Abstract
For wide applications of the lacZ gene in cellular/molecular biology, small animal investigations, and clinical assessments, the improvement of noninvasive imaging approaches to precisely assay gene expression has garnered much attention. In this study, we investigate a novel molecular platform in which alizarin 2-O-β-d-galactopyranoside AZ-1 acts as a lacZ gene/β-gal responsive 1H-MRI probe to induce significant 1H-MRI contrast changes in relaxation times T 1 and T 2 in situ as a concerted effect for the discovery of β-gal activity with the exposure of Fe3+. We also demonstrate the capability of this strategy for detecting β-gal activity with lacZ-transfected human MCF7 breast and PC3 prostate cancer cells by reaction-enhanced 1H-MRI T 1 and T 2 relaxation mapping.
Highlights
Due to various advantages such as stability, high turnover rate, and ease of conjugation, the lacZ gene-encoding β-galactosidase (β-gal) has been broadly used in cellular/molecular biology, small animal studies, clinical trials with assays of clonal insertion, transcriptional activation, and protein expression and interaction (Kruger et al, 1999; Haberkorn et al, 2005; Razgulin et al, 2011; Yang et al, 2019)
To explore the magnetic resonance imaging (MRI) signal–enhancing capability of the Fe3+–alizarin complex, the spin–lattice relaxation time than AZ-1/ FAC solution (T1) and spin–spin relaxation time T2 of the Fe3+–alizarin complex were measured with a 4.7 T MR scanner using a saturation recovery spin echo sequence and multi-spin echo sequence with varying repetition times (TRs) and echo times (TEs), respectively
The images were acquired using a 3-cm diameter solenoid coil with 4 × 4 cut section of a 96-well plate containing the different concentrations of alizarin and ferric ammonium citrate (FAC) mixed solutions in 1:1 (V/V’) dimethyl sulfoxide (DMSO)/phosphate-buffered saline (PBS) (0.1 M, pH 7.4) at 37°C
Summary
Due to various advantages such as stability, high turnover rate, and ease of conjugation, the lacZ gene-encoding β-galactosidase (β-gal) has been broadly used in cellular/molecular biology, small animal studies, clinical trials with assays of clonal insertion, transcriptional activation, and protein expression and interaction (Kruger et al, 1999; Haberkorn et al, 2005; Razgulin et al, 2011; Yang et al, 2019). In reviewing the histopathological literature, we noticed that the well-established β-gal substrate alizarin 2-O-β-D-galactopyranoside AZ-1 (Figure 1) is readily hydrolyzed by β-gal to release aglycone alizarin, which chelates with ferric iron Fe3+ to form an intense dark violet Fe complex (James et al, 2000). Upon delivery and cleavage at lacZ-transfected or β-gal–overexpressed tumor cells with the presence of Fe3+, the paramagnetic Fe complex could be spontaneously formed in situ and produced the 1H MRI contrast effect while localizing and accumulating 1H-MRI signals at the β-gal activity site. We demonstrate the use of exploiting AZ-1 to assess β-gal activity in vitro with lacZ-transfected human MCF7 breast and PC3 prostate cancer cells by 1H MRI T1 and T2 relaxation mapping
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