Human Papillomavirus Type 16 E6 Promotes Retinoblastoma Protein Phosphorylation and Cell Cycle Progression

Abstract

We show that E6 proteins from benign human papillomavirus type 1 (HPV1) and oncogenic HPV16 have the ability to alter the regulation of the G 1 /S transition of the cell cycle in primary human fibroblasts. Overexpression of both viral proteins induces cellular proliferation, retinoblastoma (pRb) phosphorylation, and accumulation of products of genes that are negatively regulated by pRb, such as p16INK4a , CDC2, E2F-1, and cyclin A. Hyperphosphorylated forms of pRb are present in E6-expressing cells even in the presence of ectopic levels of p16INK4a . The E6 proteins strongly increased the cyclin A/cyclin-dependent kinase 2 (CDK2) activity, which is involved in pRb phosphorylation. In addition, mRNA and protein levels of the CDK2 inhibitor p21 WAF1/CIP1 were strongly down-regulated in cells expressing E6 proteins. The down-regulation of the p21 WAF1/CIP1 gene appears to be independent of p53 inactivation, since HPV1 E6 and an HPV16 E6 mutant unable to target p53 were fully competent in decreasing p21 WAF1/CIP1 levels. E6 from HPV1 and HPV16 also enabled cells to overcome the G 1 arrest imposed by oncogenic ras. Immunofluorescence staining of cells coexpressing ras and E6 from either HPV16 or HPV1 revealed that antiproliferative (p16 INK4a ) and proliferative (Ki67) markers were coexpressed in the same cells. Together, these data underline a novel activity of E6 that is not mediated by inactivation of p53.Human papillomaviruses (HPVs) infect keratinocytes in the basal layer of stratified epithelia at a variety of anatomical sites (26). On the basis of their tissue tropism, the HPVs can be subdivided into cutaneous and mucosal types, which infect the skin and the mucosae, respectively. All HPV types are able to induce hyperproliferative benign lesions. Indeed, HPV-induced proliferation is an absolute requirement for completion of the viral life cycle. In addition, certain mucosal types, termed high risk, can promote transformation of a benign lesion into a malignant one (26). HPV16 is the high-risk type most frequently found in premalignant and malignant cervical lesions worldwide (2). Several studies have demonstrated that two viral early proteins, E6 and E7, play a key role in the induction of benign and malignant lesions (23) by associating with several cellular factors and altering their function (11, 13). The best-characterized property of HPV16 E6 is its binding to the p53 tumor suppressor, leading to degradation of the cellular protein via the ubiquitin pathway (20). The role of p53 is to safeguard the integrity of the genome by inducing cell cycle arrest or apoptosis in the presence of DNA damage. Therefore, its inactivation by the E6 protein leads to chromosomal instability and increases the probability of an HPV-infected cell evolving towards malignancy. Several studies have shown that HPV16 E6 is able to associate with other cellular proteins (11), indicating its involvement in additional pathways. Experiments with transgenic mice have shown that E6 can induce epithelial hyperplasia (14,19). It has recen...

Human Papillomavirus 16/18 E6 Oncoprotein Is Expressed in Lung Cancer and Related with p53 Inactivation

“…Malanchi et al (12,26) reported that HPV16 E6 could induce cellular proliferation, pRb phosphorylation, and accumulation of gene products that are negatively regulated by pRb, such as p16, cdc2, E2F-1, and cyclin A. Consistent with the hyperphosphorylated state of pRb, cyclin A/cdk2 activity is highly elevated in cells expressing E6 from either HPV16 or HPV18.…”

Reduced p21WAF1/CIP1 via Alteration of p53-DDX3 Pathway Is Associated with Poor Relapse-Free Survival in Early-Stage Human Papillomavirus–Associated Lung Cancer

“…The cyclin-dependent kinase (CDK) inhibitor p21 WAF1/CIP1 (p21) is the target of the HPV E6 oncoprotein (6,7). Induction of p21 by p53-dependent (8) or p53-independent pathways (9, 10) results in the inhibition of cyclin/CDK complexes that regulate cellular proliferation (11,12).…”

Effect of human papillomavirus on cell cycle-related proteins p16INK4A, p21waf1/cip1, p53 and cyclin D1 in sinonasal inverted papilloma and laryngeal carcinoma. An <i>in situ</i> hybridization study

“…For instance, Katori et al [27] described a significant decrease of immunoexpression of Moreover, Malanchi et al [36,37] clearly demonstrated that HPV E6 may strongly down-regulate p21waf1/cip1. In contrast to our results, Altavilla et al [26] showed stronger p21waf1/cip1 immunoexpression in HPV-positive sinonasal inverted papillomas, and Hafkamp et al [38] demonstrated that HPV positivity was strongly correlated with p21waf1/ /cip1 overexpression in tonsillar squamous cell carcinomas.…”

“…The literature data suggests that overexpression of p16INK4a and cyclin D1 is strongly related to the presence of HPV16/18. According to Malanchi et al [36,37], HPV16 E6 could induce accumulation of p16 and cellular proliferation. König et al [41] demonstrated a statistically significant relationship between the presence of HPV16/18 DNA and increased immunoexpression of p16INK4a in head and neck squamous cell cancers, but the HPV 6/11 presence was also significantly correlated with p16INK4a immunoreactivity.…”