Inhibition of DNA replication in an Escherichia coli dnaB-22 mutant failed to block quinolone-mediated lethality. Inhibition of protein synthesis by chloramphenicol inhibited nalidixic acid lethality and, to a lesser extent, ciprofloxacin lethality in both dnaB-22 and wild-type cells. Thus, major features of quinolone-mediated lethality do not depend on ongoing replication.Quinolones are broad-spectrum antibacterials that trap DNA gyrase and DNA topoisomerase IV on DNA as ternary complexes containing double-strand DNA breaks (reviewed in reference 5). The ternary complexes, which block DNA replication (8, 23), RNA synthesis (13, 24), and cell growth (7), lead to cell death by poorly understood processes. Quinolone-mediated inhibition of DNA replication, which is very rapid (19), is unlikely to account for rapid lethality because inhibition is reversible (4, 8) and because concurrent interruption of RNA or protein synthesis eliminates the lethal action of some quinolones but not their ability to block DNA replication (2, 4). A different issue is whether ongoing DNA replication is required for quinolone lethality. Such a hypothesis could be derived from studies with other topoisomerases in which collision of replication forks with ternary complexes is thought to release double-strand DNA breaks (3, 21). To address this possibility with quinolones, we blocked DNA replication by shifting a dnaB mutant of Escherichia coli to nonpermissive temperature. (DnaB is a DNA helicase [11] required for replication fork movement [1].) Subsequent treatment with quinolone killed the mutant, indicating that ongoing DNA replication is not required.E. coli strain K12SH-28 and its dnaB-22(Ts) mutant FA22 (6) were obtained from the E. coli Stock Center (Yale University) and were grown at 28°C in M9-glucose minimal medium (14) supplemented with 0.05% Casamino Acids and 1% LB medium (14). A gyrA quinolone-resistant mutant of FA22, strain KD2672, was constructed by P1-mediated transduction (22) from strain KD2346, a spontaneous ciprofloxacin-resistant derivative of KD1366 (26) that contained GyrA amino acid substitutions Ser-83 to Leu and Asp-87 to Tyr. The dnaB-22 allele was transduced into strain DM4100 (20) using a nearby tetracycline resistance marker (malF-3089:Tn10) to produce strain KD2773. Nalidixic acid, chloramphenicol, and tetracycline were products of Sigma Chemical Co. (St. Louis, MO); ciprofloxacin was obtained from Bayer Corp. (West Haven, CT). Bactericidal activity was determined by incubation in the presence of quinolone followed by dilution and growth on drug-free agar at 28°C for 1 to 2 days. The rate of DNA synthesis was measured by incubating 100 l bacterial culture with 0.1 Ci [ 3 H]thymidine for 2 min followed by determination of acid-precipitable radioactivity.When the dnaB-22 mutant was grown at 28°C and shifted to 42°C, the rate of DNA synthesis dropped by more than 95% within 10 min and was not inhibited further by subsequent nalidixic acid treatment (not shown). Under these conditions, the viable cell number r...

The research was to appraise the utility of the patient-derived tumor xenografts (PDXs) as models of estrogen receptor positive (ER+HER2− and ER+HER2+) breast cancers. We compared protein expression profiles by Reverse Phase Protein Array (RPPA) in tumors that resulted in PDXs compared to those that did not. Our overall PDX intake rate for ER+ breast cancer was 9% (9/97). The intake rate for ER+HER2+ tumors (3/16, 19%) was higher than for ER+HER2− tumors (6/81, 7%). Heat map analyses of RPPA data showed that ER+HER2− tumors were divided into 2 groups by luminal A/B signature [protein expression of ER, AR, Bcl-2, Bim (BCL2L11), GATA3 and INPP4b], and this expression signature was also associated with the rate of PDX intake. Cell survival pathways such as the PI3K/AKT signaling and RAS/ERK pathways were more activated in the specimens that could be established as PDX in both classes. Expression of the ER protein itself may have a bearing on the potential success of an ER+ PDX model. In addition, HER2 and its downstream protein expressions were up-regulated in the ER+HER2+ patient tumors that were successfully established as PDX models. Moreover, the comparison of RPPA data between original and PDX tumors suggested that the selection/adaptation process required to grow the tumors in mice is unavoidable for generation of ER+ PDX models, and we identified differences between patient tumor samples and paired PDX tumors. A better understanding of the biological characteristics of ER+PDX would be the key to using PDX models in assessing treatment strategies in a preclinical setting.

Many estrogen receptor alpha (ERα)-positive breast cancers initially respond to aromatase inhibitors (AIs), but eventually acquire resistance. Here, we report that serum-and glucocorticoid-inducible kinase 3 (SGK3), a kinase transcriptionally regulated by ERα in breast cancer, sustains ERα signaling and drives acquired AI resistance. SGK3 is up-regulated and essential for endoplasmic reticulum (EnR) homeostasis through preserving sarcoplasmic/EnR calcium ATPase 2b (SERCA2b) function in AI-resistant cells. We have further found that EnR stress response down-regulates ERα expression through the protein kinase RNA-like EnR kinase (PERK) arm, and SGK3 retains ERα expression and signaling by preventing excessive EnR stress. Our study reveals regulation of ERα expression mediated by the EnR stress response and the feed-forward regulation between SGK3 and ERα in breast cancer. Given SGK3 inhibition reduces AI-resistant cell survival by eliciting excessive EnR stress and also depletes ERα expression/function, we propose SGK3 inhibition as a potential effective treatment of acquired AI-resistant breast cancer.SGK3 | aromatase inhibitor | endoplasmic reticulum stress | estrogen receptor | SERCA2

Choroidal neovascularization (CNV) is a blinding complication of age-related macular degeneration that manifests as the growth of immature choroidal blood vessels through Bruch’s membrane, where they can leak fluid or hemorrhage under the retina. Here, we demonstrate that the histone deacetylase inhibitor (HDACi) trichostatin A (TSA) can down-regulate the pro-angiogenic hypoxia-inducible factor-1α and vascular endothelial growth factor (VEGF), and up-regulate the anti-angiogenic and neuro-protective pigment epithelium derived factor in human retinal pigment epithelial (RPE) cells. Most strikingly, TSA markedly down-regulates the expression of VEGF receptor-2 in human vascular endothelial cells and, thus, can knock down pro-angiogenic cell signaling. Additionally, TSA suppresses CNV-associated wound healing response and RPE epithelial-mesenchymal transdifferentiation. In the laser-induced model of CNV using C57Bl/6 mice, systemic administration of TSA significantly reduces fluorescein leakage and the size of CNV lesions at post—laser days 7 and 14 as well as the immunohistochemical expression of VEGF, VEGFR2, and smooth muscle actin in CNV lesions at post-laser day 7. This report suggests that TSA, and possibly HDACi’s in general, should be further evaluated for their therapeutic potential for the treatment of CNV.