVB and sacrificed 3 h later. One hour before sacrifice, BrdU was injected intraperitoneally. Proliferative cells were identified by immunohistochemical staining for BrdU (top panels [original magnification, ×20]) and Ki-67 (bottom panels [original magnification, ×40]). (B) Quantification of BrdU- and Ki-67-positive cells in skin of non-Tg and Tg mice before and after UVB irradiation. The percentage of BrdU- and Ki-67-positive cells in the epidermis was determined as described in the legend to Fig. 3B. The differences between the percentages of BrdU- or Ki-67-positive cells in the HPV38 E6/E7-Tg mice (lines 2 and 6) and the FVB/N non-Tg mice are statistically significant (P < 0.001) as determined by Student's t test.
FIG. 5.
p21WAF1 levels in the skin of non-Tg and HPV38 E6/E7-Tg mice after UVB irradiation. (A) p21WAF1 immunohistochemistry of skin of UV and non-UV irradiated mice. Skin sections were stained with antibodies against p21WAF1. Original magnification, ×40. (B) Quantification of p21WAF1-positive cells in skin from non-Tg and HPV38 E6/E7-Tg mice. The percentages of p21WAF1-positive cells in the epidermis are presented in the histogram. The percentages of BrdU- and Ki-67-positive cells in the epidermis were determined as described in the legend to Fig. 3B. The differences between the percentages of p21WAF1-positive cells in the HPV38 E6/E7-Tg animals versus the FVB/N non-Tg mice are statistically significant (P < 0.001) as determined by Student's t test. (C) Immunoblotting of Bak and p21WAF1 in skin protein extracts from non-Tg and HPV38 E6/E7-Tg animals exposed and not exposed to UV irradiation.
FIG. 6.
Tumor formation in non-Tg and HPV-Tg mice after DMPA/TPA treatment. (A) Schematic diagram of the experimental procedure of two-stage carcinogen treatment. (B) Representative pictures of non-Tg and HPV-Tg mice during DMPA/TPA treatment. Eight-week-old mice were treated with DMBA and TPA as described in Materials and Methods. Pictures of the treated area of the mice were taken at indicated times to follow the development of tumors in each group. (C) Numbers of animals with skin lesions in the group of non-Tg and HPV Tg animals. The number of animals that developed skin tumors was monitored each week until weeks 13 and 14. The difference between the curves of control and transgenic mice is statistically significant (P < 0.001, determined by log rank test for group data). (D) Average number of tumors in the groups of non-Tg and Tg mice. The number of papillomas was determined every three days. The average number of papillomas per mouse was calculated by dividing the total number of tumors in each group by the number of mice per group. Differences between the number of papillomas in the group of control and transgenic mice at days 70 and 94 are statistically significant (control versus HPV16, P < 0.00002; control versus HPV38 line 2, P < 0.0007; control versus HPV38 line 6, P < 0.0004; as determined by Student's t test). (E) Tumor development in non-Tg and Tg mice. Tumor size in mice of each group was determined at 9, 12, and 15 weeks after the beginning of DMBA/TPA treatment. Differences between the tumor sizes of non-Tg and HPV38 E6/E7-Tg or those of non-Tg and HPV16 E6/E7-expressing mice are statistically significant only at weeks 12 (P < 0.02) and 15 (P < 0.001) as determined by t test.
FIG. 7.
Histology of tumors of HPV38 E6/E7-Tg animals. Paraffin-embedded sections of tumors of HPV38 E6/E7-Tg mice were prepared and stained with HE. (A) Papilloma in HPV38 E6/E7-expressing (line 2) mouse at week 15 (original magnification, ×4) and (B) a magnified area, showing hyperkeratinization (open arrows) and hyperplasia of the squamous cell layer (closed arrows) (original magnification, ×10). (C) Representative keratoacanthoma in an HPV38 E6/E7-expressing mouse (line 2) at week 15 (original magnification, ×4) and (D) a magnified area (original magnification, ×10). Open and closed arrows indicate keratin pearls and dysplasia, respectively. (E) A SCC at week 20 observed in an HPV38 E6/E7-Tg mouse (line 6) (original magnification, ×10) (F) and a magnified area (original magnification, ×20) with invasion of the carcinoma into the underlying dermis; open arrows indicate smooth muscle tissue.
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