Pharmacological regulation of c-myc gene expression in human breast cancer cells

Previous studies demonstrated that quinidine causes G1/G0 cell cycle arrest and inhibition of proliferation in MCF-7 human breast cancer cell line (Woodfork, K. et al., 1995). The goal of studies reported here was to understand the molecular mechanisms of c-myc gene regulation by quinidine. C-myc is one of the most common oncogene aberrations in breast cancers (Deming, S. L. et al., 2000). C-myc functions include regulation of cell cycle, proliferation, differentiation, and apoptosis. The results of these studies demonstrated that quinidine causes rapid (within 1 hour) suppression of Myc protein and mRNA levels that precedes the quinidine-induced G1 cell cycle arrest point (D point) in MCF-7 cells. Additionally, the activity of c-myc promoter was suppressed by quinidine over the same range of concentrations that suppress levels of myc mRNA and protein, suggesting that changes in Myc protein and mRNA levels by quinidine may be attributed to its effect on myc promoter. A 168 by region of c-myc promoter (-100 to +68 in respect to P1) was identified as a quinidine responsive region (QRR). Suppression of Myc by quinidine was consistent with inhibition of growth and induction of more differentiated phenotype in four different breast tumor cell lines. In contrast, quinidine had minimal effect on Myc levels or proliferation in normal mammary epithelial cell lines. Furthermore, MCF-7 cells treated with c-myc antisense oligonucleotides exhibited cytoplasmic lipid droplets, similarly to the quinidine-treated cells, suggesting that suppression of Myc may play a causative role in the induction of more differentiated phenotype by quinidine in human breast cancer cells

Chemical identity and mechanisms of action and formation of a cell growth inhibitory leachable compound from disposable polycarbonate plastic vessels

This presentation reports the chemical identity and mechanisms of action and formation of a single cell growth inhibitory leachable compound from disposable polycarbonate plastic vessels. 18 leachable compounds were separated and enriched from the total extracts of the vessels. Out of these compounds, only one displayed selective inhibition of the growth of a CHO cell line. High resolution MS and NMR suggested that the leachable compound is a dinitrobisphenol A. Chemical synthesis further confirmed that this leachable compound is one of the seven possible dinitrobisphenol A. Cell assays revealed that this leachable compound acted not only as a cell cycle arrest agent without any effect on cell viability, but also as a GPR35 agonist with moderate potency. Examining the manufacturing process of polycarbonate vessels showed that the formation of this leachable compound requires the combination of three critical process parameters, high temperature molding process, gamma-irradiation, and the presence of air. This study highlights that besides the resin materials the manufacturing process also contributes to the formation of cell growth inhibitory leachable compounds

Identification of FIP200 interaction with the TSC1–TSC2 complex and its role in regulation of cell size control

FIP200 (focal adhesion kinase [FAK] family interacting protein of 200 kD) is a newly identified protein that binds to the kinase domain of FAK and inhibits its kinase activity and associated cellular functions. Here, we identify an interaction between FIP200 and the TSC1–TSC2 complex through FIP200 binding to TSC1. We found that association of FIP200 with the TSC1–TSC2 complex correlated with its ability to increase cell size and up-regulate S6 kinase phosphorylation but was not involved in the regulation of cell cycle progression. Conversely, knockdown of endogenous FIP200 by RNA interference reduced S6 kinase phosphorylation and cell size, which required TSC1 but was independent of FAK. Furthermore, overexpression of FIP200 reduced TSC1–TSC2 complex formation, although knockdown of endogenous FIP200 by RNA interference did not affect TSC1–TSC2 complex formation. Lastly, we showed that FIP200 is important in nutrient stimulation-induced, but not energy- or serum-induced, S6 kinase activation. Together, these results suggest a cellular function of FIP200 in the regulation of cell size by interaction with the TSC1–TSC2 complex

Preparation of Mouse Monoclonal Antibody for RB1CC1 and Its Clinical Application

RB1-inducible coiled-coil 1 (RB1CC1; also known as FIP200) plays important roles in several biological pathways such as cell proliferation and autophagy. Evaluation of RB1CC1 expression can provide useful clinical information on various cancers and neurodegenerative diseases. In order to realize the clinical applications, it is necessary to establish a stable supply of antibody and reproducible procedures for the laboratory examinations. In the present study, we have generated mouse monoclonal antibodies for RB1CC1, and four kinds of antibodies (N1-8, N1-216, N3-2, and N3-42) were found to be optimal for clinical applications such as ELISA and immunoblots and work as well as the pre-existing polyclonal antibodies. N1-8 monoclonal antibody provided the best recognition of RB1CC1 in the clinico-pathological examination of formalin-fixed paraffin-embedded tissues. These monoclonal antibodies will help to generate new opportunities in scientific examinations in biology and clinical medicine

Concise Review: The Evolution of human pluripotent stem cell culture: From feeder cells to synthetic coatings

Current practices to maintain human pluripotent stem cells (hPSCs), which include induced pluripotent stem cells and embryonic stem cells, in an undifferentiated state typically depend on the support of feeder cells such as mouse embryonic fibroblasts (MEFs) or an extracellular matrix such as Matrigel. Culture conditions that depend on these undefined support systems limit our ability to interpret mechanistic studies aimed at resolving how hPSCs interact with their extracellular environment to remain in a unique undifferentiated state and to make fate‐changing lineage decisions. Likewise, the xenogeneic components of MEFs and Matrigel ultimately hinder our ability to use pluripotent stem cells to treat debilitating human diseases. Many of these obstacles have been overcome by the development of synthetic coatings and bioreactors that support hPSC expansion and self‐renewal within defined culture conditions that are free from xenogeneic contamination. The establishment of defined culture conditions and synthetic matrices will facilitate studies to more precisely probe the molecular basis of pluripotent stem cell self‐renewal and differentiation. When combined with three‐dimensional cultures in bioreactors, these systems will also enable large‐scale expansion for future clinical applications. S TEM C ells 2013;31:1–7Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/94687/1/1260_ftp.pd

Feeder Cells Support the Culture of Induced Pluripotent Stem Cells Even after Chemical Fixation

Chemically fixed mouse embryonic fibroblasts (MEFs), instead of live feeder cells, were applied to the maintenance of mouse induced pluripotent stem (miPS) cells. Formaldehyde and glutaraldehyde were used for chemical fixation. The chemically fixed MEF feeders maintained the pluripotency of miPS cells, as well as their undifferentiated state. Furthermore, the chemically fixed MEF feeders were reused several times without affecting their functions. These results indicate that chemical fixation can be applied to modify biological feeders chemically, without losing their original functions. Chemically fixed MEF feeders will be applicable to other stem cell cultures as a reusable extracellular matrix candidate that can be preserved on a long-term basis