Date of Award


Document Type

Open Access Thesis


Biomedical Science

First Advisor

Lucia Pirisi-Creek


Human Papillomavirus (HPV) is a causative factor in variety of diseases ranging from simple pathologic conditions to malignant ones like cervical cancer in which HPV is the primary cause in more than 90% of cases. HPV is also associated with other tumors like head and neck squamous cell carcinoma (HNSCC) and the percentage of association varies according to the anatomical site, reaching its highest level (65%) in the oropharyngeal region. HPV proteins E6 and E7 are responsible for the transforming potential of the virus. The principal action of E6 is targeting the tumor suppressor protein p53 for degradation, while E7 inactivates the Retinoblastoma protein (Rb). It is widely believed that E6/E7 expression in HPV-positive cancers is necessary to maintain the cancer cell phenotype, and this appears to be the case in most cervical cancers, in which HPV sequences are present and expressed. However, in HPV-positive HNSCC, HPV E6/E7 may be actively expressed (HPV-active), or not (HPV-inactive). Interestingly, the gene expression profile of HPV-inactive tumor has no 'HPV signature' and at the same time it is similar to but distinct from that of HPV-negative tumors. Further, HPV positive primary tumors tend to be HPV-active while recurring HPV positive tumors usually are HPV-inactive.

Based on the previous information, we developed the hypothesis that HPV-positive inactive tumors arise as HPV-active lesions. We postulate that the virus acts as initiator inducing the early transformation changes. HPV-transformed cells may then progress to malignancy in the presence of cancer promoters like smoking and alcohol, in an HPV oncoprotein-independent pathway, giving rise to HPV-positive inactive tumors. Based upon what is known about the transforming activities of HPV E6/E7, we made the educated guess that HPV-positive tumors may progress to become inactive if p53 becomes mutated: p53 mutations are very common in HPV-negative tumors, but comparatively rare in HPV positive cancers, where inactivation of p53 is accomplished by E6.

To begin investigating this hypothesis, it was important to further investigate the behavior of p53 in normal and HPV16-immortalized cells. A previous MS student in the laboratory established that p53 levels rise in response to UV treatment not only in normal human keratinocytes (HKc) but also in HKc immortalized by HPV16 (HKc/HPV16) despite the fact that in the immortalized cells both baseline and UV-induced p53 levels are much lower than in normal HKc controls.

In this work, we found that the rise in p53 level in response to UV-induced DNA damage varies in normal HKc from different individuals in terms of both magnitude and duration. The magnitude of the response could be classified as low, moderate, and high (1, 2-3 and 5-6) folds, respectively. In addition, some samples p53 protein levels peaked at 24 h to then decrease, while others kept increasing at 48 h from ultraviolet exposure. In addition, RNA levels of p53 are induced in UV-treated cells. qRT-PCR results have demonstrated a higher p53 mRNA level in most UV-treated samples which suggests that the increase in p53 protein level is due in part to increased p53 gene expression. Morphologically, there were no noticeable differentiating features that allowed for prediction of the cells' p53 behavior from microscopic examination. Also, it was demonstrated that even if p53 expression was suppressed at mRNA level by an shRNA against p53, the level of p53 protein and mRNA increase in response to UV-induced DNA damage.

These results point to the fact that in HPV-transformed cells, the loss of p53 is not absolute and that p53 responses are still present, although severely reduced in magnitude.