Though HPV has been established as the etiologic agent for virtually all cervical cancers, the majority of cervical HPV infections do not lead to cervical cancer. Only 1% of cases progress to high grade dysplasia and cancer. The majority of cervical HPV-infections are transient non-neoplastic productive viral infections which disappear within a year. It is generally accepted that persistence of high risk HPV infection is an imperative state in the development of cervical neoplasia. The crucial factors determining persistence of infection and its correlation with an increased risk for cervical cancer in women with normal immune status are still largely unknown. These factors in women with compromised immunity such as in HIV/AIDS is also unknown, but there is an increased prevalence of cervical cancer in the HIV population. Persistent infection may only occur upon targeted infection of specific cervical cells, possibly with stem cell properties. In order to test this idea, we utilize fresh human tissue cervical epithelial tissue obtained from the Cooperative Human Tissue Network. Our laboratory successfully developed a protocol to isolate cervical epithelial cells and to identify a phenotypically distinct subpopulation with functional properties expected for stem cells. The preliminary data show that this cell fraction, originating from the basal layer of the ectocervical epithelium possesses an 10 fold increased capacity to bind papillomavirus-like particles (VLP), thus implying that these cells may express an increased number of papillomavirus binding sites facilitating viral infection if targeted by the virus. In an effort to optimize an environment that recapitulates the conditions under which stem cells function in situ, we have also developed an ectocervical organ culture model that allows us to establish and monitor HPV pseudovirus binding and infection in fresh cervical tissue. Cervical tissue maintained in culture undergoes rapid cell dissociation and exfoliation of the more differentiated, apical epithelial layers. In contrast, the basal cells remain attached to the basement membrane, where presumed epithelial stem cells reside. We have observed that only a very limited number of cells within the basal layer remains infected over time by pseudovirus using red fluorescent protein as a reporter gene. It is our plan to characterize the infected cells using FACs analysis to determine whether they are indeed cervical stem cells. We plan to use a furin cleaved pseudovirus developed in John Schiller's lab at NCI to increase the efficiency of infection. It was previously observed that the formerly quiescent basal cells could become proliferative and regenerate the entire epithelium under such whole tissue mount conditions, preserving the tissue architecture and cellular heterogeneity normally observed in vivo. It is postulated that when HPV infection occurred in vivo following a mechanical trauma to epithelial layer exposing the basal membrane, the resulting regeneration of apical layers could ultimately spark neoplasia. The conditions that lead to such development are currently under optimization and upon implementing, will further mimic in vivo HPV infection and assist our understanding of cervical carcinogenesis. We plan to further characterize the cervical stem cell population by searching for new markers using a global genetic approach which will improve stem cell isolation and to investigate the role of these cells in progression of a productive viral infection to virus induced pre-malignant lesions and cervical cancer. This study aims to improve our understanding of HPV induced cervical carcinogenesis and contribute to the development of novel strategies designed to target cervical cancer.
