E2 Transcription Research Articles

Effect of β-sitosterol on the expression of HPV E6 and p53 in cervical carcinoma cells.

Aim of the studyIn the absence of effective therapeutic strategies, cervical carcinoma continues to be second on the list of mortality rates of malignant tumours found in women. We investigated the effects of β -sitosterol, a natural product isolated from traditional Chinese herbs, on Caski and HeLa cervical carcinoma cells.Material and methodsMorphological changes were examined by light microscopy. Ultrastructures of Caski and HeLa cells treated with 20 µmol/l β-sitosterol were documented by scanning electron microscopy and transmission electron microscopy. Changes in mRNA and protein expression were quantified respectively using Real-Time qPCR and western blot methods.ResultsTreatment of Caski and HeLa cells with β-sitosterol resulted in reduced expression of PCNA, indicative of an inhibitory effect on cell proliferation. This was associated with increased p53 mRNA levels and decreased amounts of HPV E6 transcripts. Expression of p53 and HPV E6 proteins followed a similar trend as that observed for the corresponding transcripts. Caski and HeLa cells treated with β-sitosterol exhibited loss of cell surface microvilli, increased electron density of cell membrane, and decreased organelles.ConclusionsIn conclusion, treatment of Caski and HeLa cells with β-sitosterol significantly suppressed the expression of genes involved in cell proliferation and oncogenic transformation, while augmenting the expression of genes involved in apoptosis and tumour suppression. Ultrastuctural characterisation of Caski and HeLa cells treated with β-sitosterol further confirmed the anti-proliferative and anti-cancer activity of this natural product isolated from traditional Chinese herbs.

TALEN-mediated targeting of HPV oncogenes ameliorates HPV-related cervical malignancy.

Persistent HPV infection is recognized as the main etiologic factor for cervical cancer. HPV expresses the oncoproteins E6 and E7, both of which play key roles in maintaining viral infection and promoting carcinogenesis. While siRNA-mediated targeting of E6 and E7 transcripts temporarily induces apoptosis in HPV-positive cells, it does not eliminate viral DNA within the host genome, which can harbor escape mutants. Here, we demonstrated that specifically targeting E6 and E7 within host DNA with transcription activator-like effector nucleases (TALENs) induces apoptosis, inhibits growth, and reduces tumorigenicity in HPV-positive cell lines. TALEN treatment efficiently disrupted E6 and E7 oncogenes, leading to the restoration of host tumor suppressors p53 and retinoblastoma 1 (RB1), which are targeted by E6 and E7, respectively. In the K14-HPV16 transgenic mouse model of HPV-driven neoplasms, direct cervical application of HPV16-E7-targeted TALENs effectively mutated the E7 oncogene, reduced viral DNA load, and restored RB1 function and downstream targets transcription factor E2F1 and cycling-dependent kinase 2 (CDK2), thereby reversing the malignant phenotype. Together, the results from our study suggest that TALENs have potential as a therapeutic strategy for HPV infection and related cervical malignancy.

Estrogen induces estrogen receptor alpha expression and hepatocyte proliferation in the livers of male mice.

Estrogens play pivotal roles in sexual development, growth, reproduction, and sex differentiation and have been implicated in a number of physiological processes in various tissues. Most of the effects of estrogens are mediated by the estrogen receptors alpha (ERα), beta (ERβ), and G protein-coupled receptor 30 (GPR30). The liver is known to be a target tissue for estrogen signaling, but the physiological role of this signaling is not well characterized. Through analyses of an estradiol (E2)-treated hepatocyte cell line and mice, we showed that E2 signaling controls hepatocyte proliferation. Importantly, our data showed that the E2 signaling that is mediated through ERα is crucial for efficient liver regeneration after partial hepatectomy (PH). PH rapidly induced marked increases in circulating E2 and ERα transcripts in periportal hepatocytes, well before the onset of hepatocyte proliferation. Taken together, our results indicate that increased E2 is one of the initiating signals that trigger liver regeneration. We suggest that E2 treatment could be beneficial for stimulating liver regeneration in humans.

Prevalence of human papillomavirus in laryngeal and hypopharyngeal squamous cell carcinomas in northern Spain

BackgroundRecent studies support a role for human papillomavirus (HPV) in oropharyngeal squamous cell carcinomas (SCCs); however, the significance of HPV in non-oropharyngeal head and neck cancers is uncertain. The aim of this study was to determine the prevalence of HPV in a large cohort of laryngeal and hypopharyngeal SCCs in northern Spain. Materials and methodsClinical records and paraffin-embedded tumor specimens of 124 consecutive patients surgically treated for laryngeal (62 cases) and hypopharyngeal (62 cases) SCCs between 2002 and 2007 were retrieved. All cases were histologically evaluated, and presence of HPV was assessed by p16-immunohistochemistry followed by GP5+/6+-PCR-based DNA detection. Samples positive in both assays were subjected to HPV genotyping and HPV E6 transcript analysis. ResultsSeventeen cases (14%) were positive for p16 immunostaining, of which 2 (1 larynx, 1 hypopharynx, 1.6% of total series) were found positive for HPV DNA by subsequent GP5+6+-PCR. Both SCCs contained HPV type 16 and showed HPV16 E6 mRNA expression. ConclusionsHPV is only occasionally involved in laryngeal and hypopharyngeal SCC patients in northern Spain.

E6-associated transcription patterns in human papilloma virus 16-positive cervical tissues.

The change in transcription pattern induced by post-transcriptional RNA splicing is an important mechanism in the regulation of the early gene expression of human papilloma virus (HPV). The present study was conducted to establish a method to specifically amplify HPV-16 E6-associated transcripts. The E6-related transcripts from 63 HPV-16-positive cervical tumor tissue samples were amplified, consisting of eight cases of low-risk intraepithelial lesions, 38 cases of high-risk intraepithelial lesions and 17 cases of cervical cancer (CxCa). The appropriate amplified segments were recovered following agarose gel electrophoresis, and subjected to further sequencing and sequence alignment analysis. Six groups of E6 transcription patterns were identified from HPV-16-positive cervical tumor tissue, including five newly-discovered transcripts. Different HPV-16 E6-associated transcription patterns were detected during the development of CxCa. Over the course of the progression of the low-grade squamous intraepithelial lesions to CxCa, the specific HPV-16 E6-associated transcription patterns and the dominant transcripts were all different. As indicated by this study, the transcription pattern of the E6 early gene of HPV-16 was closely associated with the stages of cervical carcinogenesis, and may also be involved in the development of CxCa.

Nuclear Matrix Protein SMAR1 Represses c-Fos-mediated HPV18 E6 Transcription through Alteration of Chromatin Histone Deacetylation

Matrix attachment region (MAR)-binding proteins have been implicated in the transcriptional regulation of host as well as viral genes, but their precise role in HPV-infected cervical cancer remains unclear. Here we show that HPV18 promoter contains consensus MAR element in the LCR and E6 sequences where SMAR1 binds and reinforces HPV18 E6 transcriptional silencing. In fact, curcumin-induced up-regulation of SMAR1 ensures recruitment of SMAR1-HDAC1 repressor complex at the LCR and E6 MAR sequences, thereby decreasing histone acetylation at H3K9 and H3K18, leading to reorientation of the chromatin. As a consequence, c-Fos binding at the putative AP-1 sites on E6 promoter is inhibited. E6 depletion interrupts degradation of E6-mediated p53 and lysine acetyl transferase, Tip60. Tip60, in turn, acetylates p53, thereby restoring p53-mediated transactivation of proapoptotic genes to ensure apoptosis. This hitherto unexplained function of SMAR1 signifies the potential of this unique scaffold matrix-associated region-binding protein as a critical regulator of E6-mediated anti-apoptotic network in HPV18-infected cervical adenocarcinoma. These results also justify the candidature of curcumin for the treatment of HPV18-infected cervical carcinoma.

Abstract 4147: Mate pair sequencing reveals that human papillomavirus integration into the human genome in oropharyngeal squamous cell carcinoma is rare and different from cervical cancer

Abstract In spite of decreases in the incidence of smoking in the United States over the past several decades, the incidence of oropharyngeal squamous cell carcinoma (OPSCC) has been increasing. This is presumably due to an increase in the incidence of human papillomavirus (HPV)-positive OPSCC. The model for how HPV contributes to cancer development is based upon studies characterizing cervical cancer. In cervical cancer almost all invasive cancers have HPV integrated into the human genome with loss of episomal copies of HPV. The site of integration is usually within the HPV E2 gene which functions as a repressor of the two key HPV oncoproteins, E6 and E7. The resulting over-production of E6 and E7 inactivates two key pathways which results in genomic instability and additional down-stream alterations that eventually leads to invasive cervical cancer. In order to definitively test whether this model is indeed true for OPSCC we performed mate-pair next generation sequencing on both tumor and adjacent normal tissues from 20 OPSCC patients: 7 were HPV16-positive and had high expression of the E6 and E7 transcripts; 6 were HPV-positive but with low E6 and E7 transcription; and as a control we also analyzed 7 HPV16-negative tumors. This high throughput technology was able to successfully detect all HPV integration events into the human genome as well as HPV bridged coverage from these samples with high specificity. Just a single HPV-positive tumor with high oncogene expression had HPV integration events (two), but this patient also had a very high copy number of episomal HPV sequences. A single HPV-positive tumor with low oncogene expression had one HPV integration event, but no episomal HPV sequences present. One of the HPV16-negative tumors had a single HPV26 integration with no episomal HPV sequences. None of the matched normal samples had either HPV integrations or an appreciable amount of episomal HPV sequences present. Thus, only 21.4% of HPV-positive OPSCCs had integrations event. All four integrations were validated by PCR and Sanger sequencing which revealed that none of them interrupted the HPV E2 gene. All four of the integrations occurred next to human genes that have been found to be altered in different cancers. Our results suggest that the role that HPV plays in OPSCC may be quite distinct from what is observed in cervical cancer. The results also demonstrate that not all HPV-positive OPSCCs have HPV in the same physical state. It will thus be interesting to compare the distinct groups of HPV-positive OPSCCs with respect to overall clinical prognosis. Citation Format: Ge Gao. Mate pair sequencing reveals that human papillomavirus integration into the human genome in oropharyngeal squamous cell carcinoma is rare and different from cervical cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4147. doi:10.1158/1538-7445.AM2014-4147

The molecular response of vanadium complexes of nicotinoyl hydrazone in cervical cancers—A possible interference with HPV oncogenic markers

AimsHydrazones belonging to the class of NNO donor Schiff bases are reported to have extensive anti-viral activity and anti-neoplastic activity against certain cancers such as colon cancer, hepatocellular carcinoma and testicular cancer. Here we aim to study the possible effects of two novel nicotinoyl hydrazones on Human papillomavirus (HPV) infected cervical cancers. Main methodsThe effect of vanadium complexes on the proliferation of SiHa and HeLa cells was analyzed using MTT assay. The apoptotic potentials of the complexes were assessed by their ability to induce DNA condensation as well as loss of mitochondrial membrane potential. Caspase activity assay and DNA content analysis were performed to understand the mechanism of apoptotic induction. RT-PCR analysis of cell cycle genes, GADD45, p53, p21 and HPV specific oncogenes, E6 and E7 were used to elucidate the molecular mechanism of the complexes. Key findingsOVK 49 exhibits an increased apoptosis inducing potential when compared to OVK 89 in HPV16 positive SiHa cells compared to HPV18 positive HeLa. A down regulation for E6 and E7 mRNA transcripts along with the induction of p53 protein in SiHa cells were observed when treated with OVK 49 indicating that OVK 49 might have promising anti-cancer activity against HPV16 positive cervical cancers. SignificanceThis is the first study demonstrating that vanadium complexes could induce a p53 dependent apoptotic mechanism in high risk HPV16-positive cervical cancers.

The role of redox-sensitive cysteines in multi sub-unit transcription factors

In response to oxidative stress, cells activate numerous processes and initiate the expression of genes via redox-sensitive transcription factors including the multi sub-unit transcription factors nuclear factor-κB (NFκB), activator protein 1 (AP-1), Hypoxia-inducible factor 1 α (HIF1α), Polyoma virus enhancer-binding protein 2 (PEBP2), cAMP-responsive element-bindingprotein (CREB), Nuclear Factor I/CCAAT Transcription Factor (NFI/CTF), Nuclear transcription factor Y (NF-Y) and E4 Transcription Factor 1 - 60kDa (E4TF1-60). Thioredoxin plays a critical role in this by directly or indirectly (via redox factor 1) reducing redox-sensitive cysteine residues in these transcription factors to modify their tertiary structure, sub-cellular localisation,proteinprotein interactions or DNA-binding affinity. The identity of the redox-sensitive cysteine in E4TF1-60 has not been investigated so far. Determining which cysteines are redox-sensitive within the E4TF1-60 protein sequence together with elucidation of which property is modified will help to better understand the mechanism by which the E4TF1 complex is involved in regulating the cellular oxidative stress response.

Oncogenic viruses associated with vulva cancer in HIV-1 patients in Botswana.

BackgroundOncoviruses such as HPV, KSHV, and EBV have been reported in patients with HIV infection and AIDS. How oncovirus-associated cancers rise in AIDS patients has not been fully established. The purpose of our study was to identify the viral agents in vulvar cancer and to assess their contribution to pathogenesis.MethodWe retrospectively identified a total of 13 vulva tissue samples from HIV-1 positive and 9 vulvar samples from HIV-1 negative patients from the Botswana National Health Laboratory in Gaborone, Botswana, a Southern African country with a high incidence of HIV. We utilized PCR and IHC to identify HPV, EBV, KSHV, and JC virus in FFPE preserved tissue samples.ResultsUsing the GP5+/GP6+ primer set we detected several HPV types in tissue samples. EBV was detected in all of the positive cases (100%) and in most of the negative cases (89%). KSHV was detected in 39% of the HIV-1 positive samples and in 11% of the negative samples, and no JC virus was detected in any of the samples.Using IHC we demonstrated that LANA was expressed in 61% of the positive samples and in 44% of the negative samples. The ubiquitous EBV was more consistently expressed in negative cases (100%) than in positive cases (69%). Interestingly, the HPV-16 E6 transcript was detected in 56% of the negative samples compared to 31% of the positive samples. However, the cell cycle protein P21 used as a surrogate marker for HPV was detected in 77% of the positive samples and in 44% of the negative samples, while VEGF signals were similar in both positive (92%) and negative samples (89%).ConclusionOur study, suggests that in Botswana, vulvar squamous cell carcinoma (VSCC) is associated with oncogenic viruses present in the niche but the contribution and progression may be regulated by HPV and other immunosuppressive infections that include HIV-1.

The cellular bromodomain protein Brd4 has multiple functions in E2-mediated papillomavirus transcription activation.

The cellular bromodomain protein Brd4 functions in multiple processes of the papillomavirus life cycle, including viral replication, genome maintenance, and gene transcription through its interaction with the viral protein, E2. However, the mechanisms by which E2 and Brd4 activate viral transcription are still not completely understood. In this study, we show that recruitment of positive transcription elongation factor b (P-TEFb), a functional interaction partner of Brd4 in transcription activation, is important for E2’s transcription activation activity. Furthermore, chromatin immunoprecipitation (ChIP) analyses demonstrate that P-TEFb is recruited to the actual papillomavirus episomes. We also show that E2’s interaction with cellular chromatin through Brd4 correlates with its papillomavirus transcription activation function since JQ1(+), a bromodomain inhibitor that efficiently dissociates E2-Brd4 complexes from chromatin, potently reduces papillomavirus transcription. Our study identifies a specific function of Brd4 in papillomavirus gene transcription and highlights the potential use of bromodomain inhibitors as a method to disrupt the human papillomavirus (HPV) life cycle.

Prolyl isomerase Pin1 in cancer.

Proline-directed phosphorylation is a posttranslational modification that is instrumental in regulating signaling from the plasma membrane to the nucleus, and its dysregulation contributes to cancer development. Protein interacting with never in mitosis A1 (Pin1), which is overexpressed in many types of cancer, isomerizes specific phosphorylated Ser/Thr-Pro bonds in many substrate proteins, including glycolytic enzyme, protein kinases, protein phosphatases, methyltransferase, lipid kinase, ubiquitin E3 ligase, DNA endonuclease, RNA polymerase, and transcription activators and regulators. This Pin1-mediated isomerization alters the structures and activities of these proteins, thereby regulating cell metabolism, cell mobility, cell cycle progression, cell proliferation, cell survival, apoptosis and tumor development.

Ethyl 1,8-naphthyridone-3-carboxylates downregulate human papillomavirus-16 E6 and E7 oncogene expression.

Strong epidemiological and molecular data associate cervical cancer (CC) with high-risk human papillomavirus (HPV) infections. The carcinogenic mechanism depends mainly on the expression of E6 and E7 oncoproteins encoded by the viral genome. Using a cell-based high-throughput assay, an in-house library of compounds was screened identifying the 1,8-naphthyridone 1 that efficiently inhibited the transcription driven by the long control region of the HPV genome. A series of analogues were then synthesized, obtaining more potent derivatives able to downregulate E6 and E7 transcripts in HPV-16-positive CC CaSki cells. An unusual structural insight emerged for the C-3 position of the 1,8-naphthyridone core, where the ethyl carboxylate esters, but not the carboxylic acids, are responsible for the activity. In vitro uptake studies showed that the 3-ethyl carboxylates do not act as prodrugs. The 1,8-naphthyridones emerged as valid starting points for the development of innovative agents potentially useful for the treatment of HPV-induced CC.

Transcriptome profiling of the theca interna from bovine ovarian follicles during atresia.

The theca interna is a specialized stromal layer that envelops each growing ovarian follicle. It contains capillaries, fibroblasts, immune cells and the steroidogenic cells that synthesize androgens for conversion to estradiol by the neighboring granulosa cells. During reproductive life only a small number of follicles will grow to a sufficient size to ovulate, whereas the majority of follicles will undergo regression/atresia and phagocytosis by macrophages. To identify genes which are differentially regulated in the theca interna during follicular atresia, we undertook transcriptome profiling of the theca interna from healthy (n = 10) and antral atretic (n = 5) bovine follicles at early antral stages (<5 mm). Principal Component Analyses and hierarchical classification of the signal intensity plots for the arrays showed primary clustering into two groups, healthy and atretic. A total of 543 probe sets were differentially expressed between the atretic and healthy theca interna. Further analyses of these genes by Ingenuity Pathway Analysis and Gene Ontology Enrichment Analysis Toolkit software found most of the genes being expressed were related to cytokines, hormones and receptors as well as the cell cycle and DNA replication. Cell cycle genes which encode components of the replicating chromosome complex and mitotic spindle were down-regulated in atretic theca interna, whereas stress response and inflammation-related genes such as TP53, IKBKB and TGFB1 were up-regulated. In addition to cell cycle regulators, upstream regulators that were predicted to be inhibited included Retinoblastoma 1, E2 transcription factor 1, and hepatocyte growth factor. Our study suggests that during antral atresia of small follicles in the theca interna, arrest of cell cycle and DNA replication occurs rather than up- regulation of apoptosis-associated genes as occurs in granulosa cells.

Epidermal growth factor receptor signalling in human breast cancer cells operates parallel to estrogen receptor α signalling and results in tamoxifen insensitive proliferation.

BackgroundTamoxifen resistance is a major problem in the treatment of estrogen receptor (ER) α -positive breast cancer patients. Although the mechanisms behind tamoxifen resistance are still not completely understood, clinical data suggests that increased expression of receptor tyrosine kinases is involved. Here, we studied the estrogen and anti-estrogen sensitivity of human breast cancer MCF7 cells that have a moderate, retroviral-mediated, ectopic expression of epidermal growth factor receptor (MCF7-EGFR).MethodsProliferation of MCF7-EGFR and parental cells was induced by 17β-estradiol (E2), epidermal growth factor (EGF) or a combination of these. Inhibition of proliferation under these conditions was investigated with 4-hydroxy-tamoxifen (TAM) or fulvestrant at 10-12 to 10-6 M. Cells were lysed at different time points to determine the phosphorylation status of EGFR, MAPK1/3, AKT and the expression of ERα. Knockdown of target genes was established using smartpool siRNAs. Transcriptomics analysis was done 6 hr after stimulation with growth factors using Affymetrix HG-U133 PM array plates.ResultsWhile proliferation of parental MCF7 cells could only be induced by E2, proliferation of MCF7-EGFR cells could be induced by either E2 or EGF. Treatment with TAM or fulvestrant did significantly inhibit proliferation of MCF7-EGFR cells stimulated with E2 alone. EGF treatment of E2/TAM treated cells led to a marked cell proliferation thereby overruling the anti-estrogen-mediated inhibition of cell proliferation. Under these conditions, TAM however did still inhibit ERα- mediated transcription. While siRNA-mediated knock-down of EGFR inhibited the EGF- driven proliferation under TAM/E2/EGF condition, knock down of ERα did not. The TAM resistant cell proliferation mediated by the conditional EGFR-signaling may be dependent on the PI3K/Akt pathway but not the MEK/MAPK pathway, since a MEK inhibitor (U0126), did not block the proliferation. Transcriptomic analysis under the various E2/TAM/EGF conditions revealed that E2 and EGF dependent transcription have little overlap and rather operate in a parallel fashion.ConclusionsOur data indicate that enhanced EGFR-driven signalling is sufficient to overrule the TAM- mediated inhibition of E2-driven cell proliferation. This may have profound implications for the anti-estrogen treatment of ER-positive breast cancers that have increased levels of EGFR.

Adenovirus E1A Recruits the Human Paf1 Complex To Enhance Transcriptional Elongation

During infection by human adenovirus (HAdV), the proteins encoded by the early region 1A (E1A) gene bind and appropriate components of the cellular transcriptional machinery to activate the transcription of viral early genes. Previously, we identified roles for the human Bre1 (hBre1) and hPaf1 complexes in E1A-mediated transcriptional activation of HAdV early genes. Here we show that E1A binds hBre1 directly and that this complex targets the hPaf1 complex via the Rtf1 subunit. Depletion of hPaf1 reduces E1A-dependent activation of transcription from the E2e, E3, and E4 viral transcription units, and this does not result from a reduced ability of RNA polymerase II to be recruited to the promoter-proximal regions of these genes. In contrast, depletion of hPaf1 reduces the occupancy of RNA polymerase II across these transcription units. This is accompanied by reductions in the level of H3K36 trimethylation, a posttranslational histone modification associated with efficient transcriptional elongation, and the number of full-length transcripts from these genes. Together, these results indicate that E1A uses hBre1 to recruit the hPaf1 complex in order to optimally activate viral early transcription by enhancing transcriptional elongation. This work provides the mechanism by which the hPaf1 complex contributes to E1A-dependent activation of early gene transcription. The work also demonstrates that E1A induces gene expression by stimulating transcriptional elongation, in addition to its better-characterized effects on transcriptional initiation.

Sequence variation in the E2-binding domain of HPV16 and biological function evaluation in Tunisian cervical cancers.

HPV16 E2 variants have different effects on the transcriptional activity of the LCR. In this study, we examined the nucleotide and amino acid sequence variation within the HPV16 E2 gene and to correlate with disease progression. E2 gene disruption was detected by PCR amplification of the entire E2 gene using a single set of primers. Nucleotide variations were analyzed by bidirectional sequencing. mRNA expression patterns of E6 and E7 gene transcripts were evaluated by a reverse transcriptase-PCR method (RT-PCR). The detection of intact E2 genes was significantly higher among controls than cases (81.8% versus 37.5%, resp., P < 0.05). Among the E subgroup, variation at position 3684 C>A results in the amino acid substitution T310K and was more common among the E2 undisrupted cases (7/9; 77.7%), compared to controls (2/9; 22.2%). In addition, specific sequence variations identified in the E2 ORF at positions 3684 C>A were associated with increased viral oncogenes E6-E7 production. Besides HPV16 E2 disruption, the 3684 C>A variation within undisrupted E2 genes could be involved in an alternative mechanism for deregulating the expression of the HPV16 E6 and E7 oncogenes and appears to be a major factor contributing to the development of cervical cancer in Tunisian women.

Sp1 and Sp3 transcription factors regulate the basal expression of human microsomal epoxide hydrolase (EPHX1) through interaction with the E1b far upstream promoter

Microsomal epoxide hydrolase (mEH, EPHX1) is a critical biotransformation enzyme, catalyzing the metabolism of many xenobiotics. Human mEH is transcribed using alternative promoters. The upstream E1 promoter is active in liver while the far upstream E1b promoter drives the expression of mEH in all tissues, including liver. Although several liver-specific transcription factors have been identified in the regulation of E1 transcription, little is known regarding the mechanisms of E1b transcriptional regulation. Genome-wide mapping of DNase I hypersensitive sites revealed an open chromatin region between nucleotide −300 upstream and +400 downstream of E1b. This area coincides with a previously described promoter region responsible for maintaining high basal promoter activity. In silico analysis of this location revealed several Sp1/Sp3 binding sites. Site-directed mutagenesis of these motifs suppressed the transactivation activity of the E1b proximal promoter, indicating their importance as contributors to E1b promoter regulation. Further, E1b promoter activities were increased significantly following Sp1 and Sp3 overexpression, while Mithramycin A, a selective Sp1 inhibitor, reduced the promoter activities. EMSA studies demonstrated that Sp1 bound to two putative Sp1/Sp3 binding sites. ChIP analysis confirmed that both endogenous Sp1 and Sp3 were bound to the proximal promoter region of E1b. Knockdown of Sp1 expression using siRNA did not alter the endogenous E1b transcriptional level, while knockdown of Sp3 greatly decreased E1b expression in different human cell lines. Taken together, these results support the concept that Sp1 and Sp3 are functionally involved as transcriptional integrators regulating the basal expression of the derived mEH E1b variant transcript.

IFN-α Inhibits Telomerase in Human CD8+ T Cells by Both hTERT Downregulation and Induction of p38 MAPK Signaling

The cytokine IFN-α is secreted during viral infections and has been shown to inhibit telomerase activity and accelerate T cell differentiation in vivo. However, the mechanism for this inhibition is not clear. In this study, we show that IFN-α inhibits both the transcription and translation of human telomerase reverse transcriptase (hTERT), the catalytic component of telomerase, in activated CD8(+) T cells. This was associated with increased activity of the repressor of hTERT transcription E2 transcription factor and decreased activation of NF-κB that promotes hTERT transcription. However IFN-α did not affect the translocation of hTERT from the cytoplasm to the nucleus. IFN-α also inhibits AKT kinase activation but increases p38 MAPK activity, and both of these events have been shown previously to inhibit telomerase activity. Addition of BIRB796, an inhibitor of p38 activity, to IFN-α-treated cells reversed, in part, the inhibition of telomerase by this cytokine. Therefore, IFN-α can inhibit the enzyme telomerase in CD8(+) T cells by transcriptional and posttranslational mechanisms. Furthermore, the addition of IFN-α to CD8(+)CD27(+)CD28(+) T cells accelerates the loss of both these costimulatory molecules. This suggests that persistent viral infections may contribute to the accumulation of highly differentiated/senescent CD8(+)CD27(-)CD28(-) T cells during aging by promoting IFN-α secretion during repeated episodes of viral reactivation.

Homologous Recombination in E3 Genes of Human Adenovirus Species D

Genes within the E3 transcription unit of human adenoviruses modulate host immune responses to infection. A comprehensive genomics and bioinformatics analysis of the E3 transcription unit for 38 viruses within human adenovirus species D (HAdV-D) revealed distinct and surprising patterns of homologous recombination. Homologous recombination was identified in open reading frames for E3 CR1α, CR1β, and CR1γ, similar to that previously observed with genes encoding the three major structural capsid proteins, the penton base, hexon, and fiber.