Inhibition of Serum- and Calcium-Induced Differentiation of Human Keratinocytes by HPV16 E6 Oncoprotein: Role of p53 Inactivation

Abstract

We have recently shown that human papillomavirus (HPV16) E6 oncoprotein exhibits two separate biological activities in genital keratinocytes (PHKs). E6 protein by itself is capable of inducing colonies of proliferating cells resistant to serum and calcium-induced differentiation, whereas both E6 and E7 are required for immortalization of PHK. Using epitope-tagged E6 carboxy-terminal truncation mutants, we mapped the domain between amino acid residues 132 and 141 as being essential for the induction of differentiation resistance (L. Sherman and R. Schlegel, J. Virol. 70, 3269-3279, 1996). To determine whether E6 protein's ability to alter PHK response to serum and calcium was associated with its ability to inactivate p53, we evaluated each of the above E6 mutants and three E6 natural variants in these respective assays. Our results demonstrate that the E6 region spanning residues 132-141 is required for p53 degradation and for abrogation of p53 transactivation, suggesting a possible correlation between E6 biological activity in altering differentiation and loss of p53 function. To evaluate whether selective inactivation of p53 is sufficient for altering the response of PHK to serum and calcium we investigated the capacity of plasmids encoding a dominant mutant human p53 and human MDM-2 to functionally substitute for E6 in colony formation in PHK. Plasmids were verified for their ability to inactivate wild-type p53 by testing their capacity to abrogate the p53 transactivation function. The results obtained showed that vectors encoding human MDM-2 and mutant p53, while active in inhibition of p53-dependent transactivation and capable of expressing stable proteins in PHK, failed to induce colonies of proliferating cells resistant to serum and calcium differentiation. These data argue that p53 inactivation may not be the sole E6 function required for altering the response of PHK to serum- and calcium-triggered differentiation.

The Human Papillomavirus E6 protein and its contribution to malignant progression

“…HPV-16 E6 impairs cell di erentiation in the ocular lens of transgenic mice (Pan and Griep, 1994) and causes expansion of the undi erentiated compartment of the epithelia in K14-E6 transgenic mice and, interestingly, this activity of E6 appears to be p53-independent (Song et al, 1999). Consistent with this, HPV-16 E6 was also found to increase the resistance of human keratinocytes to serum and calcium induced di erentiation through p53-independent pathways (Sherman et al, 1997;Sherman and Schlegel, 1996), although little is known about the molecular mechanisms by which E6 does this. However, HPV-16 E6 was reported to interact with E6BP/ ERC-55 (Chen et al, 1995) which is a putative calcium binding protein localized in the endoplasmic reticulum (Weis et al, 1994).…”

Differential regulation of human papillomavirus E6 by protein kinase A: conditional degradation of human discs large protein by oncogenic E6

“…The importance of a regulated, conditional function of the E6 C-terminus most likely re¯ects the requirement for a number of ordered di erent oncogenic functions to bring about progression to late malignant stages of cervical tumours. In the case of high-risk virus infections, the calcium and serum induced terminal di erentiation (which leads to keratinization and subsequent cell death) is inhibited, and the viral E6 protein has been shown to interfere with these steps (Sherman et al, 1997;Sherman and Schlegel, 1996). It is at this stage that the loss of Dlg function could be crucial for tumour formation.…”

The E6AP Ubiquitin Ligase Is Required for Transactivation of the hTERT Promoter by the Human Papillomavirus E6 Oncoprotein

“…Transgenic mice expressing HPV-16 E6 in the basal layer of the skin display cellular hyperproliferation and epidermal hyperplasia and develop ma-lignant skin cancers, but p53-null mice do not (38). E6 also confers resistance to serum-and calcium-induced differentiation of human foreskin keratinocytes (HFKs) (39), but the analysis of E6 mutants, Mdm-2 overexpression, and dominant negative p53 constructs indicates that p53 degradation is insufficient to induce these cellular alterations (40). Evidently, there are additional E6 targets that are critical for inducing abnormal cell proliferation, and it is presumed that some of these targets require E6-E6AP interactions.…”