High-resolution in vivo imaging of gene expression is not possible in opaque animals by existing techniques. Here we present a new approach for obtaining such images by magnetic resonance imaging (MRI) using an MRI contrast agent that can indicate reporter gene expression in living animals. We have prepared MRI contrast agents in which the access of water to the first coordination sphere of a chelated paramagnetic ion is blocked with a substrate that can be removed by enzymatic cleavage. Following cleavage, the paramagnetic ion can interact directly with water protons to increase the MR signal. Here, we report an agent where galactopyranose is the blocking group. This group renders the MRI contrast agent sensitive to expression of the commonly used marker gene, beta-galactosidase. To cellular resolution, regions of higher intensity in the MR image correlate with regions expressing marker enzyme. These results offer the promise of in vivo mapping of gene expression in transgenic animals and validate a general approach for constructing a family of MRI contrast agents that respond to biological activity.
This report describes the synthesis, characterization, and in vivo testing of several bifunctional contrast-enhancing agents for optical and magnetic resonance imaging (MRI) of experimental animals. These new agents integrate the advantages of both techniques since they can be visualized simultaneously by light and MRI microscopy. Employing this strategy allows the same biological structures of a specimen to be studied at dramatically different resolutions and depths. The complexes possess a metal chelator for binding a paramagnetic ion, gadolinium (Gd3+), and a covalently attached fluorescent dye. The first class of complexes are low-molecular weight species that are composed of the macrocyclic tetraamine 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) as the metal-chelating ligand coupled to tetramethylrhodamine. The second class of MRI-enhancing agents are composed of high-molecular weight polymers that are membrane impermeable and once injected into a cell or cells are trapped inside. These complexes possess multiple copies of both the metal-chelator-diethylenetriaminepentaacetic acid (DTPA) and the tetramethylrhodamine attached to a macromolecular framework of either poly(D-lysine) (pdl) or dextran. Images acquired of single cells after injection with these bifunctional agents enabled us to follow the relative motions and reorganizations of different cell layers during amphibian gastrulation and neurulation in Xenopus laevis embryos.
In search of novel mechanisms leading to the development of antiestrogen-resistance in human breast tumors, we analyzed differences in the gene and protein expression pattern of the human breast carcinoma cell line T47D and its derivative T47D-r, which is resistant toward the pure antiestrogen ZM 182780 (Faslodex TM , fulvestrant). Affymetrix DNA chip hybridizations on the commercially available HuGeneFL and Hu95A arrays were carried out in parallel to the proteomics analysis where the total cellular protein content of T47D or T47D-r was separated on twodimensional gels. Thirty-eight proteins were found to be reproducibly up-or down-regulated more than 2-fold in T47D-r versus T47D in the proteomics analysis. Comparison with differential mRNA analysis revealed that 19 of these were up-or down-regulated in parallel with the corresponding mRNA molecules, among which are the protease cathepsin D, the GTPases Rab11a and MxA, and the secreted protein hAG-2. For 11 proteins, the corresponding mRNA was not found to be differentially expressed, and for eight proteins an inverse regulation was found at the mRNA level. In summary, mRNA expression data, when combined with proteomic information, provide a more detailed picture of how breast cancer cells are altered in their antiestrogen-resistant compared with the antiestrogen-sensitive state. Molecular & Cellular Proteomics 3:43-55, 2004.Endocrine therapy plays an important role in the management of breast cancer at various stages (1). In the clinic, tamoxifen is the most widely used nonsteroidal antiestrogen (2). It is effective in first-line and adjuvant treatments. However, the fact that tamoxifen has partial agonistic activity in the uterus and that long-term treatment was shown to result in tamoxifen-resistant breast tumors led to the development of the pure antiestrogen ZM 182780 (Faslodex TM , fulvestrant, ICI 182780), which lacks estrogen receptor (ER) 1 ␣ agonistic activity in all tissues, decreases the half-life of the ER␣ and is still effective after tamoxifen failure (3). Fulvestrant has been tested in phase III clinical trials versus tamoxifen for first line therapy of advanced breast cancer and versus the aromatase inhibitor anastrozole in patients with tamoxifen-refractory breast cancer (4). Fulvestrant was at least as effective and equally well tolerated as anastrozole for the treatment of postmenopausal women with advanced and metastatic breast cancer and gained approval from the U.S. Food and Drug Administration in 2002 (2). In cell culture and xenograft models, resistance toward ZM 182780 occurs later than after tamoxifen treatment; therefore, ZM 182780 may provide a longer duration of response compared with tamoxifen (5, 6).Breast tumors as well as breast cancer cell lines have the capability to adapt to the prevailing concentration of antiestrogen, resulting in resistance to the drug. The mechanisms that lead to antiestrogen-resistance in breast cancer patients as well as in cell culture model systems are still poorly understood to date (7).Therefore, w...
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