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Biomedical subjects

A W Prestayko

Publications and source records attributed to A W Prestayko.

At least 19 recordsLinked to original sources
PubMed1986

Formation of the thiol adducts of 4'-(9-acridinylamino)methanesulfon-m-anisidide and their binding to deoxyribonucleic acid.

We investigated the interactions of 4'-(9-acridinylamino)methanesulfon-m-anisidide (mAMSA) with thiol-containing compounds and the potential binding of the thiolytic adducts to DNA. All thiols tested (glutathione, cysteine, coenzyme A, 2-mercaptoethanol and lactate dehydrogenase) formed adducts with mAMSA as evidenced by changes in the absorption spectrum of mAMSA and induction of fluorescence. Spectral changes induced by the thiols were different, suggesting that each thiol induced specific changes in the electronic structure of the acridine nucleus. Treatment of glutathione with p-chloromercuribenzoate eliminated the absorption spectral changes and induction of fluorescence, indicating that the reduced-thiol group is involved. In high ionic strength buffer, addition of calf thymus DNA induced fluorescence-quenching of both the mAMSA-glutathione and mAMSA-cysteine adducts without spectral shift. Viscometric studies showed that mAMSA and mAMSA-glutathione intercalated into DNA and produced similar increases in the length of linear DNA.

Aminoacridines↗
PubMed1984

Platelet-derived growth factor (PDGF) induces intranuclear protein accumulation in 3T3 fibroblasts.

Quiescent 3T3 fibroblasts were grown for a short time in the presence of [3H]amino acids then treated with PDGF, and the nucleo-cytoplasmic distribution of the 3H-labelled proteins was analysed by autoradiography. There was no difference in the total amount of 3H-labelled proteins in PDGF-treated and untreated cells but PDGF induced a significant increase in intranuclear protein accumulation.

Animals↗
PubMed1982

Bifunctional intercalation of antitumor antibiotics BBM-928A and echinomycin with deoxyribonucleic acid. Effects of intercalation on deoxyribonucleic acid degradative activity of bleomycin and phleomycin.

The binding of peptide antitumor antibiotics, BBM-928A and echinomycin, to superhelical PM2 DNA and the effects of the resulting conformational changes of DNA on the DNA-degradative activity of two related antitumor antibiotics, bleomycin A2 and phleomycin D1, have been studied. The bifunctional intercalative mode of the DNA binding of BBM-928A concluded previously from viscometric and fluorometric studies has been confirmed by gel electrophoretic analysis. Under the employed electrophoretic conditions, DNA-bound BBM-928A showed little dissociation whereas echinomycin and ethidium bromide showed partial and nearly complete dissociation, respectively. BBM-928A induced neither single-strand nor double-strand breaks in DNA. Competitive binding studies by fluorescence changes suggested that binding sites on DNA molecules for BBM-928A (or echinomycin) may differ from those for ethidium bromide, since binding to DNA by the two drugs was not competitive even at saturating concentrations. The lack of such a competition between the two drugs is not consistent with the neighbor-exclusion principle. The DNA-degradative activity of both bleomycin A2 and phleomycin D1 increased with the removal of the negative superhelicity of DNA by the BBM-928A intercalation and decreased with the formation of positive superhelical turns induced by high concentrations of BBM-928A. Thus the degradative activity of both bleomycin A2 and phleomycin D1 is sensitive in a similar manner to the degree of superhelicity rather than the double helicity of DNA, although there are differences between these two drugs in interaction with DNA.

Animals↗
PubMed1982

Characterization of in vitro deoxyribonucleic acid breakage and cross-linking induced by bis-isopropylamine)-trans-dihydroxy-cis-dichloroplatinum(IV).

Bis(isopropylamine)-trans-dihydroxy-cis-dichloroplatinum(IV) (CHIP or JM-9), a derivative of Cisplatin, was found to have DNA breakage and interstrand cross-linking activities in vitro. DNA breakage was detected by alkaline and neutral sucrose gradient analysis, agarose gel electrophoresis, and alkaline ethidium bromide fluorescence assay employing covalently closed circular PM2 DNA. DNA cross-linking activity was detected by alkaline sucrose gradient analysis and by the "snap-back" assay employing PM2 DNAs. Non-sulfhydryl-containing reducing agents, e.g., NaBH4 and NADPH, stimulated both cross-linking and breakage activities. Alkaline buffers, cyanide, or sulfhydryl group containing agents inhibited both types of activities. The hydroxyl free radical scavenger sodium benzoate (100 mM) was found to inhibit 99% and 25% of DNA breakage and cross-linking activities, respectively, suggesting DNA breakage and cross-linking may be independently mediated.

Antineoplastic Agents↗
PubMed1982

Viscometric and fluorometric studies of deoxyribonucleic acid interactions of several new anthracyclines.

Interactions involved in the binding of the anthracycline analogues adriamycin, carminomycin, pyrromycin, musettamycin, marcellomycin, and aclacinomycin to calf thymus DNA and covalently closed circular PM-2 DNA have been studied. Fluorescence quenching experiments revealed that denaturation of calf thymus DNA and increasing ionic strength each resulted in a marked decrease in the DNA binding affinities of all of the anthracyclines studied. These results suggest that intercalative and electrostatic interactions are both important in the DNA binding of these analogues. Viscometric studies indicated that under high ionic strength conditions which negated electrostatic effects, all of the anthracyclines induced an unwinding--rewinding process of the closed superhelical PM-2 DNA typically observed for DNA intercalators. Relative to the 26 degrees unwinding angle of ethidium bromide, anthracyclines with a daunomycinone-like aglycon induced an unwinding angle of approximately 13 degrees. This differed slightly from the unwinding angles of 10.3 degrees-11.1 degrees which were induced by anthracyclines with a pyrromycinone-like aglycon. Increases in the length of the glycosidic side chain did not elicit significant differences in PM-2 DNA unwinding ability, implying a lack of effect of glycosidic side chain length on the anthracycline intercalation process. The unwinding angles also showed little sensitivity to decreases in ionic strength, suggesting that the fraction of bound anthracycline molecules which are in a nonintercalated state is similar to the fraction of bound ethidium bromide molecules which are in a nonintercalated state.

Antibiotics, Antineoplastic↗
PubMed1982

Structure-activity relationships involved in the site-specific fragmentation of linear duplex DNAs by talisomycin and bleomycin analogs.

The fragmentation of Hind III- and Pst I-digested PM2 DNA and of Hind III-digested pBR322 DNA by bleomycin A2 and B2 and talisomycins A, B, S2b, and S10b was investigated. As observed by electrophoresis on agarose gels, the ethidium bromide staining band patterns produced following incubation of the various restriction endonuclease-digested DNAs with the compounds were different for the bleomycin analogs and for the talisomycin analogs. Quantitation of the degree of fragmentation of various segments of linear PM2 DNA by either bleomycin A2 or talisomycin A indicated that certain segments of the PM2 genome serve as better substrates than other segments for double-strand fragmentation by either of the two antitumor antibiotics. These results show that in this system bleomycin and talisomycin analog treatment of linear PM2 or pBR322 DNA resulted in breakage of DNA, producing different-length DNA fragments, and demonstrate the importance of the two amino acids and the 4-amino-4,6-dideoxy-L-talose sugar, which are located near the bithiazole in talisomycin but absent in the bleomycin structure in conferring a different site-specificity for DNA fragmentation to talisomycin than to bleomycin.

Antibiotics, Antineoplastic↗
PubMed1982

Effects of cis-diamminedichloroplatinum (CDDP) on HeLa cell non-histone nuclear proteins.

Two-dimensional isoelectric focusing and sodium dodecyl sulfate (SDS) gel electrophoretic analyses of successive salt extracts of purified HeLa cell nuclei were used to study the effects of cis-diamminedichloroplatinum (CDDP) (cisplatin) on the synthesis and extractability of nuclear non-histone proteins. Nuclei were extracted sequentially with 0.025 M NaCl-0.075 M EDTA, 0.01 M Tris, and 0.6 M NaCl. Each fraction contained 100-400 polypeptide spots, only a few of which were affected by a 3.5-h CDDP pretreatment of the cells. The biosynthesis and/or metabolism of four polypeptide spots was significantly affected by the CDDP treatment. These polypeptides included: (a) 36K/5.8 (designated by MW/PI) in the 0.025 M NaCl-0.075 M EDTA extract, which decreased in intensity with treatment at 10 micrograms CDDP/ml; (b) polypeptide spots 50K/6.0 and 45K/5.3 in the Tris extract, which increased in intensity over a range of CDDP concentrations of 0-5 microgram CDDP/ml; and (c) a polypeptide complex at 110K/7.7 in the 0.6 M Na Cl extract, which decreased in intensity at CDDP concentrations of 0-5 microgram CDDP/ml. Scanning densitometry of the protein spots of the 0.6 M NaCl extract demonstrated that the 110K/7.7 complex decreased to half its intensity compared with non-drug-treated controls at a CDDP concentration of 0.9 microgram CDDP/ml. We have found that high-resolution two-dimensional gel electrophoretic analysis of nuclear proteins is a valuable technique for studying the effects of cytotoxic agents on the synthesis and/or extractability of specific cellular proteins.

Cell Nucleus↗
PubMed1982

Comparison of the sequences at specific sites on DNA cleaved by the antitumor antibiotics talisomycin and bleomycin.

We have investigated the site-specific cleavage of DNA by the antitumor antibiotics talisomycin and bleomycin by using 5'- or 3'-terminal 32P-labeled restriction fragments of pBR 322 DNA. Both drugs cleaved DNA preferentially at G-C and G-T sequences. However, the relative amounts of cleavage at particular cleavage sites differed between talisomycin and bleomycin at concentrations of the drugs which produced similar extents of total cleavage. In addition, talisomycin produced specific cleavages at G-A sequences which were relatively resistant to cleavage by bleomycin. Within a preferred sequence group (i.e., G-C sequences), some sites were cleaved to a greater extent relative to others by both talisomycin and bleomycin, suggesting that a greater degree of specificity than that provided by only two nucleotides is involved in the site-specific recognition and cleavage of DNA by these drugs.

Antibiotics, Antineoplastic↗
PubMed1982

Detection of a serum DNA-binding protein associated with cancer.

An accompanying report describes the purification and partial characterization of a unique DNA-binding protein (Mr 64,000; pI 5.9) that is present in human sera. This report gives the results of assays of sera from patients for the bleomycin inhibitor protein (BIP) using the Pseudomonas bacteriophage covalently closed circular DNA fluorescence technique standardized for DNA breakage induced by bleomycin. The results of the BIP assays were expressed by values of specific activity of inhibition. One arbitrary unit of inhibitory activity was defined as equivalent to the amount of serum protein required to cause 50% inhibition of DNA degradation using standard conditions of the DNA breakage assay. The mean values of specific activity of inhibition (SAI) for groups of healthy individuals (n = 26), patients with nonmalignant diseases (n = 33), and patients with malignant diseases (n = 83) were 12.60 +/- 4.69 (S.E.), 12.53 +/- 3.17, and 2.40 +/- 0.84 units/mg, respectively. Mean SAI values for patients with cancers of various types were: solid tumors (n = 46), 2.44 +/- 0.86; leukemias (n = 24), 2.59 +/- 0.96; and lymphomas (n = 18), 2.07 +/- 0.64. The decrease in BIP activity was not correlated with sex, age, or prior chemotherapy. Mean SAI values of male (n = 29) and female (n = 59) patients with cancer were 2.61 +/- 0.87 and 2.30 +/- 0.83 units/mg, respectively. Mean SAI values for different age groups were: 0 to 40 years (n = 21), 2.05 +/- 0.68 units/mg; 41 to 70 years (n = 56), 2.59 +/- 0.68 units/mg; and greater than 70 years (n = 11), 2.12 +/- 0.67 units/mg. Cancer patients with and without prior chemotherapy had mean SAI values of 2.97 +/- 0.85 (n = 23) and 2.20 +/- 0.86 units/mg (n = 65), respectively. Linear regression analysis comparing SAI values and serum protein levels showed no correlation (r = 0.21). These results suggest the decrease of the BIP is associated with malignant disease. Additional controlled studies are required before the significance of this association can be adequately assessed.

Adolescent↗
PubMed1982

Effect of anthracycline analogues on the appearance of newly synthesized total RNA and messenger RNA in the cytoplasm of erythroleukemia cells.

Effects of the structural analogues, adriamycin (ADM), daunomycin (DNM), carminomycin (CMM), 4-demethoxydaunomycin (4D-DNM), pyrromycin (PYM), marcellomycin (MCM), and aclacinomycin (ACM) upon total cell RNA synthesis and the appearance of total RNA and poly(A)+-RNA in the cytoplasm of uninduced Friend erythroleukemia cells were investigated. The anthracyclines inhibited cellular RNA synthesis with IC50 values of 1-3 microM (ADM, DNM), 0.3-0.5 microM (CMM, 4D-DNM, PYM), and 0.06 microM (MCM, ACM). IC50 values for the appearance of total RNA in the cytoplasm were consistently 2-3 times lower than those for total cell RNA synthesis for each anthracycline. IC50 values for the inhibition of poly(A)+-RNA in the cytoplasm by ADM, DNM, and CMM were equivalent to those for total RNA synthesis. The values for MCM and ACM were 2-3 times higher than those for total RNA synthesis. The kinetic actions of drug-induced inhibition of poly(A)+-RNA appearance in the cytoplasm and inhibition of total RNA synthesis were equivalent for ADM, DNM and CMM, whereas the other anthracyclines showed different kinetics. These studies confirm the greater sensitivity of nucleolar RNA synthesis to Class II anthracyclines in erythroleukemia cells and suggest that inhibition of post-transcriptional events may occur in cells exposed to PYM, MCM, and ACM but at higher concentrations than are required for inhibition of RNA synthesis.

Animals↗
PubMed1982

The antitumor effects of anthracyclines. II. The stereospecificity of the carbomethoxy group at position 10 of the class II anthracycline molecule.

The effects of marcellomycin, musettamycin, and their respective position 10 epimers mimimycin and collinemycin were compared in several systems both in vivo and in vitro. The results of these studies showed that the epimerization of the carbomethoxy-group at position 10 of the Class II anthracycline aglycone resulted in a 3-fold to 20-fold decrease in DNA-binding ability. The reduced DNA binding ability of these compounds is correlated with a 2-fold to 17-fold decrease in whole cellular nucleic acid synthesis inhibitory potency, a 2-fold to 4-fold decrease in vitro antitumor potency, and a 4-fold to 32-fold reduction in in vivo antitumor potency. These results further support the concept that a major portion of the antitumor activity of Class II anthracyclines is related to their avidity for DNA interactions and resultant disruption of normal template function. Previous studies from this laboratory have demonstrated that the 10-carbomethoxy group is essential for nucleolar RNA synthesis inhibition and Class II anthracycline antitumor activity. The current study further demonstrates that the stereochemical orientation of the carbomethoxy group at position 10 of the aglycone is also important in conferring Class II anthracycline antitumor activity.

Animals↗
PubMed1981

DNA breakage activity of the methanol extract of auromomycin.

The constituents of the antitumor agent auromomycin have been analyzed to determine their DNA-breakage activities. Spectral analysis showed that the methanol extract contained 70% of the non-peptide chromophore, whereas the residue contained 20%. Amino acid analysis of the methanol extract showed that it contained 21%-26% of the original auromomycin polypeptides. The DNA-degradation activity of the extract was 121% +/- 28% of that of the untreated auromomycin, whereas that of the residue was only 22% +/- 3.8%. Mixing of the residue and the methanol extract resulted in the loss of three-fourths of the total activity. Agarose gel electrophoretic analysis showed that the single-strand DNA breakage activity of the methanol extract was 6.5-fold greater than that of the double-strand DNA-breakage activity. The difference in the total DNA-cleavage activity of the untreated, methanol-treated, and remixed auromomycin preparations may suggest the occurrence of certain non-peptide chromophore-polypeptide interactions in both the untreated and the remixed preparations. This is consistent with the fluorescent changes observed upon mixing of the extract and residue. Fractionation of the methanol extract by Sephadex chromatography revealed that several column fractions which were enriched with non-peptide chromophore relative to the polypeptides contained in them still had significant DNA-degradation activity. These studies suggest that the non-peptide chromophore in the auromomycin preparation may contribute to most of the observed DNA breakage activity.

Amino Acids↗
PubMed1981

Inhibition of bleomycin-induced DNA breakage by superoxide dismutase.

Inhibition of bleomycin (BLM)-induced DNA breakage by superoxide dismutase (SOD) has been reported and presumed to be due to its removal of the superoxide free radicals generated by BLM in the presence of iron(II). We have studied the possibility that the inhibitory effect might result from DNA-binding of SOD. The effect of copper-zinc SOD on BLM-induced DNA degradation was investigated using the PM-2 DNA fluorescence technique. PM-2 DNA was incubated with BLM in the presence or absence of native and heat-inactivated copper-zinc SOD as determined by the epinephrine autoxidation method. The concentrations of SOD required to inhibit 50% PM-2 DNA degradation for the native and the inactivated SOD were 100 and 120 microgram/ml, respectively. Analysis of the reaction mixture by agarose gel electrophoresis confirmed the absence of DNA degradation by BLM in the presence of either form of SOD. PM-2 DNA was shown to bind native or inactivated SOD by Sephadex G-100 column chromatography, fluorescence-quenching studies, and agarose gel electrophoresis. Thus, these results indicate that SOD is able to bind to PM-2 DNA and inhibit BLM-induced degradation independently of its free radical-scavenging activity. The inhibitor was more effective against BLM than other compounds which degrade PM-2 DNA. This suggests that SOD may bind to BLM-binding and/or BLM degradation sites in PM-2 DNA, and the observed inhibition is unrelated to its effects on free radicals.

Binding Sites↗
PubMed1981

DNA supercoiling, shortening, and induction of single-strand regions by cis-diamminedichloroplatinum(II).

cis-Diamminedichloroplatinum(II) was found to bind to covalently closed circular supercoiled, covalently closed circular nonsupercoiled, and single-strand broken relaxed PM2 DNA and induce different types of DNA conformational changes. Using Kleinschmidt's technique, it was found that binding of cis-diamminedichloroplatinum(II) decreased the DNA length to 75% of the original single-strand broken relaxed DNA without inducing superhelical conformational changes. cis-Diamminedichloroplatinum(II) also shortened the length of covalently closed circular nonsupercoiled DNA before a supercoiled conformation was generated. Single strand-specific nucleases were used to detect drug-induced DNA structural alterations. Local single-strand regions or regions of denaturation were detected by S1 nuclease from Aspergillus oryzae and by BAL-31 nuclease from Alteromonas espejiana BAL-31. The single-strand regions or local denaturation regions do not seem to be related to or caused by DNA superhelical conformational changes since they were detected at drug concentrations at which no significant DNA superhelical turns were found. DNA shortening, superhelical conformational changes, and local denaturation regions can be explained by the previously proposed "DNA intrastrand cross-linking" model (Stone et al., J. Mol. Biol., 104: 793-801, 1976).

Cisplatin↗
PubMed1980

Interaction of cis-diamminedichloroplatinum(II) with PM-2 DNA.

The interaction of cis-diamminedichloroplatinum(II) (CDDP) with PM-2 DNA was studied using two techniques: (a) agarose gel electrophoresis of PM-2 DNA conformation isomers after CDDP binding; and (b) viscometric measurement of different forms of CDDP-bound PM-2 DNA. In both systems, the results indicated that the DNA isomers interacted differently with CDDP. CDDP induced a decrease of viscosity upon interacting with single-strand broken relaxed circular (Form II) and double-strand broken linear (Form III) PM-2 DNA's. These observations are consistent with a "DNA shortening effect" proposed by Cohen et al. [Science (Wash. D. C.), 203: 1014-1016, 1979] and Macquet et al. [Biochimie (Paris), 60: 901-914, 1978] When covalently closed circular (Form I) PM-2 DNA was used, increasing concentrations of CDDP induced an initial slight increase and then decrease of electrophoretic mobility to the degree that it comigrated with CDDP-bound Form II DNA. Further addition of CDDP restored the electrophoretic mobility of Form I DNA. Corresponding changes in the viscosity of CDDP-bound Form I DNA showed an initial decrease, then an increase, and a final prolonged decrease of viscosity. These effects are similar but not identical to those induced by either DNA intercalators (e.g., ethidium bromide) or certain DNA denaturating agents (e.g., formaldehyde, ultraviolet light, alkali trichloroacetate, methylmercuric hydroxide, and carbodiimide). Thus, CDP may induce a DNA superhelix-unwinding process followed either by rewinding or a denaturation process or both. Quantitative analysis of the agarose gel electrophoretic pattern plus sucrose density gradient centrifugation studies also indicated that there was little DNA strand breakage induced by CDDP treatment.

Animals↗
PubMed1980

Effects of second-generation platinum analogs on isolated PM-2 DNA and their cytotoxicity in vitro and in vivo.

Using PM-2 DNA and cytotoxicity assay systems, we have studied several second-generation platinum analogs and compared these to the parent compound cis-diamminedichloroplatinum(II). These results indicate that all planar platinum(II) congeners induced similar effects upon interaction with PM-2 DNA, i.e., alteration of the tertiary DNA conformations. The reactivity of the analogs with DNA depended on the activity of the leaving groups. Octahedral platinum(IV) compounds, however, induced breakage of covalently closed circular PM-2 DNA, and the effects were not inhibited by either chloride or ethylenediaminetetraacetate. This suggests that breakage of isolated PM-2 DNA may be related to the axial trans bonds rather than the equatorial cis bonds of the solvated platinum(IV) compounds, since the activity of the dichloroplatinum(II) compounds has been shown to be inhibited by chloride ions. Studies on the in vitro and in vivo cytotoxicity of the platinum analogs demonstrated that the reactivity of analogs against PM-2 DNA correlated with in vitro and in vivo potencies. The reactivity with PM-2 DNA appeared to depend on the characteristics of the leaving group.

Animals↗