The DASL-based expression profiling assay applied to RNA extracted from MCF-7 cells, before or after 24 h stimulation with a mitogenic dose of 17 beta-estradiol, consistently allowed to detect hormone-induced gene expression changes following extensive RNA degradation in vitro. Comparable results where obtained with tumor RNA extracted from FFPE MT biopsies (6 to 19 years old). The method proved itself sensitive, reproducible and accurate, when compared to results obtained by microarray analysis
of RNA extracted from snap-frozen tissue of the same tumor.”
“Purpose: PHA-848125 in vitro Since the late 1980s, cocaine analogues based on the phenyltropane structure, such as [C-11]CFT and [I-123]beta-CIT have been used
for the imaging of the dopamine transporter. selleck kinase inhibitor FE@CIT (fluoropropyl ester) and FP-CIT (N-fluoropropyl derivative) are further analogues. The aim of this study was to (1) evaluate and compare the metabolic stability of beta-CIT, FP-CIT and FE@CIT against carboxyl esterases and (2) evaluate selectivity of [F-18]FE@CIT compared to [I-123]beta-CIT and [I-123]FP-CIT using autoradiography.
Methods: In vitro enzymatic hydrolysis assays were performed using different concentrations of beta-CIT, FE@CIT and FP-CIT with constant concentrations of carboxyl esterase. Autoradiography was performed on coronal 20-mu m rat brain sections incubated with different radioactivity concentrations of [I-123]beta-CIT, [123 I]FP-CIT or [F-18]FE@CIT and, additionally, with 3-amino-4-(2-dimethylaminomethylphenylsulfanyl)-benzonitrile Loperamide [serotonin transporter (SERT)] and nisoxetine [norepinephrine transporter (NET)] for blocking experiments.
Results: In vitro assays showed Michaelis-Menten constants of 175 mu mol (beta-CIT), 183 mu mol (FE@CIT) and 521 mu mol (FP-CIT). Limiting velocities were 0.1005
mu mol/min (beta-CIT), 0.1418 mu mol/min (FE@CIT) and 0.1308 mu mol/min (FP-CIT). This indicates a significantly increased stability of FP-CIT, whereas carboxyl esterase stability of beta-CIT and FE@CIT showed no significant difference. Autoradiographic analyses revealed a good correlation between dopamine transporter (DAT)-rich regions and the uptake pattern of FE@CIT. Blocking experiments showed a higher DAT selectivity for [F-18]FE@CIT than for the other two tracers.
Conclusion: We found that (1) the metabolic stability of FE@CIT was comparable to that of beta-CIT, whereas FP-CIT showed higher resistance to enzymatic hydrolysis; and (2) the overall uptake pattern of [F-18]FE@CIT on brain slices was comparable to that of[I-123]beta-CIT and [I-123]FPCIT. After blocking of NET and SERT binding, a significantly higher DAT selectivity was observed for [F-18]FE@CIT. Hence, [F-18]FE@CIT may be of interest for further clinical application. (C) 2008 Elsevier Inc. All rights reserved.