Mutational analysis of human RNA polymerase II subunit 5 (RPB5): The residues critical for interactions with TFIIF subunit RAP30 and hepatitis B virus X protein

金沢大学がん研究所RNA polymerase II (RNAPII) subunit 5 (RPB5) is positioned close to DNA downstream of the initiation site and is the site of interaction with several regulators. Hepatitis B virus X protein (HBx) binds the central part of RPB5 to modulate activated transcription, and TFIIF subunit RAP30 interacts with the same part of RPB5 that is critical for the association between TFIIF and RNAPII. However the residues necessary for these interactions remain unknown. Here we report systematic mutagenesis of the central part of RPB5 using two-step alanine scanning libraries to pinpoint critical residues for its binding to RAP30 in the TFIIF complex and/or to HBx, and identified these residues in both mammalian cells and in an in vitro binding assay. Four residues, F76, I104, T111 and S113, are critical for both TFIIF- and HBx-binding, indicating the overlapping nature of the sites of interaction. In addition, V74 and N98 are required for HBx-binding, and T56 and L58 are needed for RAP30-binding. Interestingly the residues exposed to solvent, T111 and S113, are very close to the DNA, implying that two factors may modulate the interaction between DNA and RPB5. © 2005 The Japanese Biochemical Society

Quantitative Analysis of Serum Procollagen Type I C-Terminal Propeptide by Immunoassay on Microchip

BACKGROUND: Sandwich enzyme-linked immunosorbent assay (ELISA) is one of the most frequently employed assays for clinical diagnosis, since this enables the investigator to identify specific protein biomarkers. However, the conventional assay using a 96-well microtitration plate is time- and sample-consuming, and therefore is not suitable for rapid diagnosis. To overcome these drawbacks, we performed a sandwich ELISA on a microchip. METHODS AND FINDINGS: The microchip was made of cyclic olefin copolymer with straight microchannels that were 300 µm wide and 100 µm deep. For the construction of a sandwich ELISA for procollagen type I C-peptide (PICP), a biomarker for bone formation, we used a piezoelectric inkjet printing system for the deposition and fixation of the 1st anti-PICP antibody on the surface of the microchannel. After the infusion of the mixture of 2.0 µl of peroxidase-labeled 2nd anti-PICP antibody and 0.4 µl of sample to the microchannel and a 30-min incubation, the substrate for peroxidase was infused into the microchannel; and the luminescence intensity of each spot of 1st antibody was measured by CCD camera. A linear relationship was observed between PICP concentration and luminescence intensity over the range of 0 to 600 ng/ml (r(2) = 0.991), and the detection limit was 4.7 ng/ml. Blood PICP concentrations of 6 subjects estimated from microchip were compared with results obtained by the conventional method. Good correlation was observed between methods according to simple linear regression analysis (R(2) = 0.9914). The within-day and between-days reproducibilities were 3.2-7.4 and 4.4-6.8%, respectively. This assay reduced the time for the antigen-antibody reaction to 1/6, and the consumption of samples and reagents to 1/50 compared with the conventional method. CONCLUSION: This assay enabled us to determine serum PICP with accuracy, high sensitivity, time saving ability, and low consumption of sample and reagents, and thus will be applicable to clinic diagnosis

Eribulin Mesylate Targets Human Telomerase Reverse Transcriptase in Ovarian Cancer Cells

<div><p>Treatment of advanced ovarian cancer involves platinum-based chemotherapy. However, chemoresistance is a major obstacle. Cancer stem cells (CSCs) are thought to be one of the causes of chemoresistance, but the underlying mechanism remains elusive. Recently, human telomerase reverse transcriptase (hTERT) has been reported to promote CSC-like traits. In this study, we found that a mitotic inhibitor, eribulin mesylate (eribulin), effectively inhibited growth of platinum-resistant ovarian cancer cell lines. Eribulin-sensitive cells showed a higher efficiency for sphere formation, suggesting that these cells possess an enhanced CSC-like phenotype. Moreover, these cells expressed a higher level of hTERT, and suppression of hTERT expression by siRNA resulted in decreased sensitivity to eribulin, suggesting that hTERT may be a target for eribulin. Indeed, we found that eribulin directly inhibited RNA-dependent RNA polymerase (RdRP) activity, but not telomerase activity of hTERT <i>in</i><i>vitro</i>. We propose that eribulin targets the RdRP activity of hTERT and may be an effective therapeutic option for CSCs. Furthermore, hTERT may be a useful biomarker to predict clinical responses to eribulin.</p></div

Eribulin inhibits RdRP activity but not telomerase activity of hTERT.

<p>(A) RdRP activity of hTERT immune complexes prepared from HeLa cells arrested in the mitotic phase was assayed without or with 10 and 50 µM eribulin. (B) Telomerase activity in HeLa cell extracts was assayed without or with 10 and 50 µM eribulin. (C) RdRP activity of hTERT immune complexes was assayed without or with 10 and 100 µM paclitaxel (PTX).</p

Eribulin inhibits growth of cisplatin-resistant ovarian cancer cells.

<p>Cells were treated with cisplatin or eribulin for 72 h, and then cell viability was determined by MTT assays. (A) Mean IC₅₀ values for cisplatin (µM). (B) Mean IC₅₀ values for eribulin (nM). Eribulin-sensitive (Eribulin S) cell lines are shown as open bars, and eribulin-resistant (Eribulin R) cell lines are shown as closed bars. Error bars represent the SD of at least three independent experiments.</p

Suppression of hTERT expression by siRNA results in decreased sensitivity to eribulin.

<p>(A) A2780 cells expressing control siRNA (open bars), TERT siRNA1 (closed bars), or TERT siRNA2 (shaded bars) were treated with eribulin for 72 h, and then cell viability was determined by MTT assays. *p<0.05 vs. cells expressing control siRNA. (B) The level of hTERT protein expression in A2780 cells expressing control siRNA (open bars), TERT siRNA1 (closed bars), or TERT siRNA2 (shaded bars) was determined by ELISA (indicated as ng/ml). (C and D) The experiments described in (A and B) were performed in the same manner using ES-2 cells expressing control siRNA (open bars), TERT siRNA1 (closed bars), or TERT siRNA2 (shaded bars). *p<0.05 vs. cells expressing control siRNA.</p

Eribulin-sensitive ovarian cancer cells express higher levels of hTERT protein.

<p>(A) The level of hTERT protein expression was determined by ELISA (indicated as ng/ml). Eribulin S cell lines are shown as open bars, and Eribulin R cells are shown as closed bars. Each experiment was performed at least three times, and mean values ± SD are indicated. (B) The mean hTERT level of Eribulin S cell lines (n = 8) and Eribulin R cell lines (n = 6) shown in (A). Error bars indicate SD.</p

ATF7 mediates TNF-α–induced telomere shortening

Telomeres maintain the integrity of chromosome ends and telomere length is an important marker of aging. The epidemiological studies suggested that many types of stress including psychosocial stress decrease telomere length. However, it remains unknown how various stresses induce telomere shortening. Here, we report that the stress-responsive transcription factor ATF7 mediates TNF-α–induced telomere shortening. ATF7 and telomerase, an enzyme that elongates telomeres, are localized on telomeres via interactions with the Ku complex. In response to TNF-α, which is induced by various stresses including psychological stress, ATF7 was phosphorylated by p38, leading to the release of ATF7 and telomerase from telomeres. Thus, a decrease of ATF7 and telomerase on telomeres in response to stress causes telomere shortening, as observed in ATF7-deficient mice. These findings give credence to the idea that various types of stress might shorten telomere