The microculture-kinetic (MiCK) assay: the role of a drug-induced apoptosis assay in drug development and clinical care.

A drug-induced apoptosis assay, termed the microculture-kinetic (MiCK) assay, has been developed. Blinded clinical trials have shown higher response rates and longer survival in groups of patients with acute myelocytic leukemia and epithelial ovarian cancer who have been treated with drugs that show high apoptosis in the MiCK assay. Unblinded clinical trials in multiple tumor types have shown that the assay will be used frequently by clinicians to determine treatment, and when used, results in higher response rates, longer times to relapse, and longer survivals. Model economic analyses suggest possible cost savings in clinical use based on increased generic drug use and single-agent substitution for combination therapies. Two initial studies with drugs in development are promising. The assay may help reduce costs and speed time to drug approval. Correlative studies with molecular biomarkers are planned. This assay may have a role both in personalized clinical therapy and in more efficient drug development.

Calmodulin wraps around its binding domain in the plasma membrane Ca2+ pump anchored by a novel 18-1 motif.

Using solution NMR spectroscopy, we obtained the structure of Ca(2+)-calmodulin (holoCaM) in complex with peptide C28 from the binding domain of the plasma membrane Ca(2+)-ATPase (PMCA) pump isoform 4b. This provides the first atomic resolution insight into the binding mode of holoCaM to the full-length binding domain of PMCA. Structural comparison of the previously determined holoCaM.C20 complex with this holoCaM.C28 complex supports the idea that the initial binding step is represented by (holoCaM.C20) and the final bound complex by (holoCaM.C28). This affirms the existing multi-step kinetic model of PMCA4b activation by CaM. The complex exhibits a new binding motif in which holoCaM is wrapped around helical C28 peptide using two anchoring residues from the peptide at relative positions 18 and 1. The anchors correspond to Phe-1110 and Trp-1093, respectively, in full-length PMCA4b, and the peptide and CaM are oriented in an anti-parallel manner. This is a greater sequence distance between anchors than in any of the known holoCaM complexes with a helical peptide. Analysis of the geometry of holoCaM-peptide binding for the cases where the target peptide adopts an alpha(D)-helix with its anchors buried in the main hydrophobic pockets of the two CaM lobes establishes that only relative sequential positions of 10, 14, 17, and 18 are allowed for the second anchor.

Quantitation of circulating tumor cells in blood samples from ovarian and prostate cancer patients using tumor-specific fluorescent ligands.

Quantitation of circulating tumor cells (CTCs) can provide information on the stage of a malignancy, onset of disease progression and response to therapy. In an effort to more accurately quantitate CTCs, we have synthesized fluorescent conjugates of 2 high-affinity tumor-specific ligands (folate-AlexaFluor 488 and DUPA-FITC) that bind tumor cells >20-fold more efficiently than fluorescent antibodies. Here we determine whether these tumor-specific dyes can be exploited for quantitation of CTCs in peripheral blood samples from cancer patients. A CTC-enriched fraction was isolated from the peripheral blood of ovarian and prostate cancer patients by an optimized density gradient centrifugation protocol and labeled with the aforementioned fluorescent ligands. CTCs were then quantitated by flow cytometry. CTCs were detected in 18 of 20 ovarian cancer patients (mean 222 CTCs/ml; median 15 CTCs/ml; maximum 3,118 CTCs/ml), whereas CTC numbers in 16 gender-matched normal volunteers were negligible (mean 0.4 CTCs/ml; median 0.3 CTCs/ml; maximum 1.5 CTCs/ml; p < 0.001, chi(2)). CTCs were also detected in 10 of 13 prostate cancer patients (mean 26 CTCs/ml, median 14 CTCs/ml, maximum 94 CTCs/ml) but not in 18 gender-matched healthy donors (mean 0.8 CTCs/ml, median 1, maximum 3 CTC/ml; p < 0.0026, chi(2)). Tumor-specific fluorescent antibodies were much less efficient in quantitating CTCs because of their lower CTC labeling efficiency. Use of tumor-specific fluorescent ligands to label CTCs in peripheral blood can provide a simple, accurate and sensitive method for determining the number of cancer cells circulating in the bloodstream.

Immunohistochemical expression of folate receptor alpha in colorectal carcinoma: patterns and biological significance.

Folate receptor alpha (FRalpha) has emerged as a potential cancer therapy target with several folate-linked therapeutic agents currently undergoing clinical trials. In addition, FRalpha expression in tumors may offer prognostic significance. Most studies on FRalpha expression used reverse transcriptase polymerase chain reaction or cytofluorimetric assays. The applicability of such methods to paraffin-embedded tissues is limited. The aims of this study were to assess the feasibility of immunohistochemistry in detecting FRalpha expression and to assess the patterns and clinical significance of FRalpha expression in colorectal tissues. We used tissue microarrays containing 152 normal colorectal mucosa samples, 42 adenomas, 177 primary, and 52 metastatic colorectal carcinomas. Our results showed that staining for FRalpha on colorectal tissues was simple and easy to read. FRalpha positivity was more frequent in carcinomas (33% in primaries and 44% in metastases) than in normal mucosa or adenoma (7% in both) (P < .001). Positive staining in primary carcinomas correlated with younger age (n = 130) (P = .008), presence of distant metastasis (n = 130) (P = .043), and non-high-frequency microsatellite instability status (as detected by the standard polymerase chain reaction method using the 5 National Cancer Institute-recommended markers) (n = 77) (P = .006). Positive staining in primary carcinomas also correlated with a worse 5-year disease-specific survival (P = .04) on univariate but not multivariate analysis. Thus, our data show that there is selective expression of FRalpha in some colorectal cancers, providing a foundation for investigating the use of folate conjugates for imaging and therapy of colorectal tumors. Furthermore, our results suggest that a possible association exists between FRalpha expression and the microsatellite instability status in colorectal carcinoma. The significance of such an association as well as the prognostic value of FRalpha expression deserves further exploration.

Measurement of intermolecular distances for the natural agonist Peptide docked at the cholecystokinin receptor expressed in situ using fluorescence resonance energy transfer.

Fluorescence resonance energy transfer is a powerful biophysical technique used to analyze the structure of membrane proteins. Here, we used this tool to determine the distances between a distinct position within a docked agonist and a series of distinct sites within the intramembranous confluence of helices and extracellular loops of the cholecystokinin (CCK) receptor. Pseudo-wild-type CCK receptor constructs having single reactive cysteine residues inserted into each of these sites were developed. The experimental strategy included the use of the full agonist, Alexa488-CCK, bound to these receptors as donor, with Alexa568 covalently bound to the specific sites within the CCK receptor as acceptor. Site-labeling was achieved by derivatization of intact cells with a novel fluorescent methanethiosulfonate reagent. A high degree of spectral overlap was observed between receptor-bound donor and receptor-derivatized acceptors, with no transfer observed for a series of controls representing saturation of the receptor binding site with nonfluorescent ligand and use of a null-reactive CCK receptor construct. The measured distances between the fluorophore within the docked agonist and the sites within the first (residue 102) and third (residue 341) extracellular loops of the receptor were shorter than those directed to the second loop (residue 204) or to intramembranous helix two (residue 94). These distances were accommodated well within a refined molecular model of the CCK-occupied receptor that is fully consistent with all existing structure-activity and photoaffinity-labeling studies. This approach provides the initial insights into the conformation of extracellular loop regions of this receptor and establishes clear differences from analogous loops in the rhodopsin crystal structure.

Water and urea interactions with the native and unfolded forms of a beta-barrel protein.

A fundamental understanding of protein stability and the mechanism of denaturant action must ultimately rest on detailed knowledge about the structure, solvation, and energetics of the denatured state. Here, we use (17)O and (2)H magnetic relaxation dispersion (MRD) to study urea-induced denaturation of intestinal fatty acid-binding protein (I-FABP). MRD is among the few methods that can provide molecular-level information about protein solvation in native as well as denatured states, and it is used here to simultaneously monitor the interactions of urea and water with the unfolding protein. Whereas CD shows an apparently two-state transition, MRD reveals a more complex process involving at least two intermediates. At least one water molecule binds persistently (with residence time >10 nsec) to the protein even in 7.5 M urea, where the large internal binding cavity is disrupted and CD indicates a fully denatured protein. This may be the water molecule buried near the small hydrophobic folding core at the D-E turn in the native protein. The MRD data also provide insights about transient (residence time <1 nsec) interactions of urea and water with the native and denatured protein. In the denatured state, both water and urea rotation is much more retarded than for a fully solvated polypeptide. The MRD results support a picture of the denatured state where solvent penetrates relatively compact clusters of polypeptide segments.

H-bonding mediates polarization of peptide groups in folded proteins.

The carbon-nitrogen J-couplings in the hydrogen bonding chains of proteins show that H-bonding mediates peptide-group polarization, which results in the general reduction of peptide-group polarity of folded proteins in solution. The net effect is to make large regions of protein secondary structure, especially beta-sheets, intrinsically more hydrophobic, contributing thereby to overall stability of the tertiary structure.

Structural dependencies of h3JNC' scalar coupling in protein H-bond chains.

The H-bond ((h3)J(NC')) and peptide bond ((1)J(NC')) scalar couplings establish connectivity of the electronic structure in the H-bond chains of proteins. The correlated changes of (h3)J(NC') and (1)J(NC') couplings extend over several peptide groups in the chains. Consequently, the electronic structure of the H-bond chains can affect (h3)J(NC') in a manner that is independent of the local H-bond geometry. By taking this into account, and by using a more complete set of H-bond geometry parameters, we have predicted (h3)J(NC') couplings in the H-bond chains with deviations commensurate to the standard deviations of the experimentally determined values. We have created a comprehensive database of (h3)J(NC') and (1)J(NC') couplings by measuring the coupling constants in ubiquitin (alphabeta-fold) intestinal fatty acid binding protein (beta-barrel) and carp parvalbumin (alpha-helical).

Environment and mobility of a series of fluorescent reporters at the amino terminus of structurally related peptide agonists and antagonists bound to the cholecystokinin receptor.

Fluorescence is a powerful biophysical tool for the analysis of the structure and dynamics of proteins. Here, we have developed two series of new fluorescent probes of the cholecystokinin (CCK) receptor, representing structurally related peptide agonists and antagonists. Each ligand had one of three distinct fluorophores (Alexa(488), nitrobenzoxadiazolyl, or acrylodan) incorporated in analogous positions at the amino terminus just outside the hormone's pharmacophore. All of the probes bound to the CCK receptor specifically and with high affinity, and intracellular calcium signaling studies showed the chemically modified peptides to be fully biologically active. Quenching by iodide and measurement of fluorescence spectra, anisotropy, and lifetimes were used to characterize the response of the fluorescence of the probe in the peptide-receptor complex for agonists and antagonists. All three fluorescence indicators provided the same insights into differences in the environment of the same indicator in the analogous position for agonist and antagonist peptides bound to the CCK receptor. Each agonist had its fluorescence quenched more easily and showed lower anisotropy (higher mobility of the probe) and shorter lifetime than the analogous antagonist. Treatment of agonist-occupied receptors with a non-hydrolyzable GTP analogue shifted the receptor into its inactive low affinity state and increased probe fluorescence lifetimes toward values observed with antagonist probes. These data are consistent with a molecular conformational change associated with receptor activation that causes the amino terminus of the ligand (situated above transmembrane segment six) to move away from its somewhat protected environment and toward the aqueous milieu.