Repression of human papillomavirus oncogenes in HeLa cervical carcinoma cells causes the orderly reactivation of dormant tumor suppressor pathways

Abstract

Most cervical carcinomas express high-risk human papillomaviruses (HPVs) E6 and E7 proteins, which neutralize cellular tumor suppressor function. To determine the consequences of removing the E6 and E7 proteins from cervical cancer cells, we infected HeLa cells, a cervical carcinoma cell line that contains HPV18 DNA, with a recombinant virus that expresses the bovine papillomavirus E2 protein. Expression of the E2 protein resulted in rapid repression of HPV E6 and E7 expression, followed Ϸ12 h later by profound inhibition of cellular DNA synthesis. Shortly after E6͞E7 repression, there was dramatic posttranscriptional induction of p53. Two p53-responsive genes, mdm2 and p21, were induced with slightly slower kinetics than p53 and appeared to be functional, as assessed by inhibition of cyclin-dependent kinase activity and p53 destabilization. There was also dramatic posttranscriptional induction of p105 Rb and p107 after E6͞E7 repression, followed shortly thereafter by induction of p130. By 24 h after infection, only hypophosphorylated p105 Rb was detectable and transcription of several Rb͞E2F-regulated genes was dramatically repressed. Constitutive expression of the HPV16 E6͞E7 genes alleviated E2-induced growth inhibition and impaired activation of the Rb pathway and repression of E2F-responsive genes. This dynamic response strongly suggests that the p53 and Rb tumor suppressor pathways are intact in HeLa cells and that repression of HPV E6 and E7 mobilizes these pathways in an orderly fashion to deliver growth inhibitory signals to the cells. Strikingly, the major alterations in the cell cycle machinery underlying cervical carcinogenesis can be reversed by repression of the endogenous HPV oncogenes. H igh-risk human papillomaviruses (HPVs) such as HPV18 play a central role in the development of essentially all cases of cervical carcinoma (1). However, carcinoma develops infrequently even after infection by these HPV types, and it typically occurs years to decades after the initial infection. Two HPV oncogenes, E6 and E7, are expressed in cervical carcinomas and carcinoma-derived cell lines. The E6 and E7 proteins can immortalize cultured primary human keratinocytes, but these immortalized cells are not tumorigenic unless additional, undefined genetic events occur. These observations imply that the viral oncogenes do not directly induce tumor formation but rather set in motion a series of events that may ultimately result in tumorigenicity.The high-risk HPV E6 and E7 proteins exert profound effects on the tumor suppressor proteins p53 and p105 Rb (1). These tumor suppressor proteins normally control signaling pathways that regulate the cell cycle and monitor and protect the integrity of the genome. p53 is a transcription factor that activates transcription of a variety of genes including p21Waf1/CIP1/SDI1 (p21) (reviewed in ref.2). p21 directly inhibits the activity of cyclin-dependent kinase (cdk) complexes, which are required for cell cycle progression. Transcription of the mdm2 gene is also induced by p53. m...

Differential regulation of survivin by p53 contributes to cell cycle dependent apoptosis

“…HeLa cell line is well known as a cervical cancer cell line with dormant p53 pathway for its infection of human papillomavirus 18 (HPV18), in which the viral protein E6 accelerates p53 degradation by binding to p53 protein [30][31][32]. However, when HeLa cells were treated with an potent anti-tumor reagent Tripchlorolide (TC), it was found that the E6/E7 mRNA was diminished significantly after 12-h treatment and was reduced to an undetectable level after 24-h treatment (Fig.…”

Nuclear-Cytoplasmic Shuttling of the Oncogenic Mouse UNP/USP4 Deubiquitylating Enzyme

“…Thus, p130/p107 may be involved in regulating Usp4 shuttling. Of significance in this context is the fact that p130 shuttles between nucleus and cytoplasm, with its nuclear export LMB-insensitive (40) and that the levels of p130 and p107 are also depleted in HeLa and Saos-2 cells (41,42), which show exclusively nuclear Usp4 (Fig. 2).…”

Human retinoblastoma is not caused by known pRb‐inactivating human DNA tumor viruses

“…A clonal association between virus and tumor cell is usually established via viral integration, and continued expression of viral oncogenes is necessary for maintenance of the malignant phenotype. [28][29][30] The absence of viral sequences is therefore strong evidence against a role for these viruses in the etiology of retinoblastoma. These findings therefore contradict previous reports in which HPV genomic DNA was detected in a subset of bilateral (presumed germline RB1 mutation) and unilateral nonfamilial (presumed nonhereditary) retinoblastoma tumors in Mexico and South America.…”