Transcriptome signature of irreversible senescence in human papillomavirus-positive cervical cancer cells

Abstract

H igh-risk human papillomaviruses (HPVs) are a group of small DNA tumor viruses that are associated with cancers of the uterine cervix (1). More than 95% of cervical cancers contain DNA from one of the high-risk HPV types and express the viral E6 and E7 oncogenes. The major transforming E6 and E7 activities include the targeted degradation of p53 and transcriptional induction of the cellular enzyme telomerase by E6 and the inactivation of the cellular retinoblastoma protein pRB by E7 (2).Progression of HPV-positive precursor lesions to cancer usually is associated with the integration of the HPV DNA into the cellular genome with the disruption of the viral E2 ORF (3, 4). E2 can repress the transcription of the viral oncogenes E6 and E7 (5-11) and requires a functional E2 DNA-binding and transcriptional activation domain for this activity (12-14). Disruption of the E2 gene as a consequence of integration results in the deregulated expression of E6 and E7 mRNAs.Exploiting the functions of E2 represents one possibility for the treatment of HPV-associated cancers. The reexpression of E2 proteins in HPV-positive, but not HPV-negative, cervical cancer cells results in a G 1 cell cycle arrest (11,12,15,16). E2-mediated G 1 growth arrest is accompanied by the reactivation of known cellular targets for E6 and E7 and is followed by the induction of cellular senescence (17-19). As had been observed for E2-mediated growth arrest, the induction of senescence is specific for HPV-positive cancer cells and requires E6͞E7 promoter repression by E2 (19, 20). E2 expression also has been shown to induce apoptotic cell death under some circumstances (15). Direct interactions between E2 and yet unidentified cellular apoptotic regulators have been implicated in this process (21). Recent data from the DiMaio laboratory further confirm the ability of E2 to cause both apoptosis and senescence and highlight distinct mechanisms that are governed by E6 and E7, respectively (22).The term cellular or replicative senescence defines the finite replicative capacity of most somatic cells in culture, which eventually results in the complete and irreversible cessation of cellular division (for reviews, see refs. 23 and 24). To explore in detail the molecular pathways involved in E2 senescence, we used a genomic approach to analyze the transcriptional programs invoked within HeLa cells irreversibly committed to senescence. Adenovirus delivery of temperature-sensitive E2 resulted in temperature-dependent regulation of known E2 targets and the induction of senescence. Using temperature-shift experiments, we determined that senescence was irreversibly established 3 days after E2 expression. We selected this early time point for the transcriptional profiling of E2-expressing HeLa cells and have identified genes that either reflect or contribute directly to the senescence phenotype. Recombinant Vectors. The pRC-CMV-tsE2 expression plasmid encoding a temperature-sensitive bovine E2 protein has been described (12, 25). The E2ts gene was excised by using Xb...

Coordinate regulation of Fanconi anemia gene expression occurs through the Rb/E2F pathway

“…Consequences of E2 expression and reactivation of cellular E6/E7 targets have been characterized extensively, and include cell-cycle arrest and subsequent senescence induction Wells et al, 2000). We previously described the transcriptome signature of senescing HPV18-positive HeLa cervical cancer cells using Affymetrix microarrays and a temperature-sensitive E2 protein (Wells et al, 2003). Interestingly, although several FA genes including FANCD2 and FANCJ were not present on the microarray, the data indicated repression of members of the FA gene family including FANCA and FANCG, as well as the FA-associated BRCA1 and Rad51 genes.…”

“…To determine whether co-regulation of FA protein expression may occur at the level of mRNA expression, HeLa cells were treated as previously described for the microarray experiments: Adenoviral delivery of a temperature-sensitive BPV1 E2ts protein was followed by temperature shift to the permissive temperature and thus synchronized senescence induction at 32 1C in the HeLa cell population (Wells et al, 2003). Empty adenovirus (Ad)-infected control cells were treated in an identical fashion, and control infections at the restrictive temperature of 39.5 1C were included.…”

“…In the case of FANCD2, the observed repression manifested as a trend rather than with statistical significance. HeLa cells were infected with either empty adenovirus (Ad) or Ad expressing a temperature-sensitive E2 protein (AdE2ts) as described in Wells et al (2003). Total RNA was harvested on day 3 and mRNA was converted to cDNA, followed by real-time PCR using primers specific for the indicated genes.…”

“…HeLa cells were transfected with the FANCD2 reporter or control vector, superinfected with AdE2F1 and harvested on day 3 postinfection. The cells were counted, and equal cell numbers were utilized for luciferase assays as previously described (Wells et al, 2003). For reporter assays in TSUPr-1 cells, these were co-transfected with FANCD2 reporter and either p16ink4a expression or control vector.…”

A functional interaction of E7 with B-Myb-MuvB complex promotes acute cooperative transcriptional activation of both S- and M-phase genes. (129 c)

“…1 Accordingly, suppression of E6 and E7 transcription in cervical carcinoma cells leads to a corresponding transcriptional suppression of S-phase and mitotic genes. [4][5][6][7] Two prominent transcriptional activators of mitotic genes, B-Myb and FoxM1, are E2F target genes that are substantially modulated by suppression of E6/E7 transcription in HeLa cells. 7 The HPV16 E7 protein had previously been shown to specifically activate B-Myb transcription 8 and to interact with and activate FoxM1.…”