Ronald L. Felsted

Mechanisms of multidrug resistance in HL60 cells: analysis of resistance associated membrane proteins and levels of mdr gene expression

HL60 cells isolated for resistance to Adriamycin do not contain P-glycoprotein, as determined with immunological probes. These cells, however, are multidrug resistant and defective in the cellular accumulation of drug. In view of these findings, we have examined in greater detail certain properties of the HL60/Adr cells and have compared these properties to an HL60 drug-resistant isolate (HL60/Vinc) which contains high levels of P-glycoprotein. The results of these studies demonstrated that verapamil induces a major increase in cellular drug accumulation in both HL60/Adr and HL60/Vinc isolates. An 125I-labeled photoaffinity analog of verapamil labeled P-glycoprotein contained in membranes of HL60/Vinc cells. In contrast, this agent did not label any protein selectively associated with drug resistance in membranes of the HL60/Adr isolate. The photoactive dihydropyridine calcium channel blocker [3H]azidopine and [125I]NASV, a photoaffinity analog of vinblastine, labelled P-glycoprotein in membranes from HL60/Vinc cells, whereas in experiments with the HL60/Adr isolate there was no detectable labeling of a drug resistance associated membrane protein. Additional studies have been carried out to analyze membrane proteins of HL60/Adr cells labeled with the photoaffinity agent 8-azido-alpha-[32P]ATP (AzATP32). The results demonstrate that this agent labeled a resistance associated membrane protein of 190 kilodaltons (P190). P190 is essentially absent in membranes of drug-sensitive cells. Labeling of P190 with AzATP32 in membranes of resistant cells was blocked completely when incubations were carried out in the presence of excess unlabeled ATP. Additional studies were carried out to analyze mdr gene amplification and expression in sensitive and resistant cells. Experiments carried out with human 5,mdr1 (1.1 kb) and mdr3 (1.0 kb) cDNAs demonstrate that both of these sequences were highly amplified in the HL60/Vinc isolate. Only the mrd1 gene sequence however, was overexpressed. In contrast, there was no detectable amplification or overexpression of mdr1 or mdr3 sequences in HL60/Adr cells. The results of this study thus identify a new nucleotide binding protein which is overexpressed in membranes of HL60 cells isolated for resistance to Adriamycin. P190, which exhibits properties distinct from P-glycoprotein, possibly functions in the energy-dependent drug efflux system contained in the HL60/Adr resistant isolate.

Purification of the phytohemagglutinin family of proteins from red kidney beans (Phaseolus vulgaris) by affinity chromatography.

Half-gram quantities of phytohemagglutinin lectins are purified from saline extracts of red kidney beans (Phaseolus vulgaris) by affinity absorption on porcine thyroglobulin-Sepharose. All of the mitogenic and erythroagglutinin activity of the saline extract is removed by this absorbent, and 74% of the original erythroagglutinating activity elutes from the affinity absorbent representing a 25-fold purification. Five distinct proteins appear in the polyacrylamide gel electrophoresis of the affinity absorbent eluate. Although all five proteins specifically bind to porcine thyroglobulin, the cathodal migrating proteins bind more strongly than the anodal migrating proteins. The most cathodal proteins are potent erythroagglutinins. This simple, efficient method is used to prepare all the active components of the phytohemagglutinin family in large yield and high purity.

Effects of indole alkaloids on multidrug resistance and labeling of P-glycoprotein by a photoaffinity analog of vinblastine.

Multidrug resistant cells are characterized by decreased drug accumulation and retention, thought to be mediated by a high molecular weight glycoprotein, P-glycoprotein (P-gp). Agents such as verapamil have been shown to increase anticancer drug cytotoxicity and increase the amount of drug accumulated and retained by such cells. We show here that in addition to verapamil, reserpine, chloroquine, quinine, quinacrine, yohimbine, vindoline, and catharanthine also enhance the cytotoxicity of vinblastine (VLB) in a multidrug resistant, human leukemic cell line, CEM/VLB1K, described here for the first time. These cells express P-gp as a doublet that is photoaffinity labeled by the analog of VLB, N(p-azido-[3-125I]salicyl)-N-beta-aminoethylvindesine ([125I]NASV). Both reserpine and, to a lesser extent, verapamil, compete with [125I]NASV for binding to P-gp. We also found that chloroquine, quinacrine, vindoline, and catharanthine, each of which enhanced VLB cytotoxicity in CEM/VLB1K cells by 10- to 15-fold, similarly inhibited [125I]NASV labeling of P-gp. However, neither quinine nor yohimbine inhibited this labeling, and the inhibition produced by catharanthine and vindoline was the greatest or exclusively on the lower band of the P-gp doublet. Our results suggest a complex relationship between the ability of a compound to modulate MDR and its ability to compete for binding to P-gp.

Induction of a human carbonyl reductase gene located on chromosome 21

Carbonyl reductase (EC 1.1.1.184) belongs to the group of enzymes called aldo-keto reductases. It is a NADPH-dependent cytosolic protein with specificity for many carbonyl compounds including the antitumor anthracycline antibiotics, daunorubicin and doxorubicin. Human carbonyl reductase was cloned from a breast cancer cell line (MCF-7). The cDNA clone contained 1219 base paires with an open reading frame corresponding to 277 amino acids encoding a protein of Mr 30,375. Southern analysis of genomic DNA digested with several restriction enzymes and analyzed by hybridization with a labeled cDNA probe indicated that carbonyl reductase is probably coded by a single gene and does not belong to a family of structurally similar enzymes. Southern analysis of 17 mouse/human somatic cell hybrids showed that carbonyl reductase is located on chromosome 21. Carbonyl reductase mRNA could be induced 3-4-fold in 24 h with 10 microM 2,(3)-t-butyl-4-hydroxyanisole (BHA), beta-naphthoflavone or Sudan 1.

Heterogeneity of anthracycline antibiotic carbonyl reductases in mammalian livers

Abstract The anticancer antibiotics, daunorubicin and adriamycin, are reduced to their corresponding alcohol and glycol metabolites by cytoplasmic carbonyl reductases occurring in human, rabbit, rat and mouse tissues. Our data indicate that at least two different groups of pH-dependent daunorubicin reductases occur in rabbit and human liver: (1) the pH activity profile for daunorubicin reductases shows distinct optima or significant activity at both pH 6.0 and 8.5, and (2) pH 8.5-dependent daunorubicin reductases are resolved from pH 6.0-dependent daunorubicin reductases by ion exchange chromatography on DEAE-cellulose columns, gel filtration chromatography on BioGel P150, and isoelectric focusing. Ion exchange chromatography and isoelectric focusing also resolve multiple forms of each class of activities. A similar analysis suggests that a single type of pH-dependent daunorubicin reductase occurs in rat and mouse livers: (1) rat and mouse livers show a single pH optima for daunorubicin at pH 8.5, and (2) isoelectric focusing of rat and mouse preparations confirms the existence of a pH 8.5 daunorubicin and the absence of significant pH 6.0 daunorubicin activity. Although total adriamycin reduction is lower than daunorubicin reduction at any pH, significant adriamycin reduction also occurs in rabbit liver at pH 6.0 and 8.5; however, neither of these activities can be distinguished from pH 6.0 daunorubicin reductase activity by ion exchange and gel filtration chromatography and isoelectric focusing. In comparison, very low levels of pH 6.0 optimum adriamycin reductase activity are seen in human, rat and mouse livers. Thus, all species have pH 8.5 daunorubicin reductase and probably pH 8.5 adriamycin reductase, whereas rabbit and human also have pH 6.0 daunorubicin reductase(s) and rabbit has a pH 6.0 adriamycin reductase(s), which accounts for the bulk of the active anthracycline antibiotic metabolism in these species.

Comparison of Phaseolus vulgaris cultivars on the basis of isolectin differences

1. n1. The properties of lectins from 62 cultivars of the common bush bean, Phaseolus vulgaris, were examined. n n2. n2. All bean extracts agglutinated rabbit and human red cells but showed no differential preference for A-, B- or O-type human red cells. n n3. n3. Although most bean extracts were mitogenic, 13 bean varieties were apparently non-mitogenic when incubated with normal human lymphocytes. n n4. n4. Polyacrylamide gel electrophoresis of the bean extracts in acid and sodium dodecylsulfate buffers revealed 2 major different isolectin patterns. n n5. n5. Most cultivars had 5 isolectins, were strongly mitogenic, and were classified as group I beans. A second group exhibited a distinct isolectin pattern, were weakly or non-mitogenic, and were classified as group II. Finally, 2 beans with lectin electrophoretic patterns distinct from both of the above groups were classified as group III beans. n n6. n6. Representative extracts from all 3 bean groups were tested with a red kidney bean isolectin L-subunit specific radioimmunoassay. The group I beans had significantly higher levels of L-subunit crossreactive protein than the group II beans while one of the group III beans had intermediate levels.

Phytohemagglutinin isolectin subunit composition

The subunit compositions of individual phytohemagglutinin isolectins from red kidney bean Phaseolus vulgaris were examined by isoelectric focusing and sodium dodecyl sulfate electrophoresis on polyacrylamide gels. Isoelectric focusing reveals heterogeneous but unique and non-overlapping protein band patterns for each of the homotetrameric isolectins, E4 and L4. Isoelectric focusing of the intermediate isolectins which contain both subunits (E3L1, E2L2, and E1L3) show all the protein bands common to isolectins E4 or L4 in proportions relative to their suggested subunit compositions. Polyacrylamide gel electrophoresis in a continuous sodium dodecyl sulfate buffer system gives a single protein band for all of the isolectins. In contrast, a discontinuous sodium dodecyl sulfate buffer procedure resolves isolectins E4 and L4 into single major protein bands of apparent molecular weights 31 700 (+/-600) and 29 900 (+/-200), respectively. Each of the intermediate isolectins contained both protein bands and their relative proportion, as determined by absorbance scanning, confirms the phytohemagglutinin isolectin subunit compositions as E4, E3L1, E2L2, E1L3, and L4.

Synthesis and characterization of inhibitors of myristoyl-CoA:Protein N-myristoyltransferase

Several substrate and product analogs were synthesized and tested as in vitro inhibitors of bovine brain N-myristoyl-CoA:protein N-myristoyltransferase (NMT; EC 2.3.1.97). At 40 microM, the acyl CoA analog, S-(2-ketopentadecyl)-CoA, completely inhibited NMT in the presence of 80 microM myristoyl CoA. Decreasing but marked inhibition was also observed with the acyl CoA analogs, S-(2-bromo-tetradecanoyl)-CoA and S-(3-(epoxymethylene)dodecanoyl)-CoA, and the multisubstrate derivative N-(2-S-CoA-tetradecanoyl)glycinamide in the presence of 40 microM myristoyl CoA. Inhibition was also observed with the non-coenzyme A myristoyl analog, 1-bromo-2-pentadecanone. All of the above compounds exhibited reversible competitive inhibition kinetics with respect to myristoyl CoA with Ki values of 0.11 to 24 microM. Two additional acyl CoA analogs, S-(cis-3-tetradecenoyl)-CoA and S-(3-tetradecynoyl)-CoA, functioned as alternative substrates for NMT.

Cocoonases of silkworm moths: Catalytic properties and biological function

Abstract The cocoonases from several silkmoths and bovine trypsin have similar reactivities towards several small-molecule substrates and inhibitors. These include ester and amide substrates, a substrate analogue (TLCK), and a phosphate ester inhibitor (DFP). Trypsin-like substrate activation was also observed with one cocoonase. The enzymes from Antheraea polyphemus and Bombyx mori react with labelled DFP and give, on acid hydrolysis, patterns of labelled peptides similar to those from labelled DIP trypsin and chymotrypsin, thus indicating considerable homology in the active site region of the cocoonases and the pancreatic proteinases. Trypsin and A. polyphemus cocoonase have qualitatively similar but quantitatively different action on polypeptides, with trypsin being more effective. Cocoonase is necessary for effective escape of silkmoths from the cocoon, which can be accomplished from the anterior end with ease and, in a few instances, from the posterior end with difficulty.

Properties of the Antheraea mylitta cocoonase

Abstract 1. 1. The proteolytic enzyme, cocoonase, from Antheraea mylitta has been compared to other cocoonases and bovine trypsin and found to be similar in (1) molecular weight, (2) amino acid composition, (3) specificity and reactivity toward Nα-benzoyl- l -arginine ethyl ester, (4) proteolytic specificities toward glucagon, B-chain of insulin and lysozome, (5) rate of proteolysis of modified lysozyme and (6) NH2-terminal amino acid sequence. 2. 2. The A. mylitta cocoonase, however, activated chymotrypsinogen A at a rate 3200-fold slower than bovine trypsin which suggested subtle and important differences in the structures of these two enzymes.