A monofunctional derivative of melphalan: preparation, DNA alkylation products, and determination of the specificity of monoclonal antibodies that recognize melphalan-DNA adducts.

Bifunctional alkylating agents, such as those based on nitrogen mustard, form important parts of many anti-cancer chemotherapy protocols and are responsible for increased incidences of secondary tumors in successfully treated patients. These drugs generally form a majority of monofunctional DNA adducts, although the bifunctional adducts appear to be necessary for their powerful cytotoxic and antitumor effects. The relative importance of bifunctional as opposed to monofunctional adducts in the varied biological consequences of drug exposure has not been studied in detail, particularly in relation to the role and specificity of biochemical responses to therapy-related DNA damage.

DNA adducts from chemotherapeutic agents.

The guiding principle of early work was the hypothesis that the anti-cancer alkylating drugs acted through their ability to cross-link macromolecules essential for cell division. Not long afterwards, DNA was specified as the essential target, and support for the hypothesis came from evidence that the archetypal agent, mustard gas, could link guanine bases in DNA through their N-7 atoms. Quantitative correlations between alkylation of DNA and its inactivation as a template followed, with bacteriophage as a simple test object, showing that the mean lethal dose was close to a single cross-link in the genome.

Products from alkylation of DNA in cells by melphalan: human soft tissue sarcoma cell line RD and Escherichia coli WP2.

Alkylation of DNA was studied after treatment with [3H]melphalan (phenylalanine mustard; 1-2 microM) using a human tumour cell line, RD, in culture, or Escherichia coli (WP2 or WP2-uvrA strains) in growth medium. After 6 h at 37 degrees C, treated cells were isolated and re-suspended in fresh growth media. Samples were taken at times up to 48 h for isolation of DNA, and in some cases also RNA and protein (which were found to be alkylated to about the same extent as DNA).

Alkylation of DNA and its aftermath.

Current pharmacopoeias invariably refer to a category of 'alkylating drugs', still among the most widely used in cancer chemotherapy. They are described as acting through their ability to damage DNA, thus interfering with cell replication. Unfortunately, this mode of action implicates these drugs as carcinogens.

Alkylation of DNA by the nitrogen mustard bis(2-chloroethyl)methylamine.

Alkylation of DNA by the nitrogen mustard bis(2-chloroethyl)methylamine (mechlorethamine; HN2) gave four principal products, derived by mono-alkylation of guanine at N-7 and adenine at N-3 and by cross-linking of guanine to guanine or guanine to adenine at these positions. These products were isolated by hydrolysis from DNA at neutral pH, followed by ion-exchange chromatography on SP-Sephadex and reversed phase chromatography on ODS. They were characterized by identification with products from the reaction of nitrogen mustard with adenine or deoxyguanylic acid, and by their UV, mass, and proton magnetic resonance spectra.

The susceptibility of inbred mice to the production of malignant thymoma by N-methyl-N-nitrosourea.

The susceptibility of inbred mice to the induction of malignant thymoma by N-methyl-N-nitrosourea (MNU) has been quantified and compared. Strain differences emerged and this was also apparent in congenic lines of C57BL/10 mice, differing at the H-2 locus. However, different strains of inbred mice, with identical H-2 haplotypes, also showed varying susceptibility to MNU, indicating that the role of the MHC was not a simple one, but apparently depended on other, undefined genes.

Historical origins of current concepts of carcinogenesis.

The first attempts to understand the causes of cancer were based on generalizations of what might now be termed a "holistic" nature, and hereditary influences were recognized at an early stage; these views survive principally through a supposed positive connection between psychological factors such as stress and diminished ability to combat the progressive development of tumors through some form of immunologically mediated rejection of potentially cancerous cells. While evidence for immunosurveillance is generally accepted, it is now widely regarded as almost wholly confined to instances where tumor viruses are involved as causative agents. The earliest theorists drew an analogy between the processes of carcinogenesis and of evolution; the cancer cells acquired the ability to outstrip their normal counterparts in their capacity for proliferation.

From fluorescence spectra to mutational spectra, a historical overview of DNA-reactive compounds.

This historical survey has shown the emergence over a period of about 60 years of a coherent view of DNA-reactive carcinogens and their effects. The earliest workers, in the 'pre-Watson-Crick' era, probably thought that the mode of action of carcinogens, then largely comprising polycyclic aromatic hydrocarbons, would be revealed through a relationship to steroid hormones, and that they would have protein receptors. This may well apply, in a broad sense, to promoting agents in carcinogenesis.

Alkylation of DNA by melphalan with special reference to adenine derivatives and adenine-guanine cross-linking.

Alkylation of DNA by melphalan gave four principal products, derived by mono-alkylation of adenine at N-3 and guanine at N-7, and by cross-linking of adenine N-3 to guanine N-7, or of guanine N-7 to guanine N-7. Adenine-guanine cross-linking was unexpected because the two principal nucleophilic centres, N-7 of guanine and N-3 of adenine, are situated in the 'wide' and 'narrow' grooves of the DNA double helix, respectively. These products could be isolated by their hydrolysis from DNA at neutral pH, followed by chromatography of the hydrolysate in an ion-pair ODS system using a solvent containing tetrabutylammonium hydroxide as ion-pairing reagent; this gave better separation than the previously described method using SP-Sephadex.

The reaction of melphalan with deoxyguanosine and deoxyguanylic acid.

The reaction of melphalan (phenylalanine mustard, I) with 2'-deoxyguanosine, followed by removal of the sugar in acid, yielded two products. The major product was identified as 4-(N-(2-guanin-7-ylethyl)-N-(2-hydroxyethyl)amino)phenyl- alanine (II) by ultra-violet absorption, mass and NMR spectroscopy. The minor product has already been identified as the corresponding bis-guaninyl adduct III (Tilby et al.

Direct assay for O6-methylguanine-DNA methyltransferase and comparison of detection methods for the methylated enzyme in polyacrylamide gels and electroblots.

We describe in detail a direct assay for the substrate-inactivated DNA-repair enzyme, O6-methylguanine-DNA methyltransferase (O6-MT), which measures the transfer of radiolabelled methyl groups from a prepared O6-methylguanine-DNA substrate to the protein fraction of an enzyme-containing cell/tissue extract. This assay, a modification of a previously suggested method for monitoring O6-ethylguanine-DNA repair [Renard, Verly, Mehta & Ludlum (1983) Eur. J.

Purification to homogeneity and partial amino acid sequence of a fragment which includes the methyl acceptor site of the human DNA repair protein for O6-methylguanine.

DNA repair by O6-methylguanine-DNA methyltransferase (O6-MT) is accomplished by removal by the enzyme of the methyl group from premutagenic O6-methylguanine-DNA, thereby restoring native guanine in DNA. The methyl group is transferred to an acceptor site cysteine thiol group in the enzyme, which causes the irreversible inactivation of O6-MT. We detected a variety of different forms of the methylated, inactivated enzyme in crude extracts of human spleen of molecular weights higher and lower than the usually observed 21-24kDa for the human O6-MT.

Chemical induction of thymomas in AKR mice: interaction of chemical carcinogens and endogenous murine leukemia viruses. Comparison of N-methyl-N-nitrosourea and methyl methanesulphonate.

The time course of development of thymic lymphoma, which occurs spontaneously in mice of the AKR strain, is accelerated by the methylating agents N-methyl-N-nitrosourea (MNU) and methyl methanesulphonate (MMS). Since MNU is a potent mutagen inducing G----A transition mutations and MMS a relatively weak mutagen, it was of interest to examine the genetic alterations associated with each class of the chemically induced tumors and to compare these alterations with those found in the spontaneous tumors. The same spectrum of genetic alterations was found for MMS-induced and spontaneous thymomas.

Alkylation of DNA by melphalan in relation to immunoassay of melphalan-DNA adducts: characterization of mono-alkylated and cross-linked products from reaction of melphalan with dGMP and GMP.

A product expected to result from cross-linking of guanine bases in DNA by melphalan (4-(2-(di-guanin-7-yl))ethylamino-L-phenylalanine) was obtained from hydrolysis of melphalan-treated sodium deoxyguanylate at pH7 and characterized by U.V. and mass spectra.

Mutagens as carcinogens: development of current concepts.

The earliest work on reactions of mutagenic carcinogens with DNA, in which the author participated, is recalled in a personal reminiscence. Some significant consequences of this approach for studies of the mode of action of mutagenic carcinogens are briefly discussed, with regard to the types of mutation induced, and to current concepts of the involvement of somatic mutation in experimental cancer and in the aetiology of human cancer.

Increased sensitivity of lymphocytes from patients with systemic autoimmune diseases to DNA alkylation by the methylating carcinogen N-methyl-N-nitrosourea.

Lymphocytes from patients with various diseases associated with autoimmunity showed both impaired capacity to repair O6-methylguanine (a powerful, promutagenic, directly miscoding base lesion) and increased sensitivity to the cytocidal effects of cellular methylation by N-methyl-N-nitrosourea (MNU) compared with normal controls and patients with other disorders. Defective repair of O6-methylguanine was significantly associated with arthritis and myositis in the group with systemic lupus erythematosus (SLE), and increased sensitivity to the toxic action of MNU was associated with the presence of immune complexes and the administration of steroids to patients with Behçet's syndrome. The results indicate that lymphocytes from patients with the autoimmune diseases studied are more susceptible to DNA damage with possible relevance to aetiopathogenesis.

Deficient repair of O6-methylguanine in lymphocytes from rheumatoid arthritis families may be an acquired defect.

Deficient repair of the premutagenic DNA lesion O6-methylguanine (O6-MeGua) has been reported in lymphocytes from patients with autoimmune diseases. This was confirmed in the present study of probands with rheumatoid arthritis (RA) and their families. We also noted a significant deficiency in 9/19 spouses (P less than 0.

Novel uses of mass spectrometry in studies of adducts of alkylating agents with nucleic acids and proteins.

Several recent and major advances in the technology of mass spectrometry (MS) have greatly promoted use of this technique for the study of the interaction of alkylating agents with biomolecules. MS, in combination with gas chromatography (GC), may now be used to quantify adducts of carcinogens with proteins at levels down to 20 pmol/g protein. Soft ionization techniques have proved invaluable in determining the structure of carcinogen adducts with both DNA and proteins, and the newly developed tandem MS promises to be of considerable use in the characterization of complex carcinogen adduct mixtures.

Induction of thymomas by N-methyl-N-nitrosourea in AKR mice: interaction between the chemical carcinogen and endogenous murine leukaemia viruses.

AKR mice develop thymomas spontaneously when greater than 6 months old but when young AKR mice are treated with N-methyl-N-nitrosourea (MNU) they develop thymomas at 3-6 months of age. In this study the potential role of oncogene activation in the development of both the spontaneous and MNU-induced thymomas in AKR mice has been examined by DNA transfection into NIH3T3 mouse fibroblasts and by Southern analysis of tumour DNA. The results show that a high proportion of MNU-induced thymomas contain activated cellular rasK while no activated cellular ras genes were detected in spontaneous thymomas.

Repair of chemical carcinogen-induced damage in DNA of human lymphocytes and lymphoid cell lines--studies of the kinetics of removal of O6-methylguanine and 3-methyladenine.

The removal of O6-methylguanine by human lymphoid cells corresponded, with certain assumptions, to a second-order chemical reaction in any given cell. There was a spectrum of proficiency in this respect for a considerable number of cells originating from different individuals and it was found that patients with diseases associated with autoimmunity tended to fall into the less proficient groups. E-B virus-induced lymphoid cell lines, derived from proficient, but not relatively deficient, peripheral blood lymphocytes, always (in 9/9 cases) reflected the level of proficiency of the donor lymphocytes with respect to removal of O6-methylguanine.

Urinary excretion of 3-methyladenine and 1-methylnicotinamide by rats, following administration of [methyl-14C]methyl methanesulphonate and comparison with administration of [14C]methionine or formate.

Following i.p. injection of [methyl-14C]methyl methanesulphonate (MMS) into rats (100 mg/kg) 3-[14C]methyladenine was identified as a urinary product excreted mainly up to 24 h after treatment, the amount over this period being about 0.

Autoimmune haemolytic disease in mice after exposure to a methylating carcinogen.

N-Methyl-N-nitrosourea (MNU) but not methyl methanesulphonate (MMS) induced both autoimmune haemolytic anaemia and thymic lymphoma in susceptible strains of mice, particularly the C57BL/6. These effects could be positively correlated with the formation of O6-methylguanine in target DNA. All murine lymphoid cells showed lack of ability to remove O6-methylguanine from their DNA, therefore the variation of responses between different mouse strains indicated that other host factors, probably genetic, must be involved.

Defective repair of 0(6)-methylguanine in autoimmune diseases.

On the premise that somatic mutations may be involved in the pathogenesis of diseases such as systemic lupus erythematosus and rheumatoid arthritis, the proficiency of repair of 0(6)-methylguanine, a powerful, premutagenic, directly miscoding base lesion, was examined in the DNA of peripheral-blood mononuclear cells (mainly lymphocytes) from patients with such diseases. The capacity of lymphocytes to repair this lesion was impaired in many patients with autoimmune disease. In some healthy controls repair was also defective whereas in other, nonautoimmune diseases, it was not.

Mode of action of methylating carcinogens: comparative studies of murine and human cells.

Murine and human cells (mainly lymphocytes) were ethylated in vitro with either N-[14C]methyl-N-nitrosourea (MNU) or di[14C]methyl sulphate (DMS) and the extents of methylation of DNA at O-6 and N-7 of guanine and N-3 of adenine were determined. The cytotoxic action of MNU was also compared with that of DMS, as assessed by their effects on cell division following stimulation of these lymphocytes in culture by concanavalin A (Con A). The overall extent of methylation of the DNA of human cells was about 70% of that of murine cells after exposure to MNU and DMS.