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...
Although retinoic acid (RA), the active form of vitamin A, is required for normal embryonic growth and development, it is also a powerful teratogen. Infants born to mothers exposed to retinoids during pregnancy have a 25-fold increased risk for malformations, nearly exclusively of cranial neural crest-derived tissues. To characterize neural crest cell responses to RA, we exposed murine crest cultures to teratogenic levels of RA and subjected their RNA to microarray-based gene expression profile analysis using Affymetrix MG-U74Av2 GeneChips. RNAs were isolated from independent cultures treated with 10(-6) M RA for 6, 12, 24, or 48 h. Statistical analyses of gene expression profile data facilitated identification of the 205 top-ranked differentially regulated genes whose expression was reproducibly changed by RA over time. Cluster analyses of these genes across the independently treated sample series revealed distinctive kinetic patterns of altered gene expression. The largest group was transiently affected within the first 6 h of exposure, representing early responding genes. Group 2 showed sustained induction by RA over all times, whereas group 3 was characterized by the suppression of a time-dependent expression increase normally seen in untreated cells. Additional patterns demonstrated time-dependent increased or decreased expression among genes not normally regulated to a significant extent. Gene function analysis revealed that more than one-third of all RA-regulated genes were associated with developmental regulation, including both canonical and noncanonical Wnt signaling pathways. Multiple genes associated with cell adhesion and cell cycle regulation, recognized targets for the biological effects of RA, were also affected. Taken together, these results support the hypothesis that the teratogenic effects of RA derive from reprogramming gene expression of a host of genes, which play critical roles during embryonic development regulating pathways that determine subsequent differentiation of cranial neural crest cells.
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