Fluorescent probes for cancer detection.

Several new fluorescent dyes, derivatives of pyrene and of coumarin, were synthesized that have excitation and emission wavelength maxima considerably red-shifted as compared to most pyrene and coumarin compounds. These new fluorescent compounds have high extinction coefficients and high quantum yields, and they also are very environmentally sensitive, which makes them good probes of biological systems. Several of these fluorescent compounds are preferentially taken up and retained by leukemic and other cancer tissue cell lines as compared to normals, particularly 1,3-dihydroxy 6,8-pyrenedisodiumsulfonate and 3-(carboxymethylester)-7-julolidinocoumarin. These compounds may have some usefulness in cancer detection.

Tumor cell specific dark cytotoxicity of light-exposed merocyanine 540: implications for systemic therapy without light.

Merocyanine 540 (MC540) was activated by exposure to 514 nm laser light. The light-exposed MC540 was then mixed (in the dark) with tumor cells and normal cells to determine the antiproliferative activity. Treatment with light-exposed MC540 resulted in 70-90% tumor cell kill from different cell lines, while 85% of the normal human mononuclear cells and 41% of the granulocyte-macrophage colony forming cells (CFU-GM) survived the treatment. The observed cytotoxicity of light-exposed MC540 to the tumor cells was significantly greater (P less than 0.05) than the native MC540. Results show that tumor cell specificity and cytotoxicity in the light activated dye are retained for at least 30 days. Addition of catalase and mannitol decreased the cell kill by light-exposed compound, indicating that the observed effects may be due to reactive oxygen species. The electron micrographs of treated cells show a progression towards apoptosis in a majority of the cells. The life span of L1210 leukemia-bearing mice treated with light-exposed MC540 was prolonged compared to the untreated and native MC540 treated mice. High pressure liquid chromatography (HPLC) analysis of light-exposed material shows a completely different elution profile compared to the native compound. Results presented here show that light-exposed photoactive compounds can be used without further illumination and may have significant clinical applications. Photoactive mechanisms dependent on events other than short-lived transient elevations in energy or singlet oxygen must be invoked to explain the reported cytotoxicity.

Fluorescent cationic probes of mitochondria. Metrics and mechanism of interaction.

Mitochondria strongly accumulate amphiphilic cations. We report here a study of the association of respiring rat liver mitochondria with several fluorescent cationic dyes from differing structural classes. Using gravimetric and fluorometric analysis of dye partition, we find that dyes and mitochondria interact in three ways: (a) uptake with fluorescence quenching, (b) uptake without change in fluorescence intensity, and (c) lack of uptake. For dyes that quench upon uptake, the extent of quenching correlates with the degree of aggregation of the dye to dimers, as predicted by theory (Tomov, T.C. 1986. J. Biochem. Biophys. Methods. 13:29-38). Also predicted is the relationship observed between quenching and the mitochondria concentration when constant dye is titrated with mitochondria. Not predicted is the relationship observed between quenching and dye concentration when constant mitochondria are titrated with dye. Because a limit to dye uptake exists, in this case, the degree of quenching decreases as dye is added. A Langmuir isotherm analysis gives phenomenological parameters that predict quenching when it is observed as a function of dye concentration. By allowing for a decrease in membrane potential, caused by incorporation of cationic dye into the lipid bilayer, a modification of the Tomov theory predicts the dye titration data. We present a model of cationic dye-mitochondria interaction and discuss the use of these as probes of mitochondrial membrane potential.

Increased survival of normal cells during laser photodynamic therapy: implications for ex vivo autologous bone marrow purging.

Laser light-induced, dye-mediated photolysis of leukemic cells was tested in an in vitro model for its efficacy in eliminating occult tumor cells for ex vivo autologous bone marrow purging. Merocyanine 540 (MC540) was mixed with acute promyelocytic leukemia (HL-60) cells in the presence of human albumin. This cell-dye mixture was irradiated with 514 nm argon laser light. Results show that in the presence of 0.1%, 0.25% and 0.5% albumin, laser light doses of 62.4 J/cm2, 93.6 J/cm2 and 109.2 J/cm2, respectively, were required for a 5 log reduction in the survival of leukemic cells. Under identical conditions, 80% to 84% of the normal bone marrow cells and 41% of the granulocyte-macrophage colony forming cells survived. The number of surviving stromal cells was reduced (1+) compared to the untreated control (4+). Mixing of irradiated bone marrow cells with equal number of HL-60 cells did not interfere with the killing of HL-60 cells treated with MC540 and laser light. The non-specific cytotoxicity of laser light alone was less than 6% for normal bone marrow cells. These results suggest that the concentration of human albumin plays an important role in laser light-induced phototoxicity. This laser light-induced selective photolysis of leukemic cells can be used in ex vivo purging of tumor cell-contaminated bone marrow grafts to achieve very high survival rates of normal bone marrow cells and granulocyte-macrophage colony forming cells.

Elimination of leukemic cells by laser photodynamic therapy.

We studied the effects of 514-nm laser light-induced merocyanine 540 (MC540)-mediated toxicity on both leukemic and normal bone marrow (BM) cells. Acute promyelocytic leukemia (HL-60) cells were incubated with MC540 (20 micrograms/ml) and exposed to 93.6 J/cm2 irradiation at a 514-nm wavelength. Normal bone marrow cells were treated under similar conditions. At this dose, 99.9999% of the leukemic cells were killed while 55% of the BM cell survived. Of the granulocyte-macrophage colony-forming cells (CFU-GM), 27% also survived this treatment. Photosensitization of a mixture of irradiated BM cells mixed with an equal number of nonirradiated HL-60 cell did not interfere with the killing of HL-60 cells. There was no significant reduction in the viability of cells when exposed to the laser light alone. In summary, laser light-induced photosensitization with MC540 has a selective cytotoxicity to leukemic cells; therefore, this procedure may be useful for purging neoplastic cells from autologous BM.

McArdle's disease in an elderly woman.

We have described a woman who was 71 years old when McArdle's disease was first diagnosed. She had few symptoms even during severe myoglobinuria. Muscle breakdown occurred upon relatively mild exertion. Others of her kindred had elevated serum creatine kinase levels and reported stiffness and muscle weakness upon relatively mild exertion. This case demonstrates the need to be alert to relatively mild manifestations of metabolic diseases.

Calcium regulates troponin-tropomyosin binding in the reconstituted thin filament.

Steady-state fluorescence anisotropy technique was used to determine the binding constant of troponin for IAEDANS-labeled tropomyosin under various conditions. In the absence of actin, Ca does not affect the binding between troponin and tropomyosin. The presence of actin greatly strengthens troponin-tropomyosin binding in the absence of Ca. However, Ca weakens troponin-tropomyosin binding by about 2.5-fold in the reconstituted filament. It is suggested that the Ca-regulated binding may serve as a molecular switch for the troponin molecule to get "on" and "off" the actin-myosin interaction site regulating muscle contraction-relaxation cycles.

Studies on the spatial arrangement of muscle thin filament proteins using fluorescence energy transfer.

The distance between a pair of fluorophores attached to Cys-36 of beta-tropomyosin and Cys-373 of actin in reconstituted muscle thin filaments was measured by fluorescence energy transfer. Two pairs of donor/acceptor fluorophores, N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid/5-iodoacetamidofluorescein and N-(1-pyrene)maleimide/dimethylamino-4-maleimidostilbene, were covalently attached to tropomyosin and actin. The energy transfer efficiencies in various reconstituted systems were determined from decrease in donor lifetime using nanosecond pulse fluorimetry. Based on the 8 to 13% energy transfer efficiency observed for the first donor/acceptor pair labeled on tropomyosin and actin, respectively, and a calculated critical distance of 45 A (assumed kappa 2 = 2/3) the distance between Cys-373 of actin and Cys-36 of beta-tropomyosin was estimated to be 65 A. Fluorescence energy transfer experiments using other donor/acceptor pairs gave similar results. Since Cys-373 of actin is thought to be in the myosin head-binding sites, the minimal distance between tropomyosin and the myosin-binding site on actin is estimated to be about 34.5 A. These results place some constraints on possible spatial arrangements of thin filament proteins.

Characterization of beta-adrenergic receptor subtypes in androgen-induced mouse kidney hypertrophy using a new high-affinity ligand, [125I]iodocyanopindolol.

In fully developed androgen-induced hypertrophy of female mouse kidney, beta-adrenergic receptors per unit membrane protein were increased approx. 2.5-fold, as measured by the binding of [125I]iodocyanopindolol, with no change in apparent dissociation constants (Kd range 20-25 pM). Membrane protein relative to total kidney protein, Na+/K+-dependent ATPase (EC 3.6.1.3) and 5'-nucleotidase (EC 3.1.3.5) activities and cholesterol content per unit membrane protein did not differ significantly in preparations from control and treated animals. The binding of iodocyanopindolol to kidney membranes was characterized with respect to association and dissociation kinetics, and also in regard to the less-specific contributions of other major catecholamine or indolamine receptors, using mixtures of the corresponding specific competitors. beta 1-selective drugs, practolol and metoprolol, and beta 2-selective agents, IPS-339 and zinterol, were competed with iodocyanopindolol to assess the receptor type specificity, and the ensuing binding profiles were dissected by a nonlinear regression analysis as described by Munson, P.J. and Rodbard, D. (Anal. Biochem. (1982) 107, 220-239). Most of the androgen-induced beta-adrenergic receptors had the binding properties corresponding to beta 2-subtype. No consistent increase in the density of beta 1-adrenergic receptors could be shown.

Fluorescence spectroscopic studies of pyrene-actin adducts.

Reaction kinetic studies of the sulfhydryl-directed fluorescent probes N-(1-pyrene)maleimide (PM) and N-(1-pyrenyl)iodoacetamide with actin from rabbit skeletal muscle showed that there were three accessible sulfhydryl groups in actin. Fluorescence spectral studies showed energy transfer from aromatic amino acid residues to fluorophore reacted at Cys-373, as well as weak excimer fluorescence probably due to doubly labeled molecules at Cys-10 and Cys-373. These results provide further evidence that trytophan and tyrosine residues are located near the probe attached to Cys-373 or Cys-10 and the latter two thiols are in close proximity. In age PM-labeled F-actin, the succinimido ring of PM underwent intramolecular aminolysis, resulting in large emission spectral changes and increased excimer fluorescence. Solvent perturbation studies indicate that the probes were located in a hydrophobic environment; their quantum yield and spectrum properties were very sensitive to changes in the microenvironment. Nanosecond-pulse fluorimetry studies revealed complex fluorescence emission decays with three intrinsic lifetimes in adducts with low molecular weight thiols as well as in labeled proteins. Fluorescence lifetimes were 17, 48 and 111 ns for the pyrenemaleimide adduct of actin, and 3, 14 and 60 ns for the pyrenyliodoacetamide adduct. Supporting evidence is given for the argument that multiple fluorescence lifetimes are an intrinsic property of the pyrene derivatives and are not due to the presence of impurity or heterogeneity in the protein reaction sites. Because of their high sensitivity and long lifetimes, pyrene derivatives are extremely useful.

Increased beta-adrenergic receptor complement in androgen-induced mouse kidney hypertrophy.

Treatment of female mice with testosterone propionate led to a pronounced, but gradual increase in kidney beta-adrenergic receptor complement. The specific binding of [125I]iodohydroxybenzylpindolol rose 2-3-fold above the control levels after 8-12 days of the treatment. No significant changes were detected prior to the fourth day of androgen administration. No gross changes in either the binding strength or cooperativity of the binding were apparent in membrane preparations from treated animals. Averages of the high-affinity binding constant estimates were 1.3 +/- 0.3 nmol in controls, vs. 1.6 +/- 0.5 nmol in treated animals (15 groups each) in competition with pindolol, with the Hill slope factors of 0.98 +/- 0.08 for controls, and 0.91 +/- 0.07 for the treated animal membrane preparations. Scatchard estimates of the binding constants in self-competed [125I]iodohydroxybenzylpindolol binding were about 160 pmol in both control and treated animals. Competition experiments using isoproterenol also indicated similar dissociation constants (151 +/- 16 nmol) for control and treated groups. Na+/K+-activated ATPase (EC 3.6.1.3) was also found to be increased at the 12th day of the androgen treatment (to 74% above control levels).

Ribosome-membrane interactions: characterization of ribosomal proteins from loose and tight bound ribosomes.

Membrane-bound ribosomes were isolated from a post-mitochondrial supernatant fraction of mouse liver homogenate by sedimentation in a sucrose density gradient, Loose ribosomes were released from the membrane fragments with 0.5 M KCl, while tight bound ribosomes were not released. After purification of the loose and tight ribosomes subclasses, ribosomal subunit proteins were isolated and compared by two-dimensional polyacrylamide gel electrophoresis. No differences in the ribosomal protein composition was detected.

A general ellipsoid cannot always serve as a model for the rotational diffusion properties of arbitrarily shaped rigid molecules.

An ellipsoidally shaped body, or more commonly, an ellipsoid of revolution, is generally assumed to serve as a convenient model for evaluating the rotational diffusion properties of macromolecules. If Perrin's equations for the rotational diffusion coefficients of general ellipsoids can be shown to generate all possible rotational diffusion coefficients, then there would exist at least one equivalent ellipsoidal shape for every arbitrarily shaped rigid body. We investigated the problem by first generating a space, r-space, representing all possible ellipsoidal shapes. We then generated another space, D-space, representing all possible combinations of rotational diffusion coefficients. We then mapped r-space into D-space by using Perrin's equations. Ellipsoidal shapes map into diffusion space in a well-defined manner. The mapping is either 1:1, 2:1, or 3:1; several distinctly different regions of r-space map onto the same regions of D-space. Thus, for some combinations of rotational diffusion coefficients, more than one ellipsoid can be used as a model. Not all of D-space is covered by the mapping of r-space. Therefore, there are combinations of rotational diffusion coefficients that cannot be generated from ellipsoidally shaped bodies. Several examples of rigid body shapes with nonellipsoidal diffusion properties are described.

Tissue-specific ribosomal protein composition.

Membrane-bound and free polysomes from murine liver and kidney were isolated under identical conditions and their ribosomal proteins were compared by two-dimensional gel electrophoresis. The results demonstrate that these ribosome subpopulations are quantitatively and qualitatively similar except for the presence of one additional protein in the kidney-bound polysomal fraction.