Transcriptome signature of irreversible senescence in human papillomavirus-positive cervical cancer cells - PubMed (original) (raw)
Transcriptome signature of irreversible senescence in human papillomavirus-positive cervical cancer cells
Susanne I Wells et al. Proc Natl Acad Sci U S A. 2003.
Abstract
A frequent characteristic of human papillomavirus (HPV)-positive cervical cancers is the loss of viral E2 gene expression in HPV-infected cervical epithelial cells as a consequence of viral DNA integration into the cellular genome. The expression of E2 in HPV-positive cancer cells results in the repression of the viral E6/E7 oncogenes, activation of the p53 and pRB pathways, and a G1 cell cycle arrest, followed by induction of cellular senescence. The transcriptional consequences of E2-mediated cell cycle arrest that lead to senescence currently are unknown. Using conditional senescence induction in HeLa cells and microarray analysis, we describe here the expression profile of cells irreversibly committed to senescence. Our results provide insight into the molecular anatomy of senescence pathways and its regulation by HPV on-coproteins. These include the induction of the RAB vesicular transport machinery and a general down-regulation of chromatin regulatory molecules. The repression of tumor-specific G antigens during E2 senescence supports a reversal of the tumorigenic phenotype by E2 and the potential approach of tumor-specific G antigen-specific immunotherapy for cervical cancer.
Figures
Fig. 1.
Adenovirus-mediated expression of a temperature-sensitive E2 gene. (A) E2 transcriptional activity was determined at the permissive and restrictive temperatures by using an E2-dependent reporter plasmid. HeLa cells were transfected with the reporter plasmid and subsequently infected with 0.1 (white), 1 (gray), or 10 (black) plaque-forming units per cell. Luciferase activities are expressed as fold induction over baseline luciferase activities at the respective temperature. (B) HeLa cells were infected with AdE2ts and control empty Ad at the restrictive temperature. Some cultures were shifted to the permissive temperature for senescence induction, whereas control cultures were maintained at the restrictive temperature. The percentage of cells with the enlarged, flat-cell phenotype was determined on day 7 in three random fields relative to total cell counts. SDs are indicated. (C) Cells were infected with the AdE2ts virus and cultured at either the restrictive (39.5°C) or permissive (32°C) temperature. SAβ-gal staining was performed 3 weeks postsenescence induction.
Fig. 4.
Expression levels of p21CIP.(A) Northern blot analysis with p21CIP- and GAPDH-specific probes. HeLa cells were infected with empty Ad and AdE2ts followed by temperature shift as described in the Fig. 3 legend. Lanes 1 and 2 represent samples infected with empty Ad at 32°C and 39.5°C, and lanes 3 and 4 represent samples infected with AdE2ts at 32°C and 39.5°C. Blots hybridized with p21CIP probe were stripped and reprobed with GAPDH for normalization. (B) Normalized p21CIP values from A are represented relative to the Ad sample at 32°C and are compared with the data from three independent microarray experiments. (C) HeLa cells were infected as in A, protein extracts were prepared on day 3 postsenescence induction, and equal amounts of proteins were subjected to Western blot analysis by using p53- and p21CIP-specific antibodies.
Fig. 2.
Senescence irreversibility in E2-expressing HeLa cells. HeLa cells were infected on 2 consecutive days with the AdE2ts virus at 39.5°C. The cells were split the following day and placed at 32°C for synchronized senescence induction (t = 0). Control cells remained at either 39.5°C (A) or 32°C (B) for 7 days. Aliquots were shifted from the permissive temperature of 32°C to the restrictive temperature of 39.5°C on days 1–4 for the timed inactivation of E2 (C_–_F). SAβ-gal staining was performed on all samples at 7 days postsenescence induction.
Fig. 3.
Experimental design and transcriptional profiling of senescent cells. (A) RNA samples used for microarray experiments were prepared from cells infected with AdE2ts and empty Ad at the permissive and restrictive temperatures as outlined schematically. Senescence induction was observed only with the AdE2ts sample at 32°C (lane 1). Bovine papillomavirus E2 and GAPDH messages were detected by RT-PCR, using gene-specific primers. (B) Results from clustering three independent experiments (a–c) within the four treatment groups are depicted as a gene tree (experiment tree not shown). Pearson correlation was applied to the sample measurements by using the 703 gene list derived from statistical group comparisons. Transcriptionally induced genes are indicated in red; repressed genes are indicated in blue.
Fig. 5.
A window of gene expression changes during E2-mediated senescence induction. Expression of E2 in HPV-positive cells causes senescence via the repression of the viral E6/E7 oncogenes. Several gene groups that are regulated during senescence induction were categorized according to biological processes that they are known to regulate. Induced genes are depicted in red; repressed genes are depicted in blue. For each gene, the most significant observed expression change, of three independent experiments, is depicted as the ratio of expression signal obtained with AdE2ts to that obtained with Ad at 32°C. The data were normalized to the average of AdE2ts and Ad at 39.5°C as described in Materials and Methods. Expression changes for these genes in all three experiments are shown at
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