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Biomedical subjects

D R Phillips

Publications and source records attributed to D R Phillips.

At least 109 records · Page 6Linked to original sources
PubMed1995

Myeloperoxidase oxidizes mitoxantrone to metabolites which bind covalently to DNA and RNA.

The anticancer agent mitoxantrone is readily oxidized by the human haem enzyme myeloperoxidase and hydrogen peroxide. Intercalation of mitoxantrone with DNA inhibited oxidation of the drug by myeloperoxidase. However, at a physiological ionic strength, significant oxidation of the drug was evident. At a H2O2:mitoxantrone ratio of 1.0, myeloperoxidase oxidized mitoxantrone to a metabolite (product B) which associated reversibly with DNA. At greater hydrogen peroxide concentrations, two further metabolites were produced (products C and D), neither of which associated reversibly with DNA, as indicated by the absence of any spectral change in the presence of DNA. Long exposure of the products derived from the oxidation of [14C]mitoxantrone by myeloperoxidase resulted in a time-dependent covalent binding of the activated drug to both DNA and RNA. The amount of DNA adduct increased linearly with the extent of oxidation of mitoxantrone (up to a H2O2:mitoxantrone ratio of 5.0). No adducts resulted from exposure of the oxidized product B to DNA, but adducts formed following further oxidation of B by myeloperoxidase. The myeloperoxidase-catalysed oxidation of mitoxantrone to products capable of interacting covalently and non-covalently with nucleic acids may represent an important mode of action of mitoxantrone against acute myeloid leukemias since these cells (including neutrophils, monocytes and their precursors) contain high levels of myeloperoxidase.

Antineoplastic Agents↗
PubMed1994

Oxidative metabolism of mitoxantrone by the human neutrophil enzyme myeloperoxidase.

The anti-cancer drug mitoxantrone is readily oxidized by the human heme enzyme myeloperoxidase (MPO) and H2O2. Direct oxidation yielded up to three products, which depended on the ratio of H2O2 to mitoxantrone. At an H2O2: mitoxantrone ratio of 1.0, one major product was obtained, with a spectrum and HPLC retention time identical to that resulting from oxidation by horseradish peroxidase. This metabolite is a substituted hexahydronaphtho[2,3-f]quinoxaline-7,12-dione and has been discovered in the urine of patients treated with mitoxantrone, hence implicating MPO in the in vivo metabolism of mitoxantrone. At higher concentrations of H2O2, the oxidation of mitoxantrone was more complex, with two further metabolites being identified. When mitoxantrone was incubated with neutrophils that had been stimulated with phorbol myristate acetate, it was oxidized by an MPO-dependent mechanism. Therefore, it appears that MPO may play a significant role in the clinical activity displayed by mitoxantrone against acute myelogenous leukemias, as neutrophils, monocytes and their bone marrow precursors contain high levels of the enzyme.

Chromatography, High Pressure Liquid↗
PubMed1994

Molecular basis of nitrogen mustard effects on transcription processes: role of depurination.

DNA was alkylated with nitrogen mustard (HN2) and the rate of release of the alkylpurines was quantitated by HPLC. The half life of depurination of the major product (7-alkylguanine) was 9.1 h at 37 degrees C. End-labelled DNA was used to show that depurination occurred dominantly at 5'-GA, 5'-GG and 5'-GT sequences. Although extensive alkylation was observed at all 5'-GNC and 5'GNT sequences, no depurination was observed at these sites during a depurination time of 20 h at 37 degrees C. Since these sites are potential interstrand crosslinking sequences (G-adduct-G and G-adduct-A, both spanning an intervening base pair), this suggests that these regions have a greatly enhanced stability or that simultaneous depurination of both ends of the crosslink is necessary before these lesions are removed (with a predicted half-life of approximately 80 h at 37 degrees C). Depurination at the lac UV5 promoter impaired the association of Escherichia coli RNA polymerase with that promoter, while in the elongation phase two distinctly different sequence-specific processes were apparent. At 5'-GNC and 5'-GNT sequences transcriptional blockages were maintained with increasing elongation time, whereas at monoadduct sites, the blockage decreased with elongation time (predominantly at 5'-GG and 5'-GC sequences), with an average half-life of approximately 10.7 h. Collectively, these results suggest that the observed read-through past monoadduct sites is due to depurination of the DNA at those sites. E. coli RNA polymerase is therefore able to transcribe efficiently past apurinic sites and presumably does so by incorporating an incorrect base into the nascent RNA.

Alkylation↗
PubMed1994

Formation of adriamycin--DNA adducts in vitro.

Adriamycin is known to induce the formation of adducts with DNA when reacted under in vitro transcription conditions. The factors affecting the extent of adduct formation were examined in order to establish the critical components and optimal conditions required for the reaction, and to gain insight into the nature of the DNA-adduct complex. There was a strong dependence on reaction temperature (with a 40-fold increase of adducts at 40-50 degrees C compared to 10 degrees C), pH (maximum adducts at pH 7), but little dependence on the oxygen level. There was an absolute requirement for a reducing agent, with adducts detected with DTT, beta-mercaptoethanol and glutathione, maximal adducts were formed at high levels of DTT (5-10 mM). Adducts were also formed with a xanthine oxidase/NADH reducing system, with increasing amounts of adducts detected with increasing NADH; no adducts were detected in the absence of either the enzyme or NADH. Of fourteen derivatives studied, only four yielded a similar extent of adduct formation as adriamycin; there was no absolute requirement for a carbonyl at C13 or hydroxyl at C14. Adducts were also observed with ssDNA but required a longer reaction time compared to dsDNA. The sequence specificity of adduct formation with ssDNA was examined using a primer-extension assay; almost all adducts were associated with a guanine residue. Overall, the results are consistent with a two-step reaction mechanism involving reductive activation of adriamycin, with the activated species then reacting with the guanine residues of either dsDNA or ssDNA.

DNA↗
PubMed1994

Sequence specificity of (cyanomorpholino)adriamycin adducts in human cells.

The highly reiterated alpha DNA tandem repeat was extracted from HeLa cells incubated with (cyanomorpholino)adriamycin (CMA) using mild techniques and subsequently probed for drug adducts by exonuclease III. The sequence specificity of the CMA-induced blockages was compared with that for blockages induced on the same DNA fragment when reacted in vitro. The sequence specificity of the drug-induced blockages was the same on both the isolated and the intact cell alpha DNA templates, with blockages predominantly associated with GpG sequences on either strand of the DNA.

Base Sequence↗
PubMed1994

DNA sequence specificity of mitoxantrone.

An in vitro transcription assay was used to determine the sequence specificity of binding of mitoxantrone to a 497 bp fragment of DNA containing the lac UV5 promoter. Transcriptional blockages of the E. coli RNA polymerase were observed dominantly prior to 5'-CpA sequences (64% occurrence), and to a lesser extent 5'-CpG sequences (29%). Overall, 93% of all blockage sites were prior to pyrimidine (3'-5') purine sequences. An effect of flanking sequences was evident since the blockage sites contained an A/T base pair 5' prior to the consensus CpA and CpG intercalation sites. The consensus sequences for the preferred mitoxantrone intercalation sites are therefore 5'-(A/T)CA and 5'-(A/T)CG. The location of transcriptional blockages one base pair prior to the intercalation site is consistent with the fact that the mitoxantrone side chains lie in the major groove.

Base Sequence↗
PubMed1994

Does adriamycin induce interstrand cross-links in DNA?

Under nonenzymatic conditions in vitro, Adriamycin appears to form interstrand cross-links with DNA over 1-2 days. This is the first report of such Adriamycin-induced interstrand cross-links in vitro. Cross-links were measured by a fluorescence based renaturation assay and also by gel electrophoresis. Both procedures revealed an increase of cross-linking with reaction time and with increasing Adriamycin concentration and a 5-6-fold enhancement in the presence of Fe3+ ions. The cross-link contains the Adriamycin chromophore, with a lambda max of 508 nm, intercalated at the GpC site of cross-linking. Maximal stoichiometry of the cross-link was one per 11-20 bp. The cross-link appears to involve adducts of the Adriamycin chromophore linked to the N2 of guanine, with no indication that N7 of guanine is involved. Given that the mode of action of Adriamycin still remains obscure, even after 20 years of clinical use, the possibility that interstrand DNA cross-links may be associated with the clinical mechanism of action of this drug should now be fully addressed.

Animals↗
PubMed1994

Isolation and characterization of a platelet-derived macrophage-binding proteoglycan.

A macromolecule in human platelet secretory products was demonstrated previously to inhibit the binding and uptake of acetoacetylated (AcAc) low density lipoproteins (LDL) by scavenger receptors on mouse peritoneal macrophages. In the current study, this macromolecule was purified to apparent homogeneity by DEAE-Sephacel chromatography, Sephacryl S-300 chromatography, and sucrose gradient centrifugation. SDS-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of approximately 120 kDa. Chemical analysis indicated that the macromolecule (designated platelet-derived macrophage-binding proteoglycan (PDMBP)) was a chondroitin 4-sulfate proteoglycan with an approximately 32-kDa core protein. A polyclonal antibody produced against this proteoglycan identified only PDMBP on Western blots of platelet secretory products and removed all ability of these products to inhibit the binding of AcAc LDL to macrophages. Treatment of purified PDMBP with protease or chondroitinase AC or ABC abolished the ability of the proteoglycan to inhibit the binding of AcAc LDL to macrophages. Binding studies using radiolabeled PDMBP demonstrated that the proteoglycan bound directly to the macrophage cell surface and was competitively inhibited by AcAc LDL, acetyl-LDL, fucoidin, and unlabeled PDMBP. PDMBP inhibited binding of 125I-labeled AcAc LDL to macrophages but had no effect on binding to endothelial cells. The finding that PDMBP binds to the scavenger receptor on macrophages suggests a mechanism for the inhibition of foam cell formation and suggests that the receptor could be involved in the plasma clearance of chondroitin sulfate proteoglycans.

Amino Acids↗
PubMed1994

A comparison of endometrial ablation using the Nd:YAG laser or electrosurgical techniques.

STUDY OBJECTIVE: To compare the results of endometrial ablations performed either with the neodymium:yttrium-aluminum-garnet (Nd:YAG) laser or electrosurgically. STUDY DESIGN: Retrospective study, with 4-year follow-up of 82 of 98 patients. SETTING: Gynecology departments of teaching, community, and proprietary hospitals. PATIENTS: One hundred sixty-six women requiring surgical treatment for menorrhagia between March 1986 and October 1992. INTERVENTIONS: Fifty-eight women were treated with a Nd:YAG laser, 11 with a rollerball electrode, and 97 with a wire loop electrode. Concomitant resection of submucous leiomyomata was performed in 54 (32.5%) of the patients. MEASUREMENTS AND MAIN RESULTS: The mean operating times, complication rates, mean volume of fluid absorption, concomitant surgery, morbidity, duration of hospital stay, and results of laser and electrosurgical endometrial ablations were similar. Thirty-nine women (69.9%) undergoing laser endometrial ablation became amenorrheic and 54 (96.4%) had satisfactory results after 6 months. Seven (63.3%) of the women who had a rollerball ablation became amenorrheic, and 10 (90.9%) had satisfactory results. Sixty-one women (70.9%) who underwent wire loop resection became amenorrheic, with 83 (96.5%) attaining satisfactory results. Four years postoperatively, 85.4% of the patients continued to have satisfactory results. CONCLUSIONS: Laser and electrosurgical endometrial ablations are similarly effective treatment for patients suffering from chronic menorrhagia. This preliminary study should be followed by a randomized, controlled, prospective study to evaluate the two techniques more fully.

Adult↗
PubMed1994

Evaluation of a morpholinothiolporphyrin for use in photodynamic therapy.

The photonecrotic effectiveness of a morpholinothiolporphyrin derived from haematoporphyrin was measured in an animal model of cerebral glioma. The dose administered was 20 mg kg-1 and the laser dose varied from 0 to 200 J cm-2. The tumour necrosis was at least as good as that of HpD, and this therapeutic response may be attributed to the targeting of specific 'photopotent' subcellular sites.

Animals↗
PubMed1994

A modelling procedure for the analysis of dynamic drug--DNA interactions probed during active transcription of the DNA.

A multicompartment simulation analysis (CONSAM) has been used to describe the in vitro inhibition of transcription of DNA by echinomycin. The model assumes that at all drug blockage sites the fractional amount of blocked RNA polymerase is defined by the relative drug occupancy at that site (and is released at a rate defined by the drug dissociation rate), with all remaining enzyme being rapidly transferred past that site. The solution to the 48 parameters (three per drug site, 16 sites), which fully described the echinomycin-DNA transcription data set, can readily be accomplished without manual intervention within an hour on a MS-DOS, 486D-based microcomputer, compared to several months for a similar solution by Monte-Carlo simulation (requiring repeated intervention). The adequacy of the parameters to describe the model was confirmed by four independent criteria. The approach is applicable to the analysis of any enzyme system where an inhibitor of any type (interacting in either a reversible or irreversible manner) prevents the processive movement of enzyme along the template.

Base Sequence↗
PubMed1993

Effect of alkylating agents on initiation and elongation of the lac UV5 promoter.

DNA containing the lac UV5 promoter was alkylated using bifunctional sulfur and nitrogen mustards and a monofunctional sulfur mustard. The alkylation sites were mapped using Taq polymerase, and the effect of alkylation on the formation of the DNA-RNA polymerase complex was determined using gel retardation. Alkylation was observed at all G residues in the template strand. Exposure of the alkylated DNA to Escherichia coli RNA polymerase resulted in the formation of a DNA-enzyme complex that was more stable, prior to initiation, than the complex formed with nonalkylated DNA. The DNA-RNA polymerase complex formed with the alkylated DNA also demonstrated decreased ability to progress along the full length of the DNA template. These observations show that, in addition to inducing transcriptional blockages, mustards also influence the interaction between RNA polymerase and its promoter. The ability to interfere with protein-DNA interactions may contribute significantly to the effects of these compounds in eukaryotic systems with their complex array of transcription factors.

Alkylating Agents↗
PubMed1993

Cloning and characterization of the gene encoding the human platelet glycoprotein V. A member of the leucine-rich glycoprotein family cleaved during thrombin-induced platelet activation.

Glycoprotein V (GPV) is a major platelet membrane 82-kDa glycoprotein, missing in the Bernard-Soulier syndrome, that is cleaved when platelets are treated with thrombin. We report the cloning and sequencing of the GPV cDNA and gene obtained by a combination of polymerase chain reaction amplification of platelet mRNA and genomic library screening. The single-copy gene for GPV is contained within 6.5 kilobase pairs (kb) of genomic sequence and has a simple structure with a single intron of 958 base pairs in the 5'-untranslated sequence; the coding sequence is contained within a single exon. The promoter region contains a canonical TATA box, and putative GATA, Ets-1, and Sp1 cis-acting elements. Reverse transcription-polymerase chain reaction analysis on RNAs from cells of different hematopoietic origins revealed that GPV was specifically transcribed from platelets and from cells of the megakaryocytic lineage (megakaryocytes, HEL cells). A single transcript of 4.5 kb for GPV was detected in human platelets by Northern blot analysis. The entire amino acid sequence of GPV was deduced from the cDNA and genomic sequences. Mature GPV was composed of 544 amino acids which contained a single transmembrane domain, a short cytoplasmic domain (16 residues), and a large extracellular domain with 8 potential N-glycosylation sites. Analysis of the extracellular domain revealed the presence of 15 tandem Leu-rich repeats of 24 amino acids with homology to GPIb alpha and identified a cleavage site for thrombin near the COOH terminus with similarity to the A alpha chain of fibrinogen, but no hirudin-like sequence was found.

Amino Acid Sequence↗
PubMed1993

Thermal stability of DNA adducts induced by cyanomorpholinoadriamycin in vitro.

The Adriamycin derivative, cyanomorpholinoadriamycin (CMA) was reacted with DNA in vitro to form apparent interstrand crosslinks. The extent of interstrand crosslink formation was monitored by a gel electrophoresis assay and maximal crosslinking of DNA was observed within 1 hr with 5 microM of drug. The interstrand crosslinks were heat labile, with a midpoint melting temperature of 70 degrees C (10 min exposure to heat) in 45% formamide. When CMA-induced adducts were detected as blockages of lambda-exonuclease, 12 blockage sites were observed with 8 being prior to 5'-GG sequences, one prior to 5'-CC, one prior to 5'-GC and 2 at unresolved combinations of these sequences. These exonuclease-detected blockages reveal the same sites of CMA-induced crosslinking as detected by in vitro transcription footprinting and primer-extension blockages on single strand DNA, where the blockages at 5'-GG and 5'-CC were identified as sites of intrastrand crosslinking and the 5'-GC blockage as a probable site of interstrand crosslinking. The thermal stability of both types of crosslink (10 min exposure to heat) ranged from 63-70 degrees C at individual sites. High levels of adduct were detected with poly (dG-dC) but not with poly (dI-dC). These results suggest adduct formation involving an aminal linkage between the 3 position of the morpholino moiety and N2 of guanine.

Base Sequence↗
PubMed1993

The use of bidirectional transcription footprinting to detect platinum-DNA crosslinks by acridine-tethered platinum diamine complexes and cisplatin.

Bidirectional transcription footprinting has been used to probe the platination of DNA by cisplatin, and to examine the modulation of these interactions by (a) cyclisation of the non-reactive amino group by either ethyl or propyl groups, and (b) the further addition of a pendant intercalator (9-amino acridine) linked by either phenylethyl or phenylpentyl groups. Intrastrand crosslinking was detected for all derivatives at all 5'-GG and 5'-AG sequences on the template strand, but the same sites did not result in transcriptional blockages when on the non-template strand. There was little effect of cyclysation of the amino groups, but the further addition of an intercalator resulted in three responses: a time-dependent increase of the blocked transcript by one and three nucleotides; a reduction of the sequence selectivity of platination; a decrease of apparent interstrand crosslinking for these derivatives with a pendant intercalator tethered to the amino moiety of cisplatin.

Acridines↗
PubMed1993

Evidence for novel binding sites on the platelet glycoprotein IIb and IIIa subunits and immobilized fibrinogen.

The present study was designed to examine the interaction of the purified platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa or integrin alpha IIb beta 3) and the individual subunits of the complex with immobilized fibrinogen. Although 125I-GP IIb-IIIa binding to fibrinogen immobilized on Sepharose was specific, this interaction exhibited properties distinct from those of reversible fibrinogen binding to platelets: 125I-GP IIb-IIIa binding appeared irreversible, but non-covalent, Ca(2+)-independent, and was inhibited only weakly, or not at all, by the anti-(GP IIb-IIIa) monoclonal antibodies 10E5 and 7E3 and synthetic peptides from known platelet-binding domains of fibrinogen. Reversibly dissociated GP IIb or GP IIIa subunits inhibited 125I-GP IIb-IIIa binding to immobilized fibrinogen and bound directly to the fibrinogen. However, these subunits did not bind to peptides derived from known platelet-binding domains within the fibrinogen alpha- and gamma-chains, although the GP IIb-IIIa complex did. These results show that the complexed form of full-length GP IIb and GP IIIa is required for binding to these synthetic peptides, but not necessarily for binding to immobilized fibrinogen. Thus GP IIb-IIIa can bind to immobilized fibrinogen by a distinct mechanism that appears to involve novel binding sites on each subunit of the GP IIb-IIIa complex and on fibrinogen.

Amino Acid Sequence↗