Research at the Virology Division is primarily focused on the molecular mechanisms of oncogenesis by the human papillomavirus (HPV). As viral oncogenes disturb the normal cell cycle and its checkpoints and immortalize normal human cells, our studies also cover the DNA replication machinery in mammalian cells and stem cell biology.

In most cervical cancers, DNAs of the high-risk mucosotropic human papillomaviruses (HPVs), such as types 16 and 18, are maintained so as to express two viral proteins, E6 and E7, suggesting that they play important roles not only in carcinogenesis, but also in the maintenance of tumorigenic phenotypes ⁽⁶⁶⁾. The E6 protein has many cellular targets other than p53. The carboxy-terminal PDZ domain-binding motif of the E6 proteins is essential for transformation of rodent cells and induction of hyperplasia in E6-transgenic mouse skin, but not for degradation of p53. DLG4/PSD95, which is a human homologue of Dlg and contains three PDZ domains, was identified as a novel E6 target. DLG4 was expressed in normal human cells, including cervical keratinocytes, but only to a limited extent in cervical cancer cell lines. The tumorigenicity of CaSki cells was strongly inhibited by forced expression of DLG4, while their growth in culture was not inhibited at all. These results suggest that DLG4 may function as a tumor suppressor in the development of HPV-associated cancers ⁽⁶⁷⁾.

As moderate overexpression of ErbB2 is often observed in cervical cancers, the effects of E6 and E7 genes on ErbB2 expression were examined. In E6-expressing cells, HCK1T-E6, HCK1T-E6E7 and SiHa, ErbB2 expression levels increased with the cell density. Impaired contact inhibition and anchorage-independent growth of HCK1T-E6E7 were abrogated by the introduction of ErbB2-specific short hairpin RNA (shRNA) or the ErbB2-specific inhibitor, AG825. Increased ErbB2 expression in SiHa cells was diminished by the introduction of HPV16 E6- or E6AP-shRNA. At post-confluence cell densities, ErbB2 protein was stabilized in the presence of E6, whereas increased ErbB2 expression was not obvious with E6 mutants incapable of degrading p53. Furthermore, introduction of p53-shRNA into HCK1T resulted in increased ErbB2 protein stability, indicating possible ErbB2 regulation through p53. Finally, tumor formation of SiHa cells was almost abolished by the introduction of ErbB2-shRNA. Taken together, these data indicate an important role of ErbB2 regulation by HPV16 E6 in oncogenic transformation of human cervical keratinocytes ⁽⁶⁸⁾.

The E6 protein of HPV16 has been known to suppress keratinocyte differentiation through unidentified mechanisms. The Notch1 gene has been identified as a novel target of p53 and can be downregulated by E6 through p53 degradation in normal human epithelial cells. Inactivation of p53 by E6 or shRNA resulted in reduced Notch1 expression at the transcription level, and a p53-responsive element could be identified in the Notch1 promoter. As Notch1 is a determinant of keratinocyte differentiation and functions as a tumor suppressor in mammalian epidermis, the expression of E6, p53 shRNA or Notch1 shRNA suppressed both spontaneous keratinocyte differentiation in culture and the differentiation induced by DNA damage. Furthermore, the induction of Notch1 and differentiation markers as well as thickening of the epidermal layer upon UV irradiation was observed in wild-type, but not in p53-deficient mouse skin. These findings suggest a novel tumor suppressor mechanism of p53 in the development of squamous cell carcinomas, including HPV-induced tumors ⁽⁶⁹⁾.

Based on the notion that these viral proteins could be ideal molecular targets, an HLA-A24-restricted CTL (cytotoxic T lymphocyte) epitope from HPV16 E6 was identified ⁽⁷⁰⁾.

Cellular senescence was originally described as a phenomenon observed in cultured human cells. Accumulating lines of evidence now indicate that the same processes also take place in vivo, with important implications for tumour development ⁽⁷¹⁾. Many human cell types undergo senescence by activation of the p16^INK4a/pRb pathway, and introduction of Bmi-1 can inhibit p16^INK4a expression and extend the life span of human epithelial cells derived from the skin, mammary gland and lung. Introduction of p16^INK4a-specific short hairpin RNA, as well as that of Bmi-1, suppressed p16^INK4a expression in human mammary epithelial cells without promoter methylation, and extended their life span. Subsequent introduction of hTERT into cells with low p16^INK4a levels resulted in efficient immortalization of these cell types without crisis or growth arrest ⁽⁷²⁾. Such an approach has been attempted for immortalization of dental follicle cells, endometrial cells and nursing cells for primate ES cells ^(73-75), and in in-vitro carcinogenesis models ⁽⁷⁶⁾.

Genomic DNA has to be replicated completely and only once during the cell cycle. From late mitosis through the G1 phase, the MCM complex, a candidate replicative helicase, is loaded onto chromatin by ORC, CDC6 and Cdt1 proteins, and after the S phase, the actions of these MCM-loaders should be suppressed to prevent re-replication.

In human cells, Cdt1 is a very central player in such regulation and its deregulation leads to chromosomal instability. Novel human Cdt1- binding proteins, including subunits of the anaphase-promoting complex/cyclosome (APC /C), SNF2H and WSTF, topoisomerase I and IIa, and GRWD1/WDR28, were identified by a proteomics approach. Further analyses demonstrated that in addition to SCF^Skp2 and cullin4-based ubiquitin ligases, APC/C^Cdh1 is a third ubiquitin ligase that plays a crucial role in proteolytic regulation of Cdt1. Furthermore, chromatin-modifying proteins, SNF2H, topoisomerase I, and topoisomerase IIa, are associated with Cdt1 functions in loading the MCM. Cdt1 could become a molecular target for the development of new anticancer agents.

In a different screening, ORC was found to bind to TRF2, a telomere sequence-binding protein that protects telomeres. Interestingly, further analyses indicated that ORC is actually localized in telomeres and is involved in telomere homeostasis in human cells. Thus, to maintain chromosomal integrity, ORC functions in telomere homeostasis as well as in the regulation of DNA replication.