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J K Horton

Publications and source records attributed to J K Horton.

At least 37 records · Page 2Linked to original sources
PubMed1994

Multidrug resistance-associated protein gene overexpression and reduced drug sensitivity of topoisomerase II in a human breast carcinoma MCF7 cell line selected for etoposide resistance.

A human breast cancer cell line (MCF7/WT) was selected for resistance to etoposide (VP-16) by stepwise exposure to 2-fold increasing concentrations of this agent. The resulting cell line (MCF7/VP) was 28-, 21-, and 9-fold resistant to VP-16, VM-26, and doxorubicin, respectively. MCF7/VP cells also exhibited low-level cross-resistance to 4'-(9-acridinylamino)-methanesulfon-m-anisidide, mitoxantrone, and vincristine and no cross-resistance to genistein and camptothecin. Furthermore, these cells were collaterally sensitive to the alkylating agents melphalan and chlorambucil. DNA topoisomerase II levels were similar in both wild-type MCF7/WT and drug-resistant MCF7/VP cells. In contrast, topoisomerase II from MCF7/VP cells appeared to be 7-fold less sensitive to drug-induced cleavable complex formation in whole cells and 3-fold less sensitive in nuclear extracts than topoisomerase II from MCF7/WT cells. Although this suggested that the resistant cells may contain a qualitatively altered topoisomerase II, no mutations were detected in either the ATP-binding nor the putative breakage/resealing regions of either DNA topoisomerase II alpha or II beta. In addition, the steady-state intracellular VP-16 concentration was reduced by 2-fold in the resistant cells, in the absence of detectable mdr1/P-gp expression and without any change in drug efflux. In contrast, expression of the gene encoding the MRP was increased at least 10-fold in resistant MCF7/VP cells as compared to sensitive MCF7/WT cells. These results suggest that resistance to epipodophyllotoxins in MCF7/VP cells is multifactorial, involving a reduction in intracellular drug concentration, possibly as a consequence of MRP overexpression, and an altered DNA topoisomerase II drug sensitivity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Effects of phosphoramidon and pepstatin A on the secretion of endothelin-1 and big endothelin-1 by human umbilical vein endothelial cells: measurement by two-site enzyme-linked immunosorbent assays.

1. Two-site enzyme-linked immunosorbent assays have been developed for the rapid, sensitive and non-isotopic measurement of endothelin-1 and big endothelin-1. The sensitivities of detection were 0.5 and 0.3 fmol/well, with ED50 values of 13 and 12 fmol/well for the endothelin-1 and big endothelin-1 assays, respectively. Each assay is highly selective for its corresponding antigen. The ET-1 assay showed no detectable cross-reactivity with ET-1-(1-20), indicating that the assay only recognizes the 21-amino acid biologically active peptide. 2. The two assays were used to measure the effects of two classes of protease inhibitor on the basal release of enothelin-1 and big endothelin-1 from cultured first-passage human umbilical vein endothelial cells. 3. The secretion of both peptides was time-dependent over 12 h. The metalloprotease inhibitor phosphoramidon (1 x 10(-4) mol/l) significantly reduced the amount of endothelin-1 secreted into the medium (P < 0.05), with a concomitant increase in the secreted levels of big endothelin-1 (P < 0.01). The aspartyl protease inhibitor, pepstatin A, also caused a significant decrease in the secretion of endothelin (P < 0.05). However, unlike phosphoramidon, there was no increase in the levels of big ET-1 compared with the controls. At these concentrations, neither inhibitor affected the viability of the cells as indicated by Trypan Blue exclusion. 4. The two assays permit the direct measurement of endothelin-1 and its precursor, and will be of use in the elucidation of the putative human endothelin-converting enzyme(s).(ABSTRACT TRUNCATED AT 250 WORDS)

Antimalarials↗
PubMed1993

Changes in intra- or extracellular pH do not mediate P-glycoprotein-dependent multidrug resistance.

P-glycoprotein (Pgp)-mediated multidrug resistance (MDR) is thought to result from active extrusion of lipid-soluble, titratable chemotherapeutic agents. Given the lack of demonstration of coupling between ATP hydrolysis and drug transport, the resistance to chemically unrelated compounds, and findings of elevated intracellular pH (pHi), it has been proposed that reduced intracellular accumulation of drugs in MDR is due to changes in the pH difference across the plasma membrane. Elevation of pHi or decrease in local extracellular pH (pHo) could reduce the intracellular accumulation of the protonated chemotherapeutic drugs and account for Pgp-mediated MDR. Alternatively, changes in pHi or pHo could increase drug efflux by other mechanisms, such as coupled transport involving H+ or OH-, or allosteric effects on Pgp or other proteins. Both mechanisms could operate independently of the charge of the substrate. The possibility of a role of pHi in drug efflux is important to test because of the clinical significance of the phenomenon of MDR of tumors. We tested this hypothesis and found that MDR can occur in cells with low, normal, or high pHi. Further, resistant cells exhibited reduced steady-state drug accumulation and increased efflux without changes in local pHo. Finally, acute changes in pHi had no appreciable effect on Pgp-mediated drug efflux. We conclude that Pgp-mediated MDR is not a consequence of changes in pHi or pHo.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Pharmacological characterization of N-substituted phenoxazines directed toward reversing Vinca alkaloid resistance in multidrug-resistant cancer cells.

Previously we reported the synthesis and partial characterization of 21 N10-substituted phenoxazines in reversing Vinca alkaloid resistance. Here we report on a subset of these compounds; we have compared their activities in increasing Vinca alkaloid accumulation and reversing drug resistance in KB-ChR8-5 and GC3/c1 (human colon carcinoma) cell lines. Results demonstrated that 1) N-substituted phenoxazines increase accumulation of vinblastine; 2) within this series, there is little correlation or ranking of activity between the two cell lines when Vinca alkaloid accumulation is compared at equal concentrations of modulator; 3) N-substituted phenoxazines demonstrate both quantitative and qualitative differences, compared with verapamil, a standard modulator; and 4) the series includes at least two compounds, 10-[3'-[N-bis(hydroxyethyl)amino]propyl]phenoxazine and 10-(N-piperidinoacetyl)phenoxazine, which increase Vinca alkaloid accumulation but do not significantly inhibit efflux. Additionally, certain of these multidrug resistance modulators significantly enhance accumulation (8-50-fold) of Vinca alkaloids in cell lines with very low or undetectable P-glycoprotein levels, where verapamil has little activity. It is concluded that at least part of the activity of some of these N-substituted phenoxazine modulators may be mediated through a P-glycoprotein-independent mechanism.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Enzyme immunoassays for the estimation of adenosine 3',5' cyclic monophosphate and guanosine 3',5' cyclic monophosphate in biological fluids.

Simple, rapid and sensitive competitive enzyme immunoassays for the estimation of adenosine 3',5' cyclic monophosphate (cAMP) and guanosine 3',5' cyclic monophosphate (cGMP) in human plasma and urine are described. Specific antisera to each nucleotide were raised in rabbits by immunization with succinyl cyclic nucleotide--human serum albumin conjugates. For the assay, specific antibodies were incubated with a mixture of succinyl cyclic nucleotide labelled with horseradish peroxidase together with unlabelled standard or sample. The antibody-bound enzyme conjugate was separated from free hapten by anti-rabbit (IgG) sera immobilized to a microtitre plate. Activity of the bound enzyme conjugate was determined with tetramethylbenzidine. The assays were capable of detecting levels as low as 2 fmol of cAMP and cGMP. Good correlations were obtained between values generated by enzyme immunoassay and radioimmunoassay.

Animals↗
PubMed1992

Synthesis and chemical characterization of N-substituted phenoxazines directed toward reversing vinca alkaloid resistance in multidrug-resistant cancer cells.

A series of 21 N-substituted phenoxazines has been synthesized in an effort to find more specific and less toxic modulators of multidrug resistance (MDR) in cancer chemotherapy. Thus, N-(omega-chloroalkyl)- and N-(chloroacyl)phenoxazines were found to undergo iodide-catalyzed nucleophilic substitution on reaction with various secondary amines, including N,N-diethylamine, N,N-diethanolamine, morpholine, piperidine, pyrrolidine and (beta-hydroxyethyl)piperazine. Products were characterized by UV, IR, 1H-, and 13C-NMR, mass spectral data, and elemental analyses. All of the compounds were examined for cytotoxicity and for their ability to increase the accumulation of the vinca alkaloids, vincristine (VCR) and vinblastine (VLB) in multidrug-resistant GC3/Cl (human colon adenocarcinoma) and KBChR-8-5 (HeLa variant) cell lines. Compounds were compared to the standard modulator verapamil (VRP). Substitutions on the phenoxazine ring at position 10 were associated with an increase in antiproliferative and anti-MDR activities. Modification of the length of the alkyl bridge and the type of amino side chain also influenced the potency of these effects. From among the compounds examined, 10 derivatives were found to increase the accumulation of VCR and VLB in GC3/Cl and KBChR-8-5 cells relative to the effect of VRP, suggesting that with the exception of pyrrolidinyl, the tertiary amine attachments to the phenoxazine nucleus linked through a three- or four-carbon alkyl chain resulted in enhanced anti-MDR activity. On the basis of their 50% growth inhibitory (IC50) values, five of the ten compounds, namely, 10-(3'-chloropropyl)phenoxazine, 10-[3'-[N-bis(hydroxyethyl)- amino]propyl]phenoxazine, 10-(3'-N-morpholinopropyl)phenoxazine, 10-(4'-N-morpholinobutyl)phenoxazine and 10-(N-piperidinoacetyl)phenoxazine were selected as relatively nontoxic chemosensitizers. These modulators, at nontoxic concentrations, potentiated the cytotoxicity of VCR and VLB in GC3/Cl and KBChR-8-5 cells. Further, two compounds 10-(3'-N-morpholinopropyl)phenoxazine, and the butyl derivative, enhanced accumulation of VLB in GC3/Cl, KBChR8-5 and highly resistant KB-V1 cells to a level significantly greater than the maximal level achieved with VRP. Additional experiments to understand the mechanism of action of these agents in modulating MDR are in progress.

Drug Resistance↗
PubMed1990

Activation of the inflammatory response of neutrophils by Tamm-Horsfall glycoprotein.

The activation of human polymorphonuclear leukocytes (PMN) by particulate Tamm-Horsfall glycoprotein (THG) represents an interaction hitherto unrecognized. The potential pathophysiological effect of this phenomenon within the interstitium of the kidney is highlighted by the activation of the respiratory burst, as well as by comprehensive PMN degranulation. Products of the interaction are expressed in terms of phagocytosis, luminol-dependent chemiluminescence, granule marker enzyme release and arachidonic acid metabolism. Significant quantities of the primary, secondary and tertiary granule markers, myeloperoxidase, vitamin B12 binding protein and N-acetyl-beta-D-glucosaminidase, respectively, were secreted in a dose and time-dependent manner. Phagocytosis of the glycoprotein was accompanied by the generation of significant quantities of leukotriene B4. Furthermore, the ability of such a particulate ligand to activate the alternative pathway of complement clearly represents a capacity to augment the inflammatory response. Should the interaction of THG with PMN take place within the interstitium of the kidney, augmented by the deposition of complement proteins on the surface of insoluble aggregates, the resulting inflammatory response may lead to marked tissue damage and eventually result in interstitial fibrosis.

Complement Pathway, Alternative↗
PubMed1990

Structural determinants of phenoxazine type compounds required to modulate the accumulation of vinblastine and vincristine in multidrug-resistant cell lines.

Phenoxazine and seven other structurally related compounds were investigated to determine whether they would increase accumulation of Vinca alkaloids in multidrug-resistant (MDR) GC3/C1 (human colon adenocarcinoma) and KB-ChR-8-5 (HeLa variant) cell lines. Among eight compounds examined, phenoxazine caused greater accumulations of vincristine (VCR) and vinblastine (VLB) than the other chemosensitizers. The structure-activity relationship of these compounds for anti-MDR activity suggested an ideal tricyclic ring structure with a basic nitrogen atom at position 10 for modulating the accumulation of Vinca alkaloids. Addition of oxygen to position 5 of the tricyclic ring system further increased the activity, implying that a highly electronegative element with one, or more, lone pair of electrons in the nucleus opposite to heterocyclic nitrogen was a requirement for better anti-MDR activity. The relationship between the concentration of phenoxazine and the potentiation of Vinca alkaloid accumulation in comparison to verapamil was examined. For VCR in GC3/C1 cells, maximal modulation indices were: for verapamil, 1.8; phenoxazine, 8.6; and for VLB, 1.3 for verapamil compared to 3.3 for phenoxazine. In KB-ChR-8-5 cells, for VCR the maximal modulating index values were 9.0 and 4.3, respectively, and for phenoxazine and verapamil and for VLB were 5.0 and 3.7, respectively. Accumulations of VLB in GC3/C1 cells were similar in the presence of 1 microM phenoxazine or 10 mM sodium azide plus 10 mM 2-deoxyglucose. The effects of verapamil and phenoxazine on the accumulation of Vinca alkaloid were additive. Further, phenoxazine decreased the efflux of VLB by 30% in KB-ChR-8-5 cell line and 10% in GC3/C1 cells. In addition to enhancing the cytotoxicities of VCR and VLB, phenoxazine competed relatively weakly for binding to P-glycoprotein with [3H]azidopine and moderately with [3H]azidoverapamil, at equal concentrations, suggesting that the multidrug transporter may be the primary target for phenoxazine.

Adenocarcinoma↗
PubMed1989

A rapid and sensitive method for estimating low concentrations of albumin in human urine.

A chemiluminescence immunoassay has been developed for the measurement of albumin concentrations in human urine, as an indicator of diabetic nephropathy. The assay involved competition between analyte albumin and an acridinium ester labelled albumin tracer for binding to a rabbit (anti-human albumin) antibody. Immune complexes were separated using sheep (anti-rabbit immunoglobulin G) antibodies coupled to paramagnetic particles. The total incubation time was ninety minutes at room temperature followed by sedimentation and washing of the solid-phase using a magnetic rack. Chemiluminescence emission was quantified rapidly (2 s) using a commercially available luminometer. The assay was sufficiently sensitive (10 ng/ml) for the detection of microalbuminuria with the advantages of rapidity and use of stable reagents. The assay correlated well with both RIA and rate nephelometry.

Acridines↗
PubMed1989

Establishment of a melphalan-resistant rhabdomyosarcoma xenograft with cross-resistance to vincristine and enhanced sensitivity following buthionine sulfoximine-mediated glutathione depletion.

A melphalan-resistant human rhabdomyosarcoma xenograft, TE-671 MR, was established in athymic mice by serial melphalan treatment of the parent xenograft, TE-671, at the 10% lethal dosage (LD10); significant resistance was evident after ten passages of the tumor. TE-671 MR demonstrated a doubling time of 3.5 days and a latency period to 1000-mm3 tumors of 27.5 days. The glutathione level of TE-671 MR was 2.36 mumol/g tumor, wet weight, 2-fold higher than the parent line. The glutathione S-transferase activity of TE-671 MR was 117.8 mumol/min/mg protein, essentially unchanged from the parent line. Although TE-671 MR demonstrated cross-resistance to vincristine, dot blot analysis did not reveal an elevated expression of mdr1 mRNA in the resistant line. TE-671 MR demonstrated a 9.7-day growth delay following treatment with melphalan at the LD10 (compared to 20.9 days for the parent line). Treatment with L-buthionine-SR-sulfoximine (BSO) resulted in increased sensitivity to melphalan subsequently administered at 50% of the LD10 (melphalan alone, growth delays of 3.7 and 4.6 days in duplicate trials; melphalan plus BSO, growth delays of 7.2 and 9.8 days). Sensitivity to melphalan equal to that of the parent line TE-671 was not achieved, however. Treatment with BSO did not result in significantly enhanced sensitivity to subsequently administered vincristine (50% of the LD10) (vincristine alone, growth delays of 6.8 and 6.9 days in duplicate trials; vincristine plus BSO, growth delays of 10.9 and 7.5 days). These results suggest that generation of melphalan resistance may be associated with development of cross-resistance to vincristine; this resistance may be associated with (although not necessarily mediated by) glutathione elevation; this resistance may be partially overcome by BSO-mediated depletion of glutathione.

Animals↗
PubMed1989

A new and rapid method for the selection and cloning of antigen-specific hybridomas with magnetic microspheres.

A new and highly selective procedure is described for the rapid selection and cloning of antibody-secreting hybridomas with antigen-coated magnetic beads. Immune splenocytes were fused to Sp2 myeloma cells with a PEG/DMSO mixture. Cells were cultured in HAT and screened for the presence of specific antibody after 7-10 days. Hybridomas from the positive colonies were mixed with antigen-coated magnetic polymer beads and antigen-specific cells separated on a magnet. Cloning was carried out directly by limiting dilution with the magnetic beads still bound on the cells. No obvious toxic effects were observed. The antibodies established by this technique were of a high affinity (greater than 10(9) l/mol) and were generated in 50% of the usual process time. The procedure described here should greatly facilitate the process of obtaining hybridomas and expand the range of monoclonal antibodies available.

Animals↗
PubMed1989

Modulation by verapamil of vincristine pharmacokinetics and toxicity in mice bearing human tumor xenografts.

The effect of the calcium channel blocker verapamil (VRP) on the accumulation and retention of vincristine (VCR) has been examined in mice bearing xenografts of human rhabdomyosarcomas. The tumors were Rh18, moderately sensitive to VCR, and its subline, Rh18/VCR3, selected in vivo for primary resistance to VCR. Administration of VRP by i.p. bolus at dose levels above 75 mg/kg was limited by acute lethality. At this dose, the maximal concentration in plasma was 24 microM, with rapid elimination such that plasma concentrations reported to modulate resistance in vitro (approximately 5-10 microM) were maintained for less than 60 min. To sustain a 10 microM plasma concentration, mice were infused with VRP at 6.25 mg/kg/hr (150 mg/kg/day) for up to 7 days using osmotic pumps implanted in the peritoneal cavity. Steady-state plasma levels were greater than or equal to 10 microM for at least 96 hr, and this schedule demonstrated minimal toxicity. Administration of VCR 20 hr after the start of VRP infusion produced significant lethality, requiring an 8-fold reduction in the VCR dose. Pharmacokinetic studies showed that VRP markedly increased the uptake and retention of VCR in small intestine, liver and kidney of mice. In small intestine, 8-fold greater levels of VCR were determined 24 hr after VCR administration, and this was associated with in increase in T1/2 for elimination from 350 to 913 min. HPLC analysis of extracts from small intestine showed that greater than 90% of the radiolabel eluted with VCR or 4-desacetyl-VCR. Modulation of VCR retention was also related to the dose of VCR administered. The VRP-sensitive efflux pathway appeared more effective in certain tissues only at higher concentrations of VCR. In contrast, VRP did not alter significantly the uptake and retention of VCR in either the parent or VCR-resistant human xenografts. The data demonstrated that, in the mouse, VRP modulates the uptake and retention of VCR in several tissues, and this may indicate that drug efflux mediated by a VRP-sensitive mechanism (e.g. GP-170, associated with the multiple drug resistance phenotype) has a protective function against xenobiotics in these tissues.

Animals↗
PubMed1989

Can the risk of peritonitis be predicted for new continuous ambulatory peritoneal dialysis (CAPD) patients?

Serum and overnight dialysate samples were obtained from 36 adult uraemic patients at the end of their continuous ambulatory peritoneal dialysis (CAPD) training. The samples were analysed for albumin, IgG, C3, and antistaphylococcal peptidoglycan antibody. None of the dialysate measurements correlated with the risk of peritonitis during up to one year's CAPD treatment. Nineteen of the 36 patients were retested 6 to 20 months after starting CAPD. There were significant rises in serum C3 (p less than 0.02) and albumin (p less than 0.001) and a significant fall in dialysate IgG (p less than 0.02). Eight further patients were sampled at the end of training and three weeks later. They had a significant fall in dialysate IgG (p less than 0.05). During CAPD training peritoneal permeability appears to be transiently increased. Analysis of overnight dialysate samples during training does not allow prediction of those at risk of subsequent peritonitis.

Adult↗
PubMed1988

A sensitive chemiluminescence based immunoassay for antibody to staphylococcal peptidoglycan.

A sensitive chemiluminescence based immunoassay is described for measuring antibody to staphylococcal peptidoglycan in blood and dialysates from patients undergoing continuous ambulatory peritoneal dialysis (CAPD). Peptidoglycan was isolated from a strain of S. epidermidis obtained from the dialysate of a CAPD patient with peritonitis and after sonication used to coat polystyrene beads. The coated beads were incubated with standard or sample and bound IgG was detected by the addition of affinity-purified goat anti-human IgG labelled with acridinium ester. After a wash stage 0.1 M nitric acid containing 0.1% hydrogen peroxide was added to the beads. Subsequently the chemiluminescence produced following the addition of 0.3 M sodium hydroxide was measured over a 2 s time interval with an automatic luminescence analyser. Using this technique the optimum dilution of serum for detecting antibodies to peptidoglycan was found to be 1/800 and for overnight effluent from CAPD patients the dilution was 1/8. Initial values of serum and dialysate antibody levels from 34 subjects are presented. This method has the advantage that it will detect concentrations of anti-peptidoglycan which are less than 1% of those in sera, the reagents remain stable for long periods and large numbers of samples can be processed on the same day.

Antibodies, Bacterial↗
PubMed1988

Relationships between tumor responsiveness, vincristine pharmacokinetics and arrest of mitosis in human tumor xenografts.

Tumor responsiveness to vincristine (VCR) was determined in xenografts of human rhabdomyosarcoma (RMS), in sublines of RMS selected in vivo for VCR resistance, in a KB line (KB-ChR8-5) selected in vitro for colchicine resistance, and in a colon adenocarcinoma (GC3). Sensitivity to VCR was associated with prolonged retention of VCR by the tumors after a single i.p. injection, whereas in tumors with acquired or intrinsic VCR resistance the drug was eliminated more rapidly. The sensitive tumors with prolonged retention of drug also showed increased levels of mitotic accumulation for up to 72 hr following VCR administration. There were good correlations between VCR sensitivity, VCR retention and the proposed mechanism of VCR cytotoxicity-mitotic arrest. A model has been developed consistent with data obtained that can explain the responsiveness to VCR of a series of human tumor xenografts irrespective of their tissue of origin.

Animals↗
PubMed1988

A new and rapid immunochemiluminometric assay for the measurement of Tamm-Horsfall glycoprotein.

Tamm-Horsfall glycoprotein was purified to apparent homogeneity from human urine by repeated precipitation with 0.58 mol/l NaCl and gel permeation chromatography under dissociating conditions on Bio-Gel A1.5M. The protein was found to consist of a single polypeptide chain of Mr 100,000 under non-reducing conditions and Mr 75,000 under reducing conditions. Antibodies to Tamm-Horsfall glycoprotein were raised in rabbits and subsequently purified by affinity chromatography using the glycoprotein linked to Sepharose 4B. The specificity of these antibodies was confirmed by Western blotting and by indirect immunofluorescence staining of human kidney tissue. The purified antibodies were labelled with 4-(2-succinimidyloxycarbonylethyl)phenyl-10-methyl-9-acridinium carboxylate fluorosulphonate, an acridinium ester, to a specific activity of 6 X 10(5) photon counts/ng of protein, and used to establish a two-site immunochemiluminometric assay for the measurement of Tamm-Horsfall glycoprotein in serum and urine. The bound and the free fractions were separated by a second antibody to Tamm-Horsfall glycoprotein linked to paramagnetic particles. The bound antibodies were quantified by chemiluminescence. The assay had a sensitivity of detection of 2 ng/ml and a working range, as determined by inter-assay precision profiles, of 30-500 ng/ml. The range in serum samples from volunteers with normal renal function (n = 92) was 74-520 ng/ml and the mean 24-h excretion rate in healthy subjects (n = 32) was 70 +/- 26 mg.

Animals↗