Isolation of Reporter Cells That Respond to Vitamin A and/or D Using a piggyBac Transposon Promoter-Trapping Vector System

Previously, we established a highly sensitive promoter-trapping vector system using the piggyBac transposon for the efficient isolation of reporter cells. Herein, we examine whether this screening system can be applied to obtain vitamin-responsive cells. As a result, one and two reporter cells that responded to bexarotene (vitamin A) and calcitriol (vitamin D), respectively, were isolated from 4.7 × 106 seeded HeLaS3 cells. 5′ RACE analyses identified the well-known CYP24A1 gene as a calcitriol-responsive gene, as well as two new bexarotene- or calcitriol-responsive genes, BDKRB2 and TSKU, respectively. TSKU, interestingly, also responded to bexarotene. Endogenous levels of the TSKU and BDKRB2 transcripts displayed only slight changes and were not detected in the comprehensive analyses performed to date. Dose–response analyses of BDKRB2 and TSKU reporter cells in parallel revealed a differential profile in response to each vitamin A agonist, suggesting a bioanalyzer. The present study demonstrates that producing multiple reporter cells by a type of random screening can efficiently identify novel genes with unusual characteristics and be used for the profiling of the properties of vitamin compounds. Similar approaches to the method shown here may be useful for identifying new markers and for the analysis or diagnosis of nutrients, toxins, metabolites, etc

Tyrosine Kinase Inhibitor Profiling Using Multiple Forskolin-Responsive Reporter Cells

We have developed a highly sensitive promoter trap vector system using transposons to generate reporter cells with high efficiency. Using an EGFP/luciferase reporter cell clone responsive to forskolin, which is thought to activate adenylate cyclase, isolated from human chronic myelogenous leukemia cell line K562, we found several compounds unexpectedly caused reporter responses. These included tyrosine kinase inhibitors such as dasatinib and cerdulatinib, which were seemingly unrelated to the forskolin-reactive pathway. To investigate whether any other clones of forskolin-responsive cells would show the same response, nine additional forskolin-responsive clones, each with a unique integration site, were generated and quantitatively evaluated by luciferase assay. The results showed that each clone represented different response patterns to the reactive compounds. Also, it became clear that each of the reactive compounds could be profiled as a unique pattern by the 10 reporter clones. When other TKIs, mainly bcr-abl inhibitors, were evaluated using a more focused set of five reporter clones, they also showed unique profiling. Among them, dasatinib and bosutinib, and imatinib and bafetinib showed homologous profiling. The tyrosine kinase inhibitors mentioned above are approved as anticancer agents, and the system could be used for similarity evaluation, efficacy prediction, etc., in the development of new anticancer agents

Synthesis of a Chloroalkene Dipeptide Isostere-Containing Peptidomimetic and Its Biological Application

The first rapid and efficient chemical synthesis of a cyclic Arg-Gly-Asp (RGD) peptide containing a chloroalkene dipeptide isostere (CADI) is reported. By a developed synthetic method, an <i>N</i>-<i>tert</i>-butylsulfonyl protected CADI was obtained utilizing diastereoselective allylic alkylation as a key reaction. This CADI was also transformed into an <i>N</i>-Fmoc protected CADI in a few steps. The CADI was used in Fmoc-based solid-phase peptide synthesis. The first synthesis of a CADI-containing cyclic RGD peptide was successful, and the synthesized CADI-containing peptidomimetic was found to be a more potent inhibitor against integrin-mediated cell attachment than the parent cyclic peptide

Dosimetric advantages of daily adaptive strategy in IMPT for high-risk prostate cancer

Purpose To evaluate the dosimetric advantages of daily adaptive radiotherapy (DART) in intensity-modulated proton therapy (IMPT) for high-risk prostate cancer by comparing estimated doses of the conventional non-adaptive radiotherapy (NART) that irradiates according to an original treatment plan through the entire treatment and the DART that uses an adaptive treatment plan generated by using daily CT images acquired before each treatment. Methods Twenty-three patients with prostate cancer were included. A treatment plan with 63 Gy (relative biological effectiveness (RBE)) in 21 fractions was generated using treatment planning computed tomography (CT) images assuming that all patients had high-risk prostate cancer for which the clinical target volume (CTV) needs to include prostate and the seminal vesicle (SV) in our treatment protocol. Twenty-one adaptive treatment plans for each patient (total 483 data sets) were generated using daily CT images, and dose distributions were calculated. Using a 3 mm set-up uncertainty in the robust optimization, the doses to the CTV, prostate, SV, rectum, and bladder were compared. Results Estimated accumulated doses of NART and DART in the 23 patients were 60.81 +/- 3.47 Gy (RBE) and 63.24 +/- 1.04 Gy (RBE) for CTV D99 (p < 0.01), 62.99 +/- 1.28 Gy (RBE) and 63.43 +/- 1.33 Gy (RBE) for the prostate D99 (p = 0.2529), and 59.07 +/- 5.19 Gy (RBE) and 63.17 +/- 1.04 Gy (RBE) for SV D99 (p < 0.001). No significant differences were observed between NART and DART in the estimated accumulated dose for the rectum and bladder. Conclusion Compared with the NART, DART was shown to be a useful approach that can maintain the dose coverage to the target without increasing the dose to the organs at risk (OAR) using the 3 mm set-up uncertainty in the robust optimization in patients with high-risk prostate cancer