Functional Interactions Between Hpvs And Keratinocytes

As previously described, HPVs are small DNA tumor viruses that display strict epitheliotropism by infecting stratified squamous epithelia at different body sites, as reviewed previously (28,84). For stable viral maintenance to be achieved that would allow a productive viral infection to occur, the virus is presumed to infect cells of the basal layer. A candidate receptor for all HPV types, a-6 integrin, has recently been identified and is expressed on basal keratinocytes(85). However, given the association between specific viral types at particular body sites, it would appear that other factors peculiar to the host cell at that site, in addition to its epithelial origin, must also be important.

Viral Gene Regulation

A clue to the viral tropism is suggested by analysis of the different viral DNA sequences, in particular the upstream regulatory region. This region of the viral genome, which can be up to about 1 kb in length, does not code for any structural proteins, but instead contains binding sites for cellular transcription factors that are important in directing expression of the viral genes in both basal and suprabasal differentiated cell layers. Indeed virus replication is intimately linked to and dependent upon keratinocyte differentiation. Portions of the upstream regulatory region critical for epithelial specific gene expression are termed the core enhancer, usually containing binding sites for transcription factors such as AP1 and TEF-1 (86-92). It is most notable that divergent groups of HPVs have solved their individual requirements for host cell factors in different ways. Both the number and location of host transcription factor binding sites are markedly different between individual cutaneous or mucosal viruses, and individual viral types may also possess binding sites unique to that virus (93-95). In those types that are commonly found in epidermodysplasia verruciformis (EV HPV types) such as HPV-5, the upstream regulatory region contains two specific DNA motifs, termed M29 and M33, which are found only in these HPV types. In contrast, in anogential viruses such as HPV-16, this region contains elements that enable to virus to respond to glucocorticoids and progesterones (96,97). These are specific adaptations that may enable divergent viral types to survive in different epithelia.

Virally Encoded Oncogenes

Much of the recent work regarding the functions of virally encoded proteins has centered around anogenital HPV types most closely associated with the development of cervical carcinoma. In particular, much attention has focused on the E6 and E7 proteins of HPV-16 and -18 and the mechanisms by which they bring about cell transformation (98-104). In contrast, comparatively little is known about the functions of the equivalent proteins of cutaneous HPVs. Dissection of the HPV-16 viral genome revealed that both E6 and E7 were powerful oncogenes able to transform cells in culture. Subsequent functional studies showed that the E6 and E7 proteins inactivate two important tumor suppressor proteins, p53 and the retinoblastoma gene product, pRb, respectively (105-107). Studies on cutaneous HPV types have recapitulated these studies with differing results and have largely centered around HPV-5 and -8, which are associated with tumors in EV patients. Mutational studies revealed that the association of HPV-16 E7 with pRb hinged on the integrity of the LXCXE motif, and this motif also was required for the transforming activity of the protein (108). Although the majority of E7 proteins of cutaneous viruses contain this motif, many associate with pRb weakly if at all and have a low transforming potential. This association with pRb is not, however, indicative of a transforming potential of the protein, as the HPV-1 E7 protein tightly associates with pRb but fails to transform cells in culture (109). The HPV-10 E7 protein lacks this pRb binding motif entirely, suggesting that the virus uses other mechanisms to overcome this growth suppressive pathway. Furthermore, in contrast to the HPV-16 E7 protein, the E7 proteins of HPV types 8 and 47 fail to transform rodent cells (110,111). Yet, in cooperation with activated Ha-ras the HPV-5 and -8 E7 genes are able to give rise to transformed colonies, and the HPV-1 E7 gene can fully transform mouse C127 cells (112). The HPV-16 E7 protein has also been shown to associate with the p21 protein (113,114), a cyclin-dependent kinase inhibitor, whose expression is induced by p53 in response to DNA damage and in a p53-independent manner in keratinocyte differentiation. Cells expressing the HPV-16 E7 protein show altered differentiation and an inhibition of PCNA and cyclin A/E-associated kinase activity (113,114). There also is evidence that HPV-16 E6 and E7 proteins abrogate a mitotic spindle checkpoint through a p53-independent mechanism (115). Whether cutaneous E6 and E7 proteins share this ability is worthy of future investigation. Close to the pRb binding domain in the HPV-16 E7 protein is a motif that is phosphorylated by casein kinase II (CKII). Phosphorylation of E7 at this site increases the affinity of the protein for TBP, a protein component of the basal gene transcription machinery (116). Although most cutaneous virus E7 proteins lack this phosphorylation site, it appears to be conserved in HPV-77, a novel type isolated from warts and SCCs of immunocompromised individuals, suggesting a conservation of E7 function in this virus (117). It appears then that the degree of morphologic transformation induced by cutaneous E7 proteins correlates poorly with the risk of malignant conversion in vivo. In difference to the combined immortalizing capacity of the HPV-16 E6/E7 genes in human ker-atinocytes, such immortalization studies using cutaneous HPVs have been unsuccessful to date.

In contrast to the E6 gene of anogenital viruses, the E6 gene of EV HPV types 5 and 8 appears to encode the major transforming activity. In rodent cell lines, EV E6 proteins are able to induce both morphologic transformation and anchorage-independent growth which, unlike the E7 gene, is reflective of their association with carcinomas. The association of the HPV-16 E6 protein with p53 leads to the rapid degradation of the protein. This is mediated by the binding of E6 to a cellular protein, E6-AP, leading to the ubiquitination of p53 followed by its rapid degradation by the ubiquitin-depen-dent proteolysis system(11S). Although this function of E6 is believed to be important in cellular transformation, E6 also has other p53-independent transforming activities (119,120). This is highlighted by the cutaneous E6 proteins that function in transformation assays without associating with p53 or promoting its degradation (121). These combined observations point toward as yet unidentified cellular targets of the cutaneous E6 and E7 proteins. Preliminary evidence also points toward a transforming activity encoded by the HPV-8 E2 protein (110).

UV- and HPV-Associated Malignancy

In both EV and immunocompromised patients, HPV-containing tumors arise predominantly at body sites exposed to sunlight, indicating a fundamental role for UV as a cofactor in HPV-induced carcinogenesis, as well as its recognized role in skin cancer development in general (122). UV leads to a strong induction of p53 in the skin (123), playing an important role in protecting the integrity of the genome, either by inducing growth arrest allowing the repair of UV-induced DNA damage or by promoting apop-tosis (124). As noted previously, cutaneous E6 proteins fail to abrogate p53 function by degradation, yet their presence in lesions at UV-exposed sites implies that p53 responses to DNA damage have been overcome by other mechanisms. An anti-apop-totic activity of the E6 protein of anogenital HPV types has been noted (125), and such an activity encoded by cutaneous E6 may aid the survival of virally infected cells exposed to UV which in turn may facilitate the accumulation of UV-induced genetic changes.

Models of HPV Infection and Disease

The HPV viral life cycle in its natural host cell is intimately linked to and dependent upon the normal differentiation process. In basal layers, virus is maintained at low copy number in basal layers, which rises as vegetative viral DNA replication taking place in upper spinous layers. Concomitant with increased DNA replication, differential promoter usage coupled to the expression of viral genes has been detected in differentiating epidermis. The dependency of the virus upon host cell differentiation, coupled with the previous lack of an in vitro model of epidermal regeneration, has in the past proved a major obstacle in designing model systems to investigate HPV gene function under more physiologically relevant conditions. However, advances in keratinocyte biology coupled with the emergence of animal systems have gone a long way to fulfilling the necessary criteria. Much of the developmental work in the designing and testing of HPV model systems has been done using anogenital HPVs. The paucity of experimental data using cutaneous HPVs may stem from the lack of a keratinocyte immortalization assay.

Transformation of human skin was first demonstrated for HPV-11, a condylomata acuminata associated HPV type. HPV-11 viral particles were used to infect human skin that then was grafted beneath the renal capsule of athymic mice (126,127). This resulted in production of viral particles and the development of condyloma similar to those seen in patients (128). Such xenograft models were subsequently improved by the use of severe combined immunodeficiency (SCID) mice, producing larger xenografts that showed an increased rate of HPV-11 positivity (129). This work was extended to include engraftment of HPV-16-infected foreskin keratinocytes were grafted onto the skin of SCID mice. The grafted skin expressed involucrin in differentiating keratinocytes and displayed features of HPV infection including koilocytosis and production of capsid antigen (130). Direct grafting of HPV-6 or -11-containing lesions onto the skin of SCID mice also resulted in the formation of a macroscopic papillo-mata(131). The first experimental system permitting the completion of the HPV-16 life cycle was achieved by grafting an immortal HPV-16 cell line isolated from a low-grade lesion onto a vascularized granulation bed on the flanks of nude mice (132). Similar experiments using skin fragments from benign early premalignant EV lesions implanted under the kidney capsule of athymic mice led to the production of epidermal cysts displaying numerous mitoses and EV HPV DNA (133,134).

Organotypic Cell Culture Systems and Drug Effects In Vitro

Although the mouse model xenograft systems have proved useful in studying HPV infected lesions, they are technically difficult and time consuming. Changes in HPV

gene expression and induction of DNA replication can be induced by suspending keratinocytes harbouring episomal HPV DNA in semisolid medium (135). Programmed differentiation of keratinocytes in vitro can be achieved by the use of organotypic cell cultures (rafts) (136,137). In this system keratinocytes are seeded onto a dermal substitute and then raised to the air-liquid interface, allowing stratification and differentiation to occur. A variety of dermal substrates have been used, including collagen and deepidermalized human dermis. Grafting of HPV-16 immortalized keratinocytes in raft cultures leads to the reformation of epithelium; however, the epithelium exhibits parabasal crowding, enlarged nuclei in the upper layers, and features of a premalignant HPV-induced lesion (138). These features included abnormal differentiation, mitotic figures, and abnormal mitoses in upper cell layers (139,140). Biosynthesis of HPV-31 viral particles was first demonstrated by seeding onto collagen gels a cell line derived from a low-grade cervical intraepithelial neoplasia (CIN), that maintains episomal viral DNA (141,142). Such organotypic systems are most useful in studying the effects of viral genes on epithelial proliferation and differentiation, and the relative contribution of transcription factor binding sites in the upstream regulatory region to altered HPV gene expression in stratified epithelia (143). They can also be used to evaluate the effects of agents that may be important in modulating HPV gene function, such as tumor necrosis factor-a (TNF-a), interferon-y (IFN-y) (144), UV, hormones, and retinoids. Although the lack of suitable cell lines harboring cutaneous HPVs has prevented similar studies from being undertaken to date, high-efficiency gene transfer into keratinocytes can be achieved using retroviruses. Recent advances in retroviral design now allow a high transduction efficiency coupled with a sustained expression of transgenes in regenerated epithelium (145-147). The ability to use primary rather than immortalized cells for such assays has distinct advantages that will prove useful in studying HPV types with little or no inherent immortalizing potential.

Transgenic Animal Models

The use of transgenic mice is proving to be a powerful tool in studying tumor progression and dissecting the molecular events important in the multistage process of skin carcinogenesis. Although mouse skin has long been used to study tumor development and those changes important for the development of malignancy, the use of trans-genic animals offers the ability to examine in greater detail the consequences of expression of specific viral genes (for reviews see 148-150). Transgenic animals expressing one or more wild type or mutated HPV genes allows the contribution of that gene to be evaluated for its contribution towards papilloma or tumour development. For example, crossing of HPV transgenics with mice knockouts for p21 or p53 would allow the contribution of the E6 and E7 genes to perturbation of differentiation and normal cell cycle control to be tested and compared to organotypic cultures (113,151,152). HPV gene expression has been successfully targeted to the developing lens using the a-A crystallin promoter and to the epidermis using human keratin gene promoters such as K1 and K14 and bovine K10. K14-HPV-16 transgenic mice have been generated by a number of different groups (148,150,153,154). The mice show progressive squamous epithelial neoplasias that can arise at many different anatomical sites including ears, skin, anus, cervix, and vagina, perhaps modulated by autocrine factors such as TGF-a or hormones such as progesterone acting through regulatory ele ments in the upstream regulatory region (155-157). Mice transgenic for either E6 or E7 revealed the anti-apoptotic activity of the E6 protein in vivo and the proliferation-induced stimulation of apoptosis by E7 (158,159). Mutations in the E7 gene revealed the importance of specific regions, including the pRb binding domain, in the induction of epidermal hyperplasia (151). Karyotyping of primary tumors induced by bovine papillomavirus type 1 shows consistent changes on chromosomes 8 and 14 (160). This suggests that papillomavirus transgenics may be useful to study the roles of cytoge-netic changes in tumorigenesis and may also provide a model that will be helpful in evaluating potential anti-HPV agents (161). This approach will also be useful in presenting viral antigens to the immune system in a way that can be modeled to the natural infection. Such immunologic studies on HPV-16 E6/E7 transgenic mice allow immunologic responses including antibody production, induction of cytotoxic T lymphocytes against viral proteins and tolerance to be evaluated on different MCH genetic backgrounds.

THE ROLE OF HPV IN NMSC AND ORAL CANCER: CONCLUSIONS

The role of HPV in the development of skin cancer is still not clear. The viral epidemiology suggests that there is a large reservoir of latent HPV infection in the normal skin of the general population and in immunocompromised individuals. Preliminary evidence suggests that increased exposure, as in the transplant population, is associated with an increased risk of skin cancer but this needs to be confirmed in prospective studies. Malignant skin lesions in both immunocompetent and immunosuppressed patients frequently contain HPV DNA, and it is tempting to ascribe them a functional role. However, no particular HPV type predominates and it remains a possibility that HPV is merely a "passenger," present but not active in the development of skin cancer. The technology is now in place to address this, to determine the molecular epidemiology of HPV infection in keratinizing epithelia and to examine its role in skin carcinogenesis.

Similarly, because the association of HPV in oral squamous cell carcinoma is anecdotal at present, well designed, adequately controlled molecular epidemiologic studies are required to what if any role HPVs play. The epidemiologic association of HPV with OSCC and in vitro evidence of oral keratinocyte immortalization combined with growing knowledge of HPV oncogenes suggests a possible role for them in the etiology of OSCC. This putative role is not as clearcut as HPV's role appears to be in cervical carcinogenesis.

Functional studies of interactions between HPV oncogenes and keratinocytes do show that the EV HPVs transforming activity does not lie in the association of EV-HPV E6 with p53. Elucidation of the cellular targets of EV-HPVs will further add to understanding the role of these viruses in keratinocyte-derived cancers, as well as potential strategies for their eradication or prevention.

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