Julie L. Dahlheimer

Noninvasive imaging of protein-protein interactions in living animals

Protein–protein interactions control transcription, cell division, and cell proliferation as well as mediate signal transduction, oncogenic transformation, and regulation of cell death. Although a variety of methods have been used to investigate protein interactions in vitro and in cultured cells, none can analyze these interactions in intact, living animals. To enable noninvasive molecular imaging of protein–protein interactions in vivo by positron-emission tomography and fluorescence imaging, we engineered a fusion reporter gene comprising a mutant herpes simplex virus 1 thymidine kinase and green fluorescent protein for readout of a tetracycline-inducible, two-hybrid system in vivo. By using micro-positron-emission tomography, interactions between p53 tumor suppressor and the large T antigen of simian virus 40 were visualized in tumor xenografts of HeLa cells stably transfected with the imaging constructs. Imaging protein-binding partners in vivo will enable functional proteomics in whole animals and provide a tool for screening compounds targeted to specific protein–protein interactions in living animals.

Characterization of a novel 99mTc-carbonyl complex as a functional probe of MDR1 P-glycoprotein transport activity

Multidrug resistance (MDR) mediated by overexpression of MDR1 P-glycoprotein (Pgp) is one of the best characterized barriers to chemotherapy in cancer patients. Furthermore, the protective function of Pgp-mediated efflux of xenobiotics in various organs has a profound effect on the bioavailability of drugs in general. Thus, there is an expanding requirement to noninvasively interrogate Pgp transport activity in vivo. We herein report the Pgp recognition properties of a novel 99mTc(I)-tricarbonyl complex, [99mTc(CO)3(MIBI)3]+ (Tc-CO-MIBI). Tc-CO-MIBI showed 60-fold higher accumulation in drug-sensitive KB 3-1 cells compared to colchicine-selected drug-resistant KB 8-5 cells. In KB 8-5 cells, tracer enhancement was observed with the potent MDR modulator LY335979 (EC50 = 62 nM). Similar behavior was observed using drug-sensitive MCF-7 breast adenocarcinoma cells and MCF-7/MDR1 stable transfectants, confirming that Tc-CO-MIBI is specifically excluded by overexpression of MDR1 Pgp. By comparison, net accumulation in control H69 lung tumor cells was 9-fold higher than in MDR-associated protein (MRP1)-expressing H69AR cells, indicating only modest transport by MRP1. Biodistribution analysis following tail vein injection of Tc-CO-MIBI showed delayed liver clearance as well as enhanced brain uptake and retention in mdr1a/1b(-/-) gene deleted mice versus wild-type mice, directly demonstrating that Tc-CO-MIBI is a functional probe of Pgp transport activity in vivo.

In vitro and in vivo characterization of a dual-function green fluorescent protein-HSV1-thymidine kinase reporter gene driven by the human elongation factor 1α promoter

Toward the goal of monitoring activity of native mammalian promoters with molecular imaging techniques, we stably transfected DU145 prostate carcinoma cells with a fusion construct of enhanced green fluorescent protein (EGFP) and wild-type herpes simplex virus-1 thymidine kinase (HSV1-TK) as a reporter gene driven by the promoter for human elongation factor 1a a (EF-1a-EGFP-TK). Using this model system, expression of EGFP was quantified by flow cytometry and fluorescence microscopy, while the HSV1-TK component of the reporter was quantified with 8-[ 3 H]ganciclovir (8-[ 3 H]GCV). As analyzed by flow cytometry, passage of EGFP-TK-DU145 transfected cells (ETK) in vitro resulted in populations of cells with high and low expression of EGFP over time. High and low ETK cells retained 23-fold and 5-fold more GCV, respectively, than control. While differences in uptake and retention of GCV corresponded to relative expression of the reporter gene in each subpopulation of cells as determined by both flow cytometry (EGFP) and quantitative RT-PCR, the correlation was not linear. Furthermore, in high ETK cells, net retention of various radiolabeled nucleoside analogues varied; the rank order was 8-[ 3 H]GCV < 9-(4-fluoro-3-hydroxymethylbutyl)guanine ([ 18 F]FHBG) � 8-[ 3 H]penciclovir (8-[ 3 H]PCV) < 2 0 -fluoro2 0 -deoxy-5-iodouracil-beta-D-arabinofuranoside (2-[ 14 C]FIAU). Xenograft tumors of ETK cells in vivo accumulated 2.5-fold more 8-[ 3 H]GCV per gram of tissue and showed greater fluorescence from EGFP than control DU145 cells, demonstrating that the reporter gene functioned in vivo. These data extend previous reports by showing that a human promoter can be detected in vitro and in vivo with a dual-function reporter exploiting optical and radiotracer techniques. Mol Imaging (2002) 1, 65 – 73.

Reversal of multidrug resistance with LY335979: Functional analysis of P-glycoprotein-mediated transport activity and its modulation in vivo

E of multidrug resistance (MDR) is a major obstacle to successful chemotherapy of cancer. Several of the best characterized mechanisms of MDR include transporter-mediated resistance conferred by increased expression of the Mr 170,000 transmembrane glycoprotein, P-glycoprotein (Pgp), the product of the MDR1 gene, and a related Mr 190,000 membrane glycoprotein, the multidrug resistance-associated protein (MRP1). Other ATP-binding-cassette (ABC) transporters have been reported to be associated with MDR, including the breast cancer resistance protein (BCRP/MXR/ABCP; a recently cloned ABC “half-transporter”), and MRP3. Cells in culture exhibiting MDR by selection in cytotoxic drugs or transfection with these recombinant transporters generally show reduced net drug accumulation and altered intracellular drug distribution. MDR1 Pgp, MRP1, BCRP/MXR/ABCP, and related ABC transporters have been targets for cancer therapy on two fronts. First, reversal of multidrug resistance in tumor cells by nontoxic agents that block the transport activity of these ABC proteins has been an important target of pharmaceutical development. Known as MDR modulators, these agents enhance net accumulation of coadministered cytotoxic compounds within MDR tumor cells. Several high-potency modulators are now in clinical trials. Second, transgenic expression of the MDR1 gene has been explored for hematopoietic cell protection in the context of cancer chemotherapy, wherein Pgp could protect hematopoietic progenitor cells from chemotherapy-induced myelosupression. For proper use of MDR modulators as well as monitoring MDR gene therapy in chemotherapeutic protocols, identification of transporter-mediated resistance could guide the choice of agents and provide important prognostic information for cancer patients. Thus, noninvasive molecular imaging with a transport substrate serving as a surrogate marker of chemotherapeutic agents may identify those tumors and tissues in which ABC transporter proteins are expressed and active. To achieve this goal, several gamma-emitting compounds have been synthesized, validated, and characterized as transport substrates for MDR1 Pgp. One of the best characterized is hexakis(2-methoxyisobutyl isonitrile)Tc(I) ([Tc]Sestamibi), a widely available radiopharmaceutical, 2 2 6 which may enable scintigraphic analysis of MDR with imaging cameras commonly available in nuclear medicine facilities. Targeted synthesis and validation of other single-photon radiopharmaceuticals, including the commercially available agent, [1,2-bis{bis(2-ethoxyethyl)phosphino} ethane]2O2 Tc(V) ([Tc]Tetrofosmin; Myoview), and positron-emission tomography (PET) agents have been explored for imaging MDR. Most of these radiopharmaceuticals are cationic and modestly hydrophobic, which is similar to many chemotherapeutic drugs in the MDR phenotype. For example, [Tc]Sestamibi accumulates within cells in response to the physiologically negative mitochondrial and plasma membrane potentials. Thus, cellular accumulation of [Tc]Sestamibi into drug-sensitive tumor cells is high and translates into a “hot spot” on scintigraphic images or a slow washout rate from a tu-