Modulation of phorbol ester-induced HL-60 differentiation by prostaglandin E2.

When treated with phorbol tumor promoters, HL-60 cells undergo terminal differentiation evidenced by a transition from a non-phagocytic suspension culture to an attached fibroblast-like culture with high phagocytic activity. Internalization of fluorescent particles by cells exhibiting the phagocytic positive phenotype (phag+) provides a sensitive indication of promoter-induced differentiation, and the resulting fluorescent cells can be quantitatively analyzed by flow cytometry. The current study was initiated to further test the predictive power of a flow cytometry based HL-60 differentiation assay in the detection of agents associated with tumor promotion. Specifically, experiments were designed to assess the sensitivity of the test system to co-promoters which enhance promoter activity in vivo. Prostaglandin E2 (PGE2) was chosen as a model co-promoter since it has been shown to potentiate phorbol ester (i.e. 12-O-tetradecanoyl phorbol-13-acetate; TPA) induced biological effects in vivo. Results detailed in the current report indicate that PGE2 enhances TPA-induced differentiation of HL-60 cells in a dose-dependent manner. As with in vivo co-promotion experiments, PGE2 exhibited a maximum potentiating effect when administered prior to TPA. These data indicate that HL-60 cells are not only sensitive to phorbol promoters, but also to the co-promoter PGE2. These experiments support the hypothesis that a flow cytometry based HL-60 assay may prove useful for studying chemical agents or intrinsic cellular factors that are involved in the tumor promotion phase of carcinogenesis.

Development of a sensitive in vitro method for identifying tumor promoters.

The identification and characterization of nongenotoxic carcinogens represents a significant challenge to toxicologists. In vitro methods for identifying tumor promoters with suitable sensitivity and specificity have been particularly elusive. Experiments are described which suggest that the human promyelocytic leukemia cell line HL-60 provides a sensitive indicator of promoter-induced changes to gene regulation and expression. As a result of differentiation these cells undergo a transition from a non-phagocytic suspension culture to an attached fibroblast-like culture which exhibits high phagocytic activity. Fluorescent latex particles were used as sensors to highlight the phagocytic phenotype and permitted the use of flow cytometry to automatically quantitate particle internalization. To evaluate specificity, HL-60 cells were treated with a series of phorbol esters covering a range of in vivo tumor promoting activity. Results indicate that this family of compounds induces HL-60 cells to differentiate in proportion to their in vivo promoting activity. To closely assess the sensitivity of the phagocytic endpoint, HL-60 cells were treated with picogram levels of 12-O-tetradecanoyl phorbol-13-acetate (TPA), whereupon increments as low as 50 pg of TPA per ml caused statistically significant increases in phagocytic activity. The experiments described herein suggest that in vitro differentiation of HL-60 cells may reflect the promoter-dependent modifications to gene expression that are observed in vivo during the promotion phase of carcinogenesis. The described method may represent a sensitive promoter screening assay which is both rapid and economical.

Analysis of micronucleated cells by flow cytometry. 4. Kinetic analysis of cytogenetic damage in blood.

Micronucleated cells (MN cells) generated spontaneously or by clastogen action accumulate in the peripheral blood of the mouse, and their presence reflects the level of chromosome damage. Traditionally, micronucleated cells have been scored by visual inspection. With the development of flow cytometry based scoring procedures, vast numbers of cells can be analyzed, making it possible to determine the change in the number of MN cells in the total peripheral blood pool. This report describes experiments whereby initial blood samples were obtained before dosing, providing mouse-specific controls for measuring subsequent changes in MN cells. Mice were then dosed with saline (solvent control), methyl methanesulfonate, cyclophosphamide or colchicine every 48 h and bled every 96 h for 12 days. For each blood sample, one million fixed erythrocytes (RBCs) were interrogated for the presence of micronuclei, and regression analysis was used to determine the rate of MN cell influx per day for each animal or sets of animals. To evaluate the effect of treatment on MN induction, the mean slopes of solvent and chemically treated animals were compared using t-tests. The results of these experiments indicate that the kinetics of MN induction continues near the background frequency for saline dosed mice, whereas clastogenic agents or spindle poisons cause a significant influx of MN events into the blood. The results suggest that some studies may benefit from a flow cytometry based analysis of multiple blood samples, especially when the number of mice is limited, or when a weak clastogen is being investigated.

Analysis of micronucleated cells by flow cytometry. 2. Evaluating the accuracy of high-speed scoring.

Micronucleated blood cells--whether generated spontaneously or by clastogen treatment--are present in the blood and bone marrow as rare events. Historically they have been scored manually by microscopic inspection which is labor-intensive and tedious. It has been recognized by investigators that a need exists for an automated method which can accurately, objectively and quantitatively score rare micronucleated cells. In order to improve assay statistics more cells must be processed, making high-speed scoring an important objective of any automated procedure. Flow cytometry can provide the means to quantitatively analyze micronucleated cells at high speeds and with great accuracy once the chemical, biological and instrumentation conditions are optimized. Recent literature suggests that noise and fidelity of the data, as well as the sensitivity of present flow cytometers, are major obstacles that still must be overcome. Experiments are described herein which demonstrate that flow cytometry is able to score micronucleated cells under conditions where noise levels are low, and the fidelity and accuracy are high. In addition, the accuracy of scoring rare events is maintained at high speeds (e.g. 1,000,000 cells/min). A major emphasis of this manuscript is to demonstrate the means for evaluating the accuracy and sensitivity of the flow cytometer in scoring rare events. Both computer simulation and reconstruction experiments were used to gauge scoring accuracy and guided optimization experiments. These experiments demonstrate that when optimum conditions are used in conjunction with a suitable flow cytometer, it is possible to score micronucleated cells at high speeds with great precision.

Analysis of micronucleated cells by flow cytometry. 1. Achieving high resolution with a malaria model.

Micronucleated cells (MN cells) are present in the blood as rare events (i.e. about 2 MN cells/1000 total). Scoring MN cells by hand is both time-consuming and tedious, which is the primary reason why only 1000-2000 total cells (PCEs) are routinely scored for each sample. It is generally recognized that scoring larger numbers of cells would improve assay statistics and is desirable, but impractical with hand-scoring. In contrast, automated scoring methods can process large numbers of cells, thus improving statistical analysis. In order to accurately and quickly evaluate clastogenic activity, we have developed a flow cytometry based method of scoring micronucleated cells. One of the first steps in developing an automated assay is to demonstrate the ability of the method to resolve the cells of interest. In this case, micronucleated cells must be resolved from DNA-deficient red blood cells (RBCs). Since micronuclei are heterogeneous rare events which vary in both size and DNA content, we chose to use a more enriched and homogeneous biological model for optimizing the experimental variables of this assay, leading to high resolution of the rare cells. Experiments are described in which the murine malaria parasite, P. berghei, served as a micronucleus model and facilitated the development of an accurate flow cytometry based scoring method. This parasite resides in the red blood cell population and endows the cells with a homogeneous (genetically determined) DNA component in the micronucleus size range. The conditions developed with the malaria parasite are readily applied to the analysis of micronucleus events in blood samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of micronucleated cells by flow cytometry. 3. Advanced technology for detecting clastogenic activity.

Under optimum conditions, flow cytometry (FCM) can provide a powerful technology for analyzing rare micronucleated cells in the peripheral blood. Our efforts in this endeavor have been directed toward a careful and meticulous optimization of experimental conditions, in order to achieve high resolution and high accuracy before introducing biological variation. We have achieved high resolution (Tometsko et al., 1993a) wherein the micronucleus signal is moved 100-fold upfield and away from the DNA deficient red blood cell (RBC) peak. In addition, we have demonstrated the high accuracy of our flow cytometry method in scoring rare micronucleated cells (Tometsko et al., 1993b). In the course of our studies, we rigorously pursued conditions which minimized experimental noise, demanding that FCM-scoring accuracy should approach theoretical limits. Thus, we laid the foundation for detecting clastogen activity with great sensitivity. The experiments described herein extend the previous studies by using high-speed flow cytometry to detect a clastogen-induced increase in MN cells in the total erythrocyte population. Methyl methanesulfonate (MMS) served as a model clastogen in these studies. This manuscript describes our development of a suitable blood-sampling regimen, the advantages of obtaining initial blood samples before dosing, the sex-linked difference in background micronucleus levels in BALB/c mice, and the analysis of a clastogen-induced biological response in male and female mice. As described, our flow cytometry method is able to provide a quantitative analysis of the net change in micronucleated cells (delta MN) for each mouse.

In vitro system for detecting non-genotoxic carcinogens.

Chemical risk assessment has been limited by the inability of in vitro short-term assays to identify the true carcinogenic potential of many substances. Numerous methods exist for identifying mutagenic and clastogenic agents, but a practical means of identifying non-genotoxic carcinogens has remained elusive. Experiments described here suggest that some chemicals may participate in carcinogenesis by modulating the enzymatic processes of drug metabolism. The tumor promoters butylated hydroxyanisole, butylated hydroxytoluene, deoxycholic acid, reserpine, trypan blue, and 12,-O-tetradecanoyl phorbol-13-acetate were chosen as model non-genotoxic carcinogens. The enzyme-modulating action of these chemicals was measured using a modified Ames plate incorporation assay whereby the known tumor promoters were plated with a promutagen in the presence of a mammalian metabolic activation system (S9). Each of the non-genotoxic carcinogens significantly increased the mutagenic response of metabolically activated promutagen(s). These experiments suggest that the carcinogenic role of some chemicals may be attributed to their ability to modify the biochemical pathways of drug metabolism. By enhancing or inhibiting the activity of various enzymes, some tumor promoters may create an environment that increases a cell's mutational burden, thereby contributing to neoplastic transformation.

Y+- and L-system amino acid transport in normal and chronic lymphocytic leukemia lymphocytes: photoinhibition by fluoronitrophenylazide.

Three major pathways mediate amino acid transport into mammalian cells: the A-system and the ASC-system, which require a sodium gradient across the plasma membrane, and the L-system, which has no requirement for a sodium gradient. We have found that the lymphocytes from patients with B-cell chronic lymphocytic leukemia (CLL) have a marked reduction in the L-system of amino acid transport when compared to normal human B-lymphocytes from blood or tonsils. Transport by the A- and ASC-systems was not decreased in CLL B-lymphocytes. Because of the specific defect of the sodium-independent L-system amino acid transport in CLL cells, we have examined the activity of another sodium-independent transport system, the Y+-system, in human lymphocytes. The Y+-system favors the transport of dibasic, cationic amino acids such as lysine, ornithine, and arginine, which carry a positively charged group on their side chains. Our studies indicate that there is a large nonsaturable component of amino acid transport by the Y+-system in human lymphocytes. Using a multicomponent mathematical analysis, we have determined that the saturable component of Y+-transport is similar in T- (thymus-derived) and B- (bone-marrow-derived) lymphocytes and is unimpaired in CLL B-lymphocytes. Further, fluoronitrophenylazide, which was thought to be a specific inhibitor of the Y+-system when photoactivated, also inhibits A-, and L-system transport in CLL, T-, and B-lymphocytes.

Promutagen activation with mammalian and avian S9 liver microsomes.

Metabolic activation activity of Aroclor 1254 induced rat liver microsomes (S9) were compared with uninduced microsomes obtained from beef, pig, sheep and chicken liver. Using the Ames Salmonella typhimurium mutagenesis assay (strains TA98 and TA100), in conjunction with benzo(a)pyrene and 2-aminoanthracene, the highest promutagen activation levels were obtained with chicken liver S9. The S9 from the other domestic animals gave results which were close to those of the induced rat S9 values. Enhanced promutagen activation activity was observed with chicken S9 following freeze drying and reconstitution of the S9 preparation.

Separation of d-(+)-Nicotine from a Racemic Mixture by Stereospecific Degradation of the l-(-) Isomer with Pseudomonas putida.

Incubation of racemic mixtures of dl-(+/-)-nicotine with Pseudomonas putida resulted in a complete stereoselective degradation of the l-(-) isomer. Unnatural d-(+)-nicotine, which is of pharmacological interest for stereochemical studies of various nicotine-responsive systems, was not affected by the bacterium and was recovered by extraction.

Application of light sensitive chemicals to probe protein structure and function.

These results suggest that important information regarding the conformation of complementary binding sites and the behavior of various charged states of inhibitors can be obtained from studies of the pH dependence of photoaffinity labeling experiments. This information may assist in identifying the pKas of key residues controlling the binding interaction. This technique should prove very useful in probing binding site structure on nonfunctioning targets, such as enzyme zymogens, as well as complicated receptor interactions.

Inhibition of promutagen activation by the antioxidants butylated hydroxyanisole and butylated hydroxytoluene.

Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) exhibited antimutagenic activity in the Salmonella typhimurium reversion test. Both BHA and BHT reduced reversion induced by chemicals requiring metabolic activation for effectiveness. However, they did not affect reversion induced by direct-acting mutagens. These results suggested that BHA and BHT may inhibit the metabolic activation processes and demonstrated that the S. typhimurium reversion test may be used to identify inhibitors of the neoplastic process.

Photolytic inhibition and labeling of proteins with aryl diazonium compounds.

In the course of preparing aryl azide derivatives for use as photoprobes, we have observed significant light sensitivity in the precursor aryl diazonium compounds. The photosensitive properties of this class of compounds are of interest since they will seek out cationic binding sites in biological targets, and can be employed to inhibit complementary targets at acid pH. The relationship between photolytic change in the structure of diazonium compounds and the corresponding change in function of a biological target are presented. Experiments are described in which the dark and light sensitive properties of a model diazonium compound, diazobenzene sulfonate (DBS), were determined. The ultraviolet spectra were used to evaluate the dark stability and light sensitivity of DBS. Chymotrypsin and trypsin served as functioning targets for further evaluation of the photochemical properties. Both enzymes are stable to the probe in the dark at acid pH. A rapid loss of enzyme activity was observed following flash photolysis of DBS-enzyme solutions. Photolytic incorporation of radioactive DBS into chymotrypsin was observed. Aryl diazonium salts can be employed to probe the availability of complementary sites in biological targets at different acid pH values.

Characterization of azidobenzamidines as photoaffinity labels for trypsin.

Meta- and para-azidobenzamidine have been prepared and evaluated as photoaffinity labels. The compounds inhibit trypsin reversible in the dark and are competitive with substrate binding. Upon photolysis, irreversible noncompetitive inhibition is observed and is dependent upon concentration, photolysis time, and pH. Specificity of the probes is indicated by experiments in which p-tosyl-l-arginine methyl ester, a trypsin substrate, is used to protect against photoinactivation. Maximum inactivation of trypsin is achieved at pH 6.2 using either azidobenzamidine derivative. Evaluation of the pH dependence of photoaffinity labeling may provide a sensitive tool for probing conformational changes in inhibitor binding sites. These studies provide a basis for the use of azidobenzamidines as photoaffinity analogs of lysine and arginine side chains.

Preparation and characterization of calcium-binding and other hydrophobic proteins from synaptic membranes.

A number of hydrophobic proteins have been separated and purified to varying degrees from synaptic membranes derived from bovine brain. The proteins, which have been obtained using preparative acrylamide gel electrophoresis, have been analyzed for molecular weight, amino acid composition, peptide mapping, N-terminal amino acids, and for their ability to bind calcium and ATP. A number of the proteins bound calcium, the greatest binding being associated with a component having a molecular weight of 1.6 - 10(4), a binding capacity of 4 calcium/molecule, and a Km of 1.5 - 10(-5) M. An acidic tryptic peptide derived from this protein was evidently responsible for the calcium-binding. ATP binding appeared to be confined largely to the higher molecular weight proteins. From the peptide mapping there appears to be a similar acidic component in a number of the proteins exhibiting calcium-binding. ATP-binding was associated mainly with the high molecular weight proteins, particularly those which consisted of numerous basic tryptic peptides.