In the majority of cervical cancers, DNAs of high-risk mucosotpropic human papillomaviruses (HPVs), such as type 16, are maintained so as to express two viral proteins, E6 and E7, suggesting an essential importance to carcinogenesis. The high-risk HPV E6 proteins are known to inactivate p53 tumor suppressor protein but appear to have an additional, molecularly unknown function(s). In this study, we demonstrate that these E6 proteins can bind to the second PDZ domain of the human homologue of the Drosophila discs large tumor suppressor protein (hDLG) through their C-terminal XS/TXV/L (where X represents any amino acid, S/T serine or threonine, and V/L valine or leucine) motif. This finding is similar to the interaction between the adenomatous polyposis coli gene product and hDLG. E6 mutants losing the ability to bind to hDLG are no longer able to induce E6-dependent transformation of rodent cells. These results suggest an intriguing possibility that interaction between the E6 protein and hDLG or other PDZ domain-containing proteins could be an underlying mechanism in the development of HPV-associated cancers.

Histone H3 (H3) phosphorylation at Ser¹⁰ occurs during mitosis in eukaryotes and was recently shown to play an important role in chromosome condensation inTetrahymena. When producing monoclonal antibodies that recognize glial fibrillary acidic protein phosphorylation at Thr⁷, we obtained some monoclonal antibodies that cross-reacted with early mitotic chromosomes. They reacted with 15-kDa phosphoprotein specifically in mitotic cell lysate. With microsequencing, this phosphoprotein was proved to be H3. Mutational analysis revealed that they recognized H3 Ser²⁸phosphorylation. Then we produced a monoclonal antibody, HTA28, using a phosphopeptide corresponding to phosphorylated H3 Ser²⁸. This antibody specifically recognized the phosphorylation of H3 Ser²⁸ but not that of glial fibrillary acidic protein Thr⁷. Immunocytochemical studies with HTA28 revealed that Ser²⁸ phosphorylation occurred in chromosomes predominantly during early mitosis and coincided with the initiation of mitotic chromosome condensation. Biochemical analyses using³²P-labeled mitotic cells also confirmed that H3 is phosphorylated at Ser²⁸ during early mitosis. In addition, we found that H3 is phosphorylated at Ser²⁸ as well as Ser¹⁰ when premature chromosome condensation was induced in tsBN2 cells. These observations suggest that H3 phosphorylation at Ser²⁸, together with Ser¹⁰, is a conserved event and is likely to be involved in mitotic chromosome condensation.

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When exposed to genotoxic stress, eukaryotic cells demonstrate a DNA damage response with delay or arrest of cell-cycle progression, providing time for DNA repair. Induction of the Epstein-Barr virus (EBV) lytic program elicited a cellular DNA damage response, with activation of the ataxia telangiectasia-mutated (ATM) signal transduction pathway. Activation of the ATM-Rad3-related (ATR) replication checkpoint pathway, in contrast, was minimal. The DNA damage sensor Mre11-Rad50-Nbs1 (MRN) complex and phosphorylated ATM were recruited and retained in viral replication compartments, recognizing newly synthesized viral DNAs as abnormal DNA structures. Phosphorylated p53 also became concentrated in replication compartments and physically interacted with viral BZLF1 protein. Despite the activation of ATM checkpoint signaling, p53-downstream signaling was blocked, with rather high S-phase CDK activity associated with progression of lytic infection. Therefore, although host cells activate ATM checkpoint signaling with response to the lytic viral DNA synthesis, the virus can skillfully evade this host checkpoint security system and actively promote an S-phase-like environment advantageous for viral lytic replication.

The E6 protein of cervical cancer-associated human papillomaviruses (HPVs) is known to suppress keratinocyte differentiation through unidentified mechanisms. Notch1 is a determinant of keratinocyte differentiation and functions as a tumor suppressor in mammalian epidermis. Here, we report that the Notch1 gene is a novel target of p53 and can be down-regulated by E6 through p53 degradation in normal human epithelial cells. Thus, inactivation of p53 by E6 or short-hairpin RNA (shRNA) resulted in reduced Notch1 expression at the transcription level, and a p53-responsive element could be identified in the Notch1 promoter. The expression of E6, p53 shRNA, or Notch1 shRNA suppressed both spontaneous keratinocyte differentiation in culture and its induction upon DNA damage. Furthermore, the induction of Notch1 and differentiation makers as well as thickening of the epidermal layer upon UV irradiation was observed in wild-type but not in p53-deficient mouse skin. Together, our findings not only demonstrate a novel link between p53 and Notch1 in keratinocyte differentiation upon genotoxic stress but also suggest a novel tumor suppressor mechanism of p53 in the development of squamous cell carcinomas, including HPV-induced tumors.