Role of deoxyribonucleic acid topology in altering the site/sequence specificity of cleavage of deoxyribonucleic acid by bleomycin and talisomycin.
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Base Sequence↗
Biomedical subjects
S T Crooke
Publications and source records attributed to S T Crooke.
Effects of DNA superhelical changes induced by ethidium bromide on the DNA-degrading activity of two antitumor antibiotics, bleomycin and phleomycin.
The effects of changes in the conformational state of DNA on the single-strand and double-strand breakage activity of two antitumor antibiotics, bleomycin (BLM) A2 and phleomycin D1, have been studied by the gel electrophoretic analysis of the drug-degraded PM2 phage superhelical DNA pretreated with an intercalating agent, ethidium bromide (EB). Both the single-strand and double-strand breakage activities of BLM A2 increased as the negatively superhelical turns of native PM2 DNA were gradually removed by intercalation with increasing EB concentrations. The activities peaked when DNA was completely relaxed and gradually decreased as the higher concentrations of EB twisted DNA into the positively superhelical form. The decrease in breakage activity was not due to any inhibitory effect of EB at higher concentrations, since treatment of the relaxed Form I0 DNA with low EB concentrations also reduced the activity. In contrast to BLM A2, phleomycin D1 responded minimally to DNA conformational changes, which suggested further that the two drugs may react with DNA differently. The differential responses of BLM A2 activity towards different DNA conformational states may have biological implications, since DNA in cells may exist in different conformational states relating to various gene functions. The current study may serve as a model for studying combined effects of intercalative and nonintercalative antitumor antibiotics which are used frequently in combination treatments of cancer.
Animals↗
Intermolecular cross-linking of DNA through bifunctional intercalation of an antitumor antibiotic, luzopeptin A (BBM-928A).
A bifunctional intercalator may intercalate with DNA in at least two ways. Both intercalating moieties may intercalate with the same DNA molecule (type I, intramolecular cross-linking) or with two separate DNA molecules (type II, intermolecular cross-linking). Production of type I is often assumed. Type II biintercalation has been suggested, but no direct evidence has been reported. In the present study, endonuclease-restricted PM2 phage or pBR322 plasmid DNA fragments were treated with the bifunctional intercalative antitumor antibiotics, luzopeptin A (BBM-928A) and echinomycin, and analyzed by agarose gel electrophoresis. Luzopeptin A treatment produced additional DNA bands which were the products of type II biintercalation. The types of restriction fragments involved were identified. Maximal type II biintercalation occurred at a luzopeptin A/DNA range of 0.14 to 0.18, at which more than 50% of the total DNA molecules were involved. Type II products were converted gradually to type I products upon prolonged incubation at 37 degrees, probably due to the tendency for intermolecular bonds to disrupt. Echinomycin treatment failed to produce type II products, probably because of a DNA-binding affinity weaker than that of luzopeptin A. Thus, it is possible to use the present gel system to demonstrate the type II biintercalation for strong biintercalators, but milder systems are needed for weak biintercalators.
Antibiotics, Antineoplastic↗
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↗
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↗
Cyclophosphamide, doxorubicin, vincristine, and low-dose continuous infusion bleomycin in non-Hodgkin's lymphoma: Cancer and Leukemia Group B Study No. 7804.
Fifty-eight evaluable patients with non-Hodgkin's lymphoma were treated with a low dose, 120-hour continuous intravenous infusion of bleomycin in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone. Pharmacokinetic data, obtained in six patients, confirm that steady-state plasma concentrations of bleomycin can be attained even with the administration of 2 units/day of the drug. Neither clinical pulmonary toxicity nor subclinical pulmonary changes, as determined by serial measurement of the single breath carbon monoxide-diffusing capacity, were observed. Compared to similar chemotherapeutic programs utilizing bolus administration of bleomycin, pulmonary toxicity may be reduced. Response frequencies among 37 previously untreated patients were similar to those obtained using the same chemotherapeutic agents but with intravenous bolus administration of bleomycin. In addition, 18 of 21 patients who had received prior chemotherapy responded. Low dose, continuous intravenous infusion of bleomycin may improve the therapeutic index of combination chemotherapy programs for non-Hodgkin's lymphoma.
Adult↗
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↗
Dibromodulcitol plus bleomycin compared with bleomycin alone in head and neck cancer.
Advanced recurrent squamous cell head and neck cancer patients were prospectively randomized to receive or not receive dibromodulcitol 10 mg/kg PO weekly for 8 consecutive weeks in addition to bleomycin chemotherapy. Patients initially entered in the study received bleomycin 15 mu/m2 three times weekly for 8 weeks. This was later changed to 15 mu/m2 twice weekly for 8 weeks because of unacceptable stomatitis. Most patients had relapsed following surgery and/or radiotherapy, but none had received prior chemotherapy. A2 : 1 randomization in favor of the dibromodulcitol-containing therapy was used. There were 12 partial responses in the 44 evaluable patients receiving the combination (27%), and 4 partial responses in the 18 patients receiving single-agent bleomycin chemotherapy (22%). This difference was not statistically significant. Response durations were also relatively short for both therapies. Within the limitations of this study, we were unable to demonstrate that patient benefit resulted from the addition of dibromodulcitol to bleomycin chemotherapy for this patient population.
Adult↗
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↗
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↗
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↗
Inhibition of PM-2 DNA degradation by a human serum protein.
A unique DNA-binding protein was detected that inhibited DNA degradation induced by bleomycin and was decreased in sera of cancer patients. The protein from normal human serum was purified to homogeneity by ammonium sulfate precipitation and DEAE-cellulose and DNA-cellulose column chromatography. Two-dimensional isoelectric focusing gel electrophoresis revealed a single protein spot with a molecular weight of 64,000 and a pI at pH 5.9. The NH2 terminus was lysine, and the ratio of acidic to basic residues was 1.2. DNA binding was demonstrated by column chromatography, agarose gel electrophoresis, fluorescence quenching, and circular dichroism. The inhibitory activity was abolished by treatment with Pronase but not by RNase or DNase I. FeCl2 caused a partial loss of inhibitory activity. The inhibition of DNA degradation was more effective for breakage induced by bleomycin than neocarzinostatin, macromomycin, or DNase I. Evidence from DNA-binding studies suggests the inhibition is due to binding of the protein to sites on DNA preferred by bleomycin. Thus, the protein could be useful for studies on the mechanism of action of bleomycin and other antitumor agents, the cytotoxic effects of which are due primarily to damage of cellular DNA. The protein was decreased significantly in sera of cancer patients, and its potential use as a diagnostic tool for neoplasias is being investigated further.
Bleomycin↗
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↗
Bleomycin and talisomycin sequence-specific strand scission of DNA: a mechanism of double-strand cleavage.
Computer analyses of DNA sequencing data obtained using various restriction fragments of pBR 322 DNA indicate that a trinucleotide sequence (-Pyr-G-C-) is the most preferred site for cleavage by the antitumor antibiotic bleomycin A2. Talisomycin A, a structurally related bleomycin analog, cleaved at the sequences -G-T/A- most preferentially. However, the presence of a pyrimidine at the 5' side of guanine at the cleavage site did not increase the probability of that site being cleaved by talisomycin. Using denaturing and nondenaturing polyacrylamide gel analyses of the drug-DNA reaction products. The sites of both single- and double-strand breaks have been localized and differentiated. The results indicate that a major determinant for location of a site-specific double-strand break is the production of two closely spaced sequence-specific single-strand breaks by the drugs on opposite strands of the DNA. A four-base pair sequence is proposed for the optimal sequence for bleomycin-induced double-strand breaks.
Base Sequence↗
In vivo resistance towards anthracyclines, etoposide, and cis-diamminedichloroplatinum(II).
From a single wild-type strain of Ehrlich ascites tumor, sublines resistant to daunorubicin, etoposide, and cis-diamminedichloroplatinum(II) have been developed in vivo. Different levels of resistance were achieved after 4 to 8 months for anthracyclines (greater than 32-fold), cis-diamminedichloroplatinum(II) (4-fold), and etoposide (greater than 6-fold). Anthracycline resistance was associated with decreased nuclear steady-state concentration of anthracyclines, increased content of high-molecular-weight membrane glycoproteins, and glucose-dependent drug extrusion after metabolic blockade with sodium azide. A similar "pump" system which was apparently not drug specific was also documented in etoposide resistance. Resistance towards cis-diamminedichloroplatinum(II) was accompanied by decreased cis-diamminedichloroplatinum(II)-induced DNA damage in vitro when proteinase K-resistant interstrand cross-links were measured by alkaline elution. Parallel in vivo studies revealed cross-resistance of various degrees among a number of anthracycline analogs, complete cross-resistance among daunorubicin, doxorubicin, and 4'-(9-acridinylamino)methanesulfon-M-anisidine (amsacrine), and partial cross-resistance between daunorubicin and etoposide. However, cis-diamminedichloroplatinum(II) was curative in anthracycline- and etoposide-resistant cells, as daunorubicin and etoposide were curative in acquired resistance towards cis-diamminedichloroplatinum(II). cis-Diamminedichloroplatinum(II) resistance was also overcome by the derivative 1,2,diaminocyclohexylplatinum malonate. The Vinca alkaloid vindesine, although only marginally active in the control tumor, was highly active in cells selected for cis-diamminedichloroplatinum(II) resistance. These in vivo patterns of cross-resistance and collateral sensitivity may be related to observations in clinical chemotherapy.
Animals↗
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↗
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↗
A phase I and clinical pharmacology study of intravenously administered carminomycin in cancer patients in the United States.
Carminomycin (CMN) was administered i.v. to 44 patients with a variety of nonhematological cancers every 4 weeks at doses of 15, 20, 22.5, and 25 mg/sq m. Granulocytopenia was the dose-limiting toxicity. The median granulocyte count for previously untreated patients receiving 22.5 mg/sq m was 0.962 cells/microliters, and for previously treated patients receiving 20 mg/sq m it was 0.420 cell/microliters. Moderate to severe phlebitis was associated with drug administration in 50% of cases. Nausea, vomiting, and alopecia were mild. Three of nine patients who received a total CMN dose of greater than or equal to 100 mg/sq m (mean, 132 mg/sq m) developed unexplained decreases in radionuclide cardiac ejection fraction, with one patient developing decreased QRS amplitude and congestive heart failure at a total dose of 160 mg/sq m. CMN is rapidly metabolized to carminomycinol. The elimination half-lives of CMN and carminomycinol are 6 to 10 and 50 hr, respectively. CMN was found to be a more potent inhibitor of human granulocyte-macrophage colony-forming units than was carminomycinol. Objective partial responses were seen in two of seven previously untreated patients with non-small cell lung cancer and one of three patients with squamous cell carcinoma of the head and neck previously untreated with chemotherapy.
Adult↗