Progressive squamous epithelial neoplasia in K14-human papillomavirus type 16 transgenic mice.

To model human papillomavirus-induced neoplastic progression, expression of the early region of human papillomavirus type 16 (HPV16) was targeted to the basal cells of the squamous epithelium in transgenic mice, using a human keratin 14 (K14) enhancer/promoter. Twenty-one transgenic founder mice were produced, and eight lines carrying either wild-type or mutant HPV16 early regions that did not express the E1 or E2 genes were established. As is characteristic of human cancers, the E6 and E7 genes remained intact in these mutants. The absence of E1 or E2 function did not influence the severity of the phenotype that eventually developed in the transgenic mice. Hyperplasia, papillomatosis, and dysplasia appeared at multiple epidermal and squamous mucosal sites, including ear and truncal skin, face, snout and eyelids, and anus. The ears were the most consistently affected site, with pathology being present in all lines with 100% penetrance. This phenotype also progressed through discernible stages. An initial mild hyperplasia was followed by hyperplasia, which further progressed to dysplasia and papillomatosis. During histopathological progression, there was an incremental increase in cellular DNA synthesis, determined by 5-bromo-2'-deoxyuridine incorporation, and a profound perturbation in keratinocyte terminal differentiation, as revealed by immunohistochemistry to K5, K14, and K10 and filaggrin. These K14-HPV16 transgenic mice present an opportunity to study the role of the HPV16 oncogenes in the neoplastic progression of squamous epithelium and provide a model with which to identify genetic and epigenetic factors necessary for carcinogenesis.     

Squamous epithelial hyperplasia and carcinoma in mice transgenic for the human papillomavirus type 16 E7 oncogene.

The human papillomavirus type 16 (HPV-16) genome is commonly present in human cervical carcinoma, in which a subset of the viral genes, E6 and E7, are expressed. The HPV-16 E6 and E7 gene products can associated with and inactivate the tumor suppressor proteins p53 and Rb (the retinoblastoma susceptibility gene product), and in tissue culture cells, these viral genes display oncogenic properties. These findings have led to the hypothesis that E6 and E7 contribute to cervical carcinogenesis. This hypothesis has recently been tested by using transgenic mice as an animal model. HPV-16 E6 and E7 together were found to induce cancers in multiple tissues in which they were expressed, including squamous cell carcinoma, the cancer type most commonly associated with HPV-16 in the human cervix. We have extended these studies to investigate the in vivo activities of HPV-16 E7 when expressed in squamous epithelia of transgenic mice. Grossly, E7 transgenic mice had multiple phenotypes, including wrinkled skin that was apparent prior to the appearance of hair on neonates, thickened ears, and loss of hair in adults. In lines of mice expressing higher levels of E7, we observed stunted growth and mortality at an early age, potentially caused by an incapacity to feed. Histological analysis demonstrated that E7 causes epidermal hyperplasia in multiple transgenic lineages with high penetrance. This epithelial hyperplasia was characterized by an expansion of the proliferating compartment and an expansion of the keratin 10-positive layer of cells and was associated with hyperkeratosis. Hyperplasia was found at multiple sites in the animals in addition to the skin, including the mouth palate, esophagus, forestomach, and exocervix. In multiple transgenic lineages, adult animals developed skin tumors late in life with low penetrance. These tumors arose from the squamous epithelia and from sebaceous glands and were characterized histologically to be highly differentiated, locally invasive, and aggressive in their growth properties. On the basis of these phenotypes, we conclude that HPV-16 E7 can alter epithelial cell growth parameters sufficiently to potentiate tumorigenesis in mice.     

Tumorigenicity by human papillomavirus type 16 E6 and E7 in transgenic mice correlates with alterations in epithelial cell growth and differentiation.

The human papillomavirus type 16 (HPV-16) E6 and E7 oncogenes are thought to play a role in the development of most human cervical cancers. These E6 and E7 oncoproteins affect cell growth control at least in part through their association with and inactivation of the cellular tumor suppressor gene products, p53 and Rb. To study the biological activities of the HPV-16 E6 and E7 genes in epithelial cells in vivo, transgenic mice were generated in which expression of E6 and E7 was targeted to the ocular lens. Expression of the transgenes correlated with bilateral microphthalmia and cataracts (100% penetrance) resulting from an efficient impairment of lens fiber cell differentiation and coincident induction of cell proliferation. Lens tumors formed in 40% of adult mice from the mouse lineage with the highest level of E6 and E7 expression. Additionally, when lens cells from neonatal transgenic animals were placed in tissue culture, immortalized cell populations grew out and acquired a tumorigenic phenotype with continuous passage. These observations indicate that genetic changes in addition to the transgenes are likely necessary for tumor formation. These transgenic mice and cell lines provide the basis for further studies into the mechanism of action of E6 and E7 in eliciting the observed pathology and into the genetic alterations required for HPV-16-associated tumor progression.     

Expression of human papillomavirus type 16 L1 protein in transgenic tobacco plants

To develop a plant expression system for the production of the human papillomavirus type 16 (HPV16) vaccine, we investigated whether the HPV16 L1 protein can be expressed in tobacco plants and whether it can be used as the cheapest form of edible vaccine. The HPV16 L1 coding sequence was amplified by PCR using specific primers from the plasmid pGEM-T-HPV16 containing the template sequence, and subcloned into the intermediate vector pUCmT and binary vector pBI121 consecutively to obtain the plant expression plasmid pBI-L1. The T-DNA regions of the pBI-L1 binary vector contained the constitutive Cauliflower mosaic virus (CaMV) 35S promoter and the neomycin phosphotransferase npt Ⅱ gene, which allowed the selection of transformed plants using kanamycin. The tobacco plants were transformed by cocultivating them, using the leaf disc method, with Agrobacterium tumefaciens LBA4404, which harbored the plant expression plasmid. The regenerated transgenic tobacco plants were selected using kanamycin, and confirmed by PCR. The results of the Southern blot assay also showed that the HPV16 L1 gene was integrated stably into the genome of the transformed tobacco plants. The Western blot analysis showed that the transformed tobacco leaves could express the HPV 16 L1 protein. Furthermore, it was demonstrated by ELISA assay that the expressed protein accounted for 0.034%-0.076% of the total soluble leaf protein, was able to form 55nm virus-like particles compatible with HPV virus-like particle (VLP), and induced mouse erythrocyte hemagglutination in vitro. The present results indicate that the HPV 16 L1 protein can be expressed in transgenic tobacco plants and the expressed protein possesses the natural features of the HPV16 L1 protein, implying that the HPV16 L1 transgenic plants can be potentially used as an edible vaccine.     

Production of human papillomavirus type 16 virions in a keratinocyte cell line.

Human papillomavirus type 16 (HPV-16) is strongly associated with carcinoma of the cervix, but the complete life cycle of the virus cannot be studied because no experimental system is available in which HPV-16 progeny are produced, and there is currently no source of HPV-16 virus particles. Most cell lines that harbor HPV-16 DNA contain the viral genome as integrated or concatenated DNA in which open reading frames are disrupted or deleted, but a human cervical keratinocyte cell line has been described which maintains HPV-16 DNA in monomeric episomal form (M.A. Stanley, H.M. Brown, M.W. Appleby, and A.C. Minson, Int. J. Cancer 43:672-676, 1989). This cell line was induced to form a stratified differentiating epithelium by grafting onto nude mice. Long-term grafts displayed the histological features of a low-grade cervical dysplasia, and terminally differentiated cells contained amplified levels of HPV-16 DNA, virus capsid antigen, and virus particles. This experimental system appears to permit the completion of the HPV-16 life cycle in virus-containing keratinocytes.     

人乳头瘤病毒16型L1蛋白的克隆及表达

采用PCR技术从宫颈癌组织中扩增人乳头瘤病毒16型(Human papillomavirus type16,HPV16)L1全长基因片段,目的片段克隆到pMD18T载体后经酶切鉴定及测序确认。构建重组原核表达质粒pGEX4T1-L1,转化大肠杆菌E.coliBL21,IPTG诱导表达出以非可溶性蛋白形式存在的表达蛋白,该重组蛋白的表达量占菌体总蛋白的17%,免疫印迹检测表明,表达蛋白与宫颈癌病人血清出现特异性反应。成功构建了重组原核表达质粒pGEX4T1-L1,并且在原核细胞中得到表达,为进一步研究L1蛋白的免疫学活性及疫苗的研发奠定了基础。     

Production of human papillomavirus type 16 early proteins in Bac-To-Bac Expression System (GibcoBRL)

Human papillomavirus type 16 (HPV16) is a major agent in cervical cancer etiology. Its early proteins are responsible for virus persistence, replication and initiation of neoplastic disease. In the present study we describe a use of baculovirus-insect cell expression system for production and study of HPV16 E2 and E4 proteins. The E2 protein binds specifically to viral DNA and E4 protein shows characteristic cytopathic effects on cells.     

Increased incidence of squamous cell carcinomas in Mastomys natalensis papillomavirus E6 transgenic mice during two-stage skin carcinogenesis

Papillomaviruses cause certain forms of human cancers, most notably carcinomas of the uterine cervix. In contrast to the well-established involvement of papillomavirus infection in the etiology of cervical carcinomas and in carcinomas of a rare hereditary condition, epidermodysplasia verruciformis, a causative role for cutaneous human papillomavirus types in the development of nonmelanoma skin cancer has not been proven. In order to better understand the functions of individual genes of cutaneous papillomavirus types, we generated transgenic mice carrying oncogene E6 of the Mastomys natalensis papillomavirus (MnPV), which causes keratoacanthomas of the skin in its natural host. In the present study, we demonstrate that under conditions of experimental two-stage skin carcinogenesis, fast-paced squamous cell carcinomas develop in nearly 100% of MnPV E6 transgenic mice in comparison to 10% in their nontransgenic littermates (log rank test; P < 0.0001). Therefore, we conclude that the MnPV E6 transgene favors the malignant progression of chemically induced tumors. Whereas an activating H-ras mutation is a consistent feature in benign and malignant tumors in wild-type mice, the majority of papillomas and keratoacanthomas and all squamous cell carcinomas obtained in MnPV E6 transgenic mice contain nonmutated ras alleles. These results indicate that the development of squamous cell carcinomas in MnPV E6 transgenic mice does not depend on an activated H-ras oncogene.     

Identification of a transforming gene of human papillomavirus type 16.

Previously, we observed sequential two-step alteration, growth stimulation, and progression to a more malignant state in NIH 3T3 cells transfected by human papillomavirus type 16 (HPV-16) DNA. In this study, we prepared a cDNA library from RNA extracted from cells transfected with the HPV-16 DNA and isolated cDNA clones which had growth-stimulating activity. Analysis of these cDNA clones indicated that the E7 open reading frame alone is responsible for inducing both steps of this cell transformation.     

Mutational analysis of human papillomavirus type 16 E7 functions.

The human papillomavirus type 16 E7 gene encodes a nuclear oncoprotein (98 amino acids [AAs] long) consisting of three regions: regions 1 (AAs 1 to 20) and 2 (AAs 21 to 40), which show high homology to the sequences of conserved domains 1 and 2, respectively, of adenovirus E1A; and region 3 (AAs 41 to 98) containing two metal-binding motifs Cys-X-X-Cys (AAs 58 and 91 to 94). We constructed AA deletion (substitution) mutants and single-AA substitution mutants of E7 placed under the control of the simian virus 40 promoter and examined their biological functions. Stable expression of E7 protein in monkey COS-1 cells required almost the entire length of E7 and was markedly lowered by the mutations in region 3. Transactivation of the adenovirus E2 promoter in monkey CV-1 cells was lowered by the mutations. It was abolished by changing Cys-24 to Gly and markedly decreased by a mutation at His-2 or at the metal-binding motifs in region 3. Focal transformation of rat 3Y1 cells by E7 was eliminated by changing His-2 to Asp or Cys-24 to Gly and was greatly impaired by changing Cys-61 or Cys-94 to Gly. The transforming function survived mutations at Leu-13 and Cys-68 and deletion of Asp-Ser-Ser (AAs 30 to 32). The data suggest that regions 1 to 3 are required for its functions and that the meta-binding motifs in region 3 are required to maintain a stable or functional structure of the E7 protein.     

Characterization of a human papillomavirus type 16 variant-dependent neutralizing epitope.

We have determined that three type-specific and conformationally dependent monoclonal antibodies, H16.E70, H16.U4, and H16.V5, neutralize pseudotype human papillomavirus type 16 (HPV16) virions in vitro. H16.U4 and H16.V5 neutralized pseudotype virions derived from the German HPV16 variant 114K and the Zairian variant Z-1194 with equal efficiency. In contrast, neutralization of Z-1194 pseudotype virions by H16.E70 was two orders of magnitude weaker than neutralization of 114K pseudotype virions. This difference correlated with enzyme-linked immunosorbent assay reactivity of H16.E70 to L1 virus-like particles of the two variants. A substitution at residue 282 of L1 was responsible for this differential reactivity, suggesting that this residue constitutes part of the H16.E70 epitope.     

alpha(6) Integrin is the main receptor of human papillomavirus type 16 VLP

The present study was performed to determine the specific receptor of type HPV-16 using recombinant human papillomavirus-like particle (HPV-16 L1-VLP). The expression levels of alpha(6), beta(1), and beta(4) integrins were determined and compared with the amount of HPV-VLP binding in ten cell lines by flow cytometry. Our results show that the amount of VLP binding and the expression level of alpha(6) integrin are correlated, which was confirmed by an inhibition experiment using antibodies and by immunocytochemistry. Both the expression level of alpha(6) integrin and the amount of HPV-VLP binding were high in cervical cancer cell lines, as the type HPV-16 is the main cause of cervical cancer. The degree of binding of HPV-VLP matched the alpha(6) integrin expression level in cell lines but was not correlated with beta(1) and beta(4) levels, which suggests that alpha(6) integrin is the main receptor of HPV type 16.     

Induction of an antibody response in mice against human papillomavirus (HPV) type 16 after immunization with HPV recombinant Salmonella strains

 Human papillomaviruses (HPV) are present in approximately 95% of all cervical carcinomas and the HPV E6 and E7 genes are continuously expressed in these lesions. There is also circumstantial evidence that often natural immunity against HPV is generated and that this is of influence on HPV-induced lesions. Stimulation of the immune system by proper presentation of relevant HPV antigens might, therefore, lead to a prophylactic or therapeutic immunological intervention for HPV-induced lesions. For this purpose we have expressed the E6 and E7 protein of HPV 16 in an attenuated strain of Salmonella typhimurium (SL3261, aroA mutation), which has been used extensively as a live vector. Live recombinant Salmonella vaccines have the ability to elicit humoral, secretory and cell-mediated immune responses, including cytotoxic T cells, against the heterologous antigens they express. This report describes the construction of recombinant Salmonella strains expressing the HPV 16 E6 and E7 proteins, and the induction of an HPV-16-specific immune response in mice after immunization with these live vectors. Received: 25 June 1996 / Accepted: 6 August 1996     

Immunological ignorance of an E7-encoded cytolytic T-lymphocyte epitope in transgenic mice expressing the E7 and E6 oncogenes of human papillomavirus type 16.

Certain human papillomaviruses (HPV) have been implicated in the etiology of cervical malignancies, and the E7 and E6 gene products of HPV type 16 are frequently expressed in these lesions. However, cytolytic T-lymphocyte (CTL)-mediated responses to HPV are rarely detectable in patients with cervical cancer. To examine whether the T-cell response is deficient during the HPV-induced transformation, we produced lines of transgenic (Tg) mice that expressed the E6 and E7 oncogenes in keratinized epithelia. The mice developed severe hypertrophy of all keratinized epithelia, but no malignancies were observed. Although epithelial cells from Tg mice could present at least an E7-encoded CTL epitope (E7 49-57), CTLs from these mice were neither primed to nor made tolerant of this epitope. No quantitative or qualitative differences were seen in the CTL responses of the Tg mice compared to those of their littermates following immunization with the peptide E7 49-57. Immunization of Tg mice with the E7 49-57 peptide protected them against a subcutaneous challenge with tumor cells expressing a transfected E7 gene, yet the skin was unaffected, although the cultured skin epithelial cells from Tg mice expressed E7. Our results suggest that the Tg mice were immunologically ignorant of HPV oncoproteins with respect to a CTL response and that a similar type of ignorance may explain why HPV-associated cervical cancer cells can escape immunological destruction.     

High-frequency germ line gene conversion in transgenic mice.

Gene conversion is the nonreciprocal transfer of genetic information between two related genes or DNA sequences. It can influence the evolution of gene families, having the capacity to generate both diversity and homogeneity. The potential evolutionary significance of this process is directly related to its frequency in the germ line. While measurement of meiotic inter- and intrachromosomal gene conversion frequency is routine in fungal systems, it has hitherto been impractical in mammals. We have designed a system for identifying and quantitating germ line gene conversion in mice by analyzing transgenic male gametes for a contrived recombination event. Spermatids which undergo the designed intrachromosomal gene conversion produce functional beta-galactosidase (encoded by the lacZ gene), which is visualized by histochemical staining. We observed a high incidence of lacZ-positive spermatids (approximately 2%), which were produced by a combination of meiotic and mitotic conversion events. These results demonstrate that gene conversion in mice is an active recombinational process leading to nonparental gametic haplotypes. This high frequency of intrachromosomal gene conversion seems incompatible with the evolutionary divergence of newly duplicated genes. Hence, a process may exist to uncouple gene pairs from frequent conversion-mediated homogenization.