Adeno-Associated Virus Type 2 Increases Proteosome-Dependent Degradation of p21^WAF1in a Human Papillomavirus Type 31b-Positive Cervical Carcinoma Line

Abstract

Adeno-associated virus type 2 (AAV2) seropositivity is negatively correlated with the development of human papillomavirus (HPV)-associated cervical cancer. We have begun analysis of the molecular mechanisms underlying AAV2-mediated oncosuppression through cell cycle regulation in HPV-infected keratinocytes isolated from a low-grade cervical lesion. AAV2 superinfection of HPV type 31b (HPV31b)-positive cells at early times postinfection resulted in degradation of the cyclin-dependent kinase (CDK) inhibitor p21 WAF1 protein in a proteosome-dependent manner. Downstream consequences of lowering p21 WAF1 levels included a proportional loss of cyclin E/CDK2 complexes bound to p21 WAF1 . The loss of stable p21 WAF1 /cyclin E/CDK2 complexes coincided with an increase in CDK2-associated kinase activity and cyclin E levels. Both events have the potential to enhance the G 1 /S transition point mediated by active cyclin E/CDK2 complexes. Concurrently, cyclin A and E2F levels were decreased, conditions reminiscent of delayed entrance into the S phase of the cell cycle. On the other hand, infection of primary human foreskin keratinocytes with AAV2 resulted in upregulation of p21 WAF1 protein levels, reminiscent of a block in G 1 phase progression. We propose that by downregulating p21 WAF1 , AAV2 initiates cell cycle activities leading to enhanced G 1 /S phase-like conditions which may be favorable for AAV2-specific functions and may lead to downstream interference with HPV-associated cervical cancer progression.Infection of the mucosal epithelium of the anogenital region by high-risk human papillomavirus (HPV) types is positively associated with the development and neoplastic progression of cervical carcinoma (12,44,83,84). Specifically, high-risk HPV types 16, 18, 31, 33, and 45 are associated with over 95% of all cervical cancers (28,81,85). The tumorigenicity of HPV has been attributed to the E6 and E7 oncogenes, which interfere with the functions of cellular tumor suppressors p53 (61) and pRb (14, 42), respectively. Both oncoproteins are involved in the release of the G 1 cell cycle block in differentiating keratinocytes, thus deregulating key cell cycle checkpoints and promoting viral DNA replication (25). On the other hand, infection with the adenoassociated virus type 2 (AAV2), a helper-dependent human parvovirus, may have a negative effect against the development of cervical cancer (46). Seroepidemiological studies have demonstrated that women with HPV-associated cervical carcinoma have antibodies to AAV less frequently compared to matched controls (27,46,68,71). More recent epidemiological studies have also suggested that the presence of AAV DNA in cervical tissue (19) and AAV2 seropositivity (68) are associated with a reduced risk of developing high-grade cervical squamous intraepithelial lesions. Thus, AAV has been proposed to antagonize the development of HPV-related carcinoma (45,62).The antiproliferative and tumor-suppressive properties of AAV have been suggested to result from its ability to target oncogenic v...

Downregulation of Cdc2/CDK1 Kinase Activity Induces the Synthesis of Noninfectious Human Papillomavirus Type 31b Virions in Organotypic Tissues Exposed to Benzo[ a ]pyrene

“…For immunoprecipitating p53 proteins, raft cell extracts were prepared from 12-day raft tissues using a modification of methods previously described (3). Protein extracts were prepared from raft tissues as follows.…”

“…Total protein extracts from BaP-treated and mock monolayer cultures were prepared as we have previously described (3). Determination of total protein levels using Western blot analysis was performed as previously described (3).…”

“…Determination of total protein levels using Western blot analysis was performed as previously described (3).…”

Replication of Minute Virus of Mice in Murine Cells Is Facilitated by Virally Induced Depletion of p21

“…2D, compare lanes 3 and 4]). Adeno-associated virus type 2 (AAV2) has previously been shown to increase proteasome-dependent degradation of p21 in human papillomavirus-positive cell lines (4).…”

The Cigarette Smoke Carcinogen Benzo[ a ]pyrene Enhances Human Papillomavirus Synthesis

“…A 100-l aliquot of the virus prep was transferred into a 1.5-ml microcentrifuge tube, followed by the addition of 100 l buffer A as described previously (1), followed by the addition of 200 l of buffer B, as described previously (1), and boiled in a water bath for 8 min. To each sample, 100 l of a 5ϫ sample loading buffer was added as described previously (1). Total protein concentrations were measured by using the Lowry method as described previously (34).…”