Hepatitis B virus X protein (HBx) transactivates viral and cellular genes through a wide variety of cis-elements. However, the mechanism is still obscure. Our finding that HBx directly interacts with RNA polymerase II subunit 5 (RPB5), a common subunit of RNA polymerases, implies that HBx directly modulates the function of RNA polymerase (Cheong, J. H., Yi, M., Lin, Y., and Murakami, S. (1995) EMBO J. 14, 142-150). In this context, we examined the possibility that HBx and RPB5 interact with other general transcription factors. HBx and RPB5 specifically bound to transcription factor IIB (TFIIB) in vitro, both of which were detected by either far-Western blotting or the glutathione S-transferaseresin pull-down assay. Delineation of the binding regions of these three proteins revealed that HBx, RPB5, and TFIIB each has two binding regions for the other two proteins. Co-immunoprecipitation using HepG2 cell lysates that express HBx demonstrated trimeric interaction in vivo. Some HBx substitution mutants, which had severely impaired transacting activity, exhibited reduced binding affinity with either TFIIB or RPB5 in a mutually exclusive manner, suggesting that HBx transactivation requires the interactions of both RPB5 and TFIIB. These results indicated that HBx is a novel virus modulator that facilitates transcriptional initiation by stabilizing the association between RNA polymerase and TFIIB through communication with RPB5 and TFIIB.
APE1 is the major nuclease for excising abasic (AP) sites and particular 3′-obstructive termini from DNA, and is an integral participant in the base excision repair (BER) pathway. BER capacity plays a prominent role in dictating responsiveness to agents that generate oxidative or alkylation DNA damage, as well as certain chain-terminating nucleoside analogs and 5-fluorouracil. We describe within the development of a robust, 1536-well automated screening assay that employs a deoxyoligonucleotide substrate operating in the red-shifted fluorescence spectral region to identify APE1 endonuclease inhibitors. This AP site incision assay was used in a titration-based high-throughput screen of the Library of Pharmacologically Active Compounds (LOPAC1280), a collection of well-characterized, drug-like molecules representing all major target classes. Prioritized hits were authenticated and characterized via two high-throughput screening assays – a Thiazole Orange fluorophore-DNA displacement test and an E. coli endonuclease IV counterscreen – and a conventional, gel-based radiotracer incision assay. The top, validated compounds, i.e. 6-hydroxy-DL-DOPA, Reactive Blue 2 and myricetin, were shown to inhibit AP site cleavage activity of whole cell protein extracts from HEK 293T and HeLa cell lines, and to enhance the cytotoxic and genotoxic potency of the alkylating agent methylmethane sulfonate. The studies herein report on the identification of novel, small molecule APE1-targeted bioactive inhibitor probes, which represent initial chemotypes towards the development of potential pharmaceuticals.
To modulate transcription, regulatory factors communicate with basal transcription factors and/or RNA polymerases in a variety of ways. Previously, it has been reported that RNA polymerase II subunit 5 (RPB5) is one of the targets of hepatitis B virus X protein (HBx) and that both HBx and RPB5 specifically interact with general transcription factor IIB (TFIIB), implying that RPB5 is one of the communicating subunits of RNA polymerase II involved in transcriptional regulation. In this context, we screened for a host protein(s) that interacts with RPB5. By far-Western blot screening, we cloned a novel gene encoding a 508-amino-acid-residue RPB5-binding protein from a HepG2 cDNA library and designated it RPB5-mediating protein (RMP). In eukaryotes, nuclear RNA polymerases I, II, and III are highly conserved multisubunit enzymes involved in the synthesis of rRNAs, mRNAs, and tRNAs, respectively (48). All these nuclear RNA polymerases share the function of RNA synthesis but utilize different promoters and require different transcription factors. For example, promoter-specific transcription initiation from protein-coding genes requires the concerted action of a complex array of factors involving RNA polymerase II and general transcriptional factors (TFIID, TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH) (14,46). Transcriptional activators and repressors bind distal elements of a promoter and modulate transcription through communication with components of a preinitiation complex, such as TATA-binding protein (TBP) and its binding proteins, TFIIB, TFIIA, and TFIIH (18,19,24,42). Recently, another group of proteins, cofactors (also called mediators), have been demonstrated to affect transcription positively or negatively by communicating with promoterspecific regulatory factors and the transcriptional machinery. These proteins include global coactivator p300/CBP (20, 25, 31, 34), nuclear silence mediators (2, 12, 52), and a large number of mediator proteins, or SRBs (10,28,39,47,54). RNA polymerase subunits may be additional targets for transcriptional regulators, because RNA polymerases are the ultimate target of transcriptional modulation. In this context, several subunits have been recently reported to interact with the regulators (8, 9), in addition to the well-documented regulatory role of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (28, 30).Hepatitis B virus (HBV) X protein (HBx) is essential for HBV infection and plays an important role in HBV-associated hepatocellular carcinoma (11,17,26,51). Many reports have shown that HBx transactivates viral and cellular genes through a wide variety of cis-acting elements. However, the mechanism of this effect has not been well elucidated (3,5,7,15,16,22,23,50). It has been previously demonstrated that HBx directly interacts with RNA polymerase II subunit 5 (RPB5), a common RNA polymerase subunit (13), that both RPB5 and HBx communicate with TFIIB but through different sites (21), and that the trimeric interaction of these three factors is involved in HBx t...
FEN1 has key roles in Okazaki fragment maturation during replication, long patch base excision repair, rescue of stalled replication forks, maintenance of telomere stability and apoptosis. FEN1 may be dysregulated in breast and ovarian cancers and have clinicopathological significance in patients. We comprehensively investigated FEN1 mRNA expression in multiple cohorts of breast cancer [training set (128), test set (249), external validation (1952)]. FEN1 protein expression was evaluated in 568 oestrogen receptor (ER) negative breast cancers, 894 ER positive breast cancers and 156 ovarian epithelial cancers. FEN1 mRNA overexpression was highly significantly associated with high grade (p=4.89 × 10−57), high mitotic index (p=5.25 × 10−28), pleomorphism (p=6.31 × 10−19), ER negative (p=9.02 × 10−35), PR negative (p=9.24 × 10−24), triple negative phenotype (p=6.67 × 10−21), PAM50.Her2 (p=5.19 × 10−13), PAM50.Basal (p=2.7 × 10−41), PAM50.LumB (p=1.56 × 10−26), integrative molecular cluster 1 (intClust.1) (p=7.47 × 10−12), intClust.5 (p=4.05 × 10−12) and intClust. 10 (p=7.59 × 10−38) breast cancers. FEN1 mRNA overexpression is associated with poor breast cancer specific survival in univariate (p= 4.4 × 10−16) and multivariate analysis (p= 9.19 × 10−7). At the protein level, in ER positive tumours, FEN1 overexpression remains significantly linked to high grade, high mitotic index and pleomorphism (ps<0.01). In ER negative tumours, high FEN1 is significantly associated with pleomorphism, tumour type, lymphovascular invasion, triple negative phenotype, EGFR and HER2 expression (ps<0.05). In ER positive as well as in ER negative tumours, FEN1 protein overexpression is associated with poor survival in univariate and multivariate analysis (ps<0.01). In ovarian epithelial cancers, similarly, FEN1 overexpression is associated with high grade, high stage and poor survival (ps<0.05). We conclude that FEN1 is a promising biomarker in breast and ovarian epithelial cancer.
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