Amplification of the antitumor activity of phleomycins and bleomycins in rats and mice by caffeine.

While having no antitumor effect per se, caffeine substantially enhanced the antitumor effects of the phleomycins PLM-CHP and PLM-PEP, and the bleomycins BLM-CHP and Blenoxane in rats carrying Walker 256 carcinosarcoma and/or mice carrying Ehrlich ascites tumor, even at doses of phleomycin and bleomycin below the minimum effective level. Positive but less conclusive results were also obtained with PLM-A4A4G and PLM-G.

A general method for selecting strains of Escherichia coli having altered spontaneous or induced mutabilities.

A simple method is described for selecting mutant strains of E. coli differing from their parent in the ability to spontaneously mutate via any mechanism. The method detects hypo- and hyper-mutators. It relies on the detection as papillae on thy colonies of second mutations either at the dra or drm locus.

The origins of the back-mutation assay method: a personal recollection.

The back-mutation assay method for determining the mutagenicity of various treatments was first developed a little over 50 years ago and has been in continuous use ever since. Shortly after the method was first used it became evident that certain factors of cell density, composition of media, etc., had to be carefully controlled to preserve an acceptable reliability of the method. A factor of particular importance was the suppression of growth of back-mutant prototrophic cells by the large number of auxotrophic cells present, a phenomenon which later became known as the "Grigg Effect." This review describes the origins of the back-mutation method and of the confounding competitive suppression phenomenon, the cause of competitive suppression, methods of diagnosing whether it is likely to bias the interpretation of a particular back-mutation experiment, and an experimental design which removes it entirely as a possible source of error. A number of other phenomena, such as phenotypic lag and coincident mutation associated with back-mutation, are also discussed as possible sources of error.

DNA methylation and mutation.

5-Methylcytosine (5mC) in DNA is produced by post-synthetic modification of cytosine residues, and it occurs primarily in CpG doublets in the mammalian genome. 5mC is a mutable site, because it can undergo spontaneous deamination to thymine. There is a repair mechanism which specifically recognises G.T mispairs, and replaces thymine with cytosine. However, this repair is not fully efficient, because the 5mC-->T transition mutation occurs about 10 times as frequently as other transitions. Such mutations are frequently seen in inherited diseases, and mutations in the p53 gene in tumours are also very commonly in 5mCpG doublets. As well as mutations, there can also be heritable changes in DNA methylation, known as epimutations, which may be of particular significance in somatic cells. Whereas the pattern of DNA methylation is very constant for any one cell type, the pattern becomes very variable in tumour cells. The breakdown of the normal controls of DNA methylation in tumorigenesis can lead to increased gene expression or to gene silencing. DNA damage increases not only mutation, but also heritable changes in methylation. At present, little is known about the ability of DNA repair to preserve the normal pattern of methylation in somatic cells.

Sulphydryl-mediated DNA breakage by phlemomycin in Escherichia coli.

Sulphydryl-mediated DNA breakage, which is induced by the antibiotic phleomycin in vitro, has been found to contribute significantly to the DNA damage produced by phleomycin in Escherichia coli. The effect of pleomycin was inhibited in vivo, as in vitro, by chelating agents, sulphydryl blocking agents and antioxidants. An increase in the intracellular concentration of free sulphydryl resulted in an increased response to phleomycin, while mutants containing very low levels of free sulphydryl due to a defect in glutathione synthesis showed greatly reduced DNA breakage, particularly at low phleomycin concentrations. In spheroplasts of these gshA mutants, restoration of the response to phleomycin of dithiothreitol. Sulphydryl-mediated breakage appears to be the principal mechanism for DNA damage in E. coli at libly enzymic, operates at higher phleomycin concentrations.

Amplification of the antibiotic effects of the bleomycins, phleomycins and tallysomycins: its dependence on the nature of the variable basic groups.

The bleomycins, phleomycins and tallysomycins are structurally similar glycopeptide antibiotics. Within each class, individual members differ only in the structure of a basic group. The antibiotic effect of phleomycin (Bristol batch A9331-648) against Escherichia coli is amplified substantially by a number of simple heterocyclic and aromatic compounds. In this paper a sample of 26 such compounds were tested for this property with 25 different phleomycins, bleomycins and tallysomycins. The nature of the variable basic group of the phleomycins, bleomycins and tallysomycins determined the response obtained with all amplifiers, although variation of response was much less marked with caffeine which potentiated the cytotoxic effects of all the phleomycins, bleomycins and tallysomycins tested. Phleomycins and bleomycins having two or three guanidino groups in the variable basic group, or phleomycins having a secondary amino group within a methylene chain and a terminal 2-phenylethyl substituent, were amplified by most compounds, whereas the cytotoxicity of others was enhanced little or not at all. Similar phleomycins, having a secondary amino and a terminal guanidino group and no 2-phenylethyl substituent showed little enhancement, and in these cases the inclusion of a 2-phenylethyl substituent had a major influence in determining amplifiability. Bleomycins and phleomycins having identical basic groups were amplified to similar extents by the sample of 26 amplifying agents used.

Amplification of the antitumor activity of phleomycins in rats and mice by heterocyclic analogues of purines.

Significant and often substantial enhancement of the antitumor properties of several individual phleomycins , by co- administration via intraperitoneal injection of a number of purine analogues, is demonstrated in rats and mice having three diverse tumors. It is evident that the dose levels of both the phleomycin and the amplifier are very significant and that optimal levels vary widely with the actual agents used. Constant serum levels of amplifier can be maintained for several days by administration via silastic-pellet implantation rather than injection, and this route of administration is an effective alternative for amplifiers of low solubility.

Amplifications of phleomycin and bleomycin-induced antibiotic activity in Escherichia coli by aromatic cationic compounds.

A wide range of aromatic compounds has been shown to amplify phleomycin-induced cell killing in Escherichia coli. They include acridines, acridinium chlorides, dihydroanthracenes, anthracenes, dianthracenes, phenanthridinium salts, phenazinium chlorides, phenoxazones, triphenyl methane dyes, benzoquinolizinium chloride, diphenylmethane derivatives, stilbene and diphenyl derivatives. Low concentrations of these amplifiers also amplified the DNA breakage and degradation effects of phleomycin. The minimum structural specification for activity as an amplifying agent is suggested. A representative sample of compounds effective as amplifiers of phleomycin also amplified the antibiotic effects of bleomycins B4 and B6. The amplifiers described are known to vary in their ability to penetrate and accumulate in different organisms or tissues. This suggests the possibility of developing a series of antibiotic regimes using these amplifiers (or the large number of derivative compounds also likely to be active) where the therapeutic index is determined by the properties of the amplifier chosen rather than of the phleomycin or the bleomycin.

Sequencing 5-methylcytosine residues by the bisulphite method.

Measuring patterns of cytosine methylation in genomic DNA is most efficiently accomplished by use of the bisulphite method. This method depends on the large difference in reactivity of cytosines relative to 5-Methyl cytosines in genomic DNA to bisulphite. The chemistry and history of the method and recent developments which greatly increase its sensitivity, simplicity and reliability are described. An updated protocol to guide users is appended.

Selective breakage of DNA alongside 5-bromodeoxyuridine nucleotide residues by high temperature hydrolysis.

The substitution of thymine mucleotides (pT) in oligodeoxynucleotides by bromouracil nucleotides (pBU) changes the properties of the oligonucleotides in two ways: (1) It alters their mobility during DEAE-Cellulose homochromatography1. (2) It substantially enhances their sensitivity to high temperature hydrolysis under mildly alkaline conditions (pH 8.9). The resultant breaks occur adjacent to pBU residues and leave terminal phosphates on the breakage products. With more extreme conditions some loss of terminal phosphates can occur. Heating at 100 degrees for 16 hr at pH 8.9 produces cleavage at about half of the pBU residues with minimal loss of terminal phosphates. The properties described here may explain the thermal sensitivity of bacteria grown in 5BU2 and may have a use in DNA sequencing technology.

The mechanism of sensitivity to phleomycin in growing Escherichia coli cells.

Stationary-phase Escherichia coli B cells transferred to new growth medium are initially resistant to net DNA breakage by low concentrations of phleomycin, and become sensitive as DNA replication commences. From studies with inhibitors of various stages of the DNA replication cycle it is evident that it is not DNA synthesis itself that is required for induction of DNA breakage by phleomycin, but events associated with the initiation of DNA replication. Termination of replication in the absence of further initiaiton results in resistance to phleomycin. The cellular change responsible for changes in sensitivity to phleomycin could be the attachment of the bacterial chromosome to the cell membrane at initiation and detachment on termination of replication, suggesting an alteration in the balance between cellular DNA breakage and repair processes for membrane-associated compared with non-membrane-associated DNA.

An Escherichia coli mutant resistant to phleomycin, bleomycin, and heat inactivation is defective in ubiquinone synthesis.

A mutant of Escherichia coli, selected for resistance to the antibiotic and antitumor agent phleomycin, has been characterized, and the phleomycin resistance determinant has been identified. The mutant is equally resistant to bleomycins. The resistance to phleomycin is strongly dependent on the nature of the C-terminal amine of the drug, with the greatest resistance being shown to phleomycins and bleomycins with the most basic terminal amines. The mutation also confers resistance to the lethal effects of heating at 52 degrees C. Other characteristics of the phleomycin-resistant strain include a slow growth rate, an inability to grow on succinate as the sole carbon source (Suc- phenotype), cross resistance to aminoglycoside antibiotics, and a slight sensitivity to hydrogen peroxide, methyl methanesulfonate, and gamma-irradiation. Some of these characteristics, together with mapping data, suggested that the phleomycin resistance and Suc- determinant probably lies within the ubiF gene coding for an enzyme effecting a step in the biosynthesis of ubiquinone. The phenotypes of known mutants defective in this and other steps of the ubiquinone pathway were found to be closely similar to those of the original phleomycin-resistant strain.