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. A simple method is described for the preparation of useful quantities of a pure monofunctional derivative of the nitrogen mustard-based drug melphalan. Monohydroxymelphalan was prepared by partial hydrolysis, purified by reversed phase chromatography, and characterized by MS, NMR, and HPLC. Contamination with melphalan was

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. This conclusion applied to either mustard gas or the more recently introduced platinum drugs. Although both inter- and intra-strand cross-links were effective, it was thought that in cells the inter-strand cross-link would, by preventing the separation of the strands necessary for cell division, and by being more difficult to repair, constitute the more effectively lethal lesion. With repair-deficient bacteria, it also emerged that a single cross-link in the genome was lethal, but proficient bacteria could remove about 20 cross-links through excision repair. Mono-7-alkylguanines were not removed and were evidently inert. Thus, only a few percent of the total alkylation products were the most effective lesions. Parallel studies with cultured mammalian cells gave a rather different picture, in that the mean lethal doses of even hypersensitive cell lines were around 20 or more cross-links per genome, about the same as for resistant strains of bacteria. Most cells could withstand several hundreds of cross-links per genome, and although adducts were removed, there was incomplete removal of cross-links. Some, but not all, sensitive cell lines were deficient in excision repair. Methods were devised for measuring the extents of alkylation of DNA in cells of patients treated with chemotherapeutic drugs; these are mainly immunoassays, and were applied generally to peripheral blood leukocytes, although some tumours were studied. Extents of alkylation of leukocyte DNA were generally of the same order as, or rather less than the mean lethal doses of cultured cells of the 'normal' type, but in some reports for cisplatin-treated patients, very wide variability between individuals was found. A positive correlation between adduct levels, and particularly a very minor adduct recognised specifically by one antibody, and favourable therapeutic outcome was discerned, and suggested to have a pharmacogenetic basis. In several instances, extents of alkylation of tumours were significantly higher than the average for leukocytes; for ovarian and a testicular tumour for cisplatin, and for a plasma cell tumour for melphalan. Nevertheless, these favourable examples would not constitute more than three or four mean lethal doses in the tumour cells, assuming that they had the same sensitivity as 'normal' cell lines: the therapeutic effect would of course be much more favourable if the tumour cells resembled 'sensitive' cell lines. This lack of a favourable difference between extents of alkylation in DNA of patients and the mean lethal dose for normal cells was particularly obvious with the methylating drugs dacarbazine and procarbazine. These considerations stress the need for higher extents of alkylation to be achieved in target tumour DNA for successful chemotherapy. One approach is to give a higher overall dose, and to 'rescue' the bone marrow (known from the earliest report on mustard gas to be the most susceptible tissue) by autologous transplantation. The second, which has yet to reach the clinic, is to convert unreactive prodrugs through enzymic activation into alkylating agents specifically in tumours (see Bagshawe, 1994).

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). Alkylated DNA was analysed as previously described (M.R. Osborne and P.D. Lawley, Chem.-Biol. Interact 89 (1993) 49-60). The four principal products, mono-7-alkylguanine (G-M-OH); mono-3-alkyladenine (A-M-OH); and the cross-linked products G-M-G and A-M-G, were identified in DNA from melphalan treated cells, and quantitatively determined. In each case, alkylation of cellular macromolecules was maximal after about 6 h. In DNA of the human tumour cell line, the relative amounts of adenine products decreased with time, most markedly with A-M-OH to 42% of the 2-h value after 48 h. In DNA of both bacterial strains, A-M-OH was virtually undetectable even at early times. Comparisons between the time course of relative decreases in amounts of these alkylpurine products and the corresponding values for alkylated DNA in vitro suggest that the adenine products are subject to removal by repair enzyme action in E. coli of either strain.

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. Thus the early studies recalled in this essay proved to be relevant to our understanding of both the main problems with which cancer research concerns itself: the causation of cancer and possible methods of treatment of this group of diseases.

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. Proficiency of repair of O6-methylguanine by thymic tissue was not responsible for these strain differences and the increased resistance to induction of thymoma by MNU in older mice was due to age-dependent changes within the thymus gland. Interestingly, the thymus of older mice, grafted with neonatal thymic tissue, still provided a suitable environment for the development of thymic tumours.

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. Demonstration of the mutagenicity of a chemical, mustard gas, shifted attention to alkylating agents as carcinogens, and to the concept of mutagens, carcinogens and cytotoxic agents as 'radiomimetic'. Alkylating carcinogens were shown to react with DNA in vitro and in vivo in ways consistent with their action as mutagens, particularly as inducers of base substitutions, GC-->AT transitions. Carcinogenic hydrocarbons were subsequently shown to react with DNA of their target tissue, mouse skin, to extents positively correlated with their carcinogenic potency. They were found to react through aralkylating metabolites to give products that can block DNA polymerase, but can also cause base substitutions of the transversion type, mainly GC-->TA. Current interest centres on correlating the observed base substitutions that activate oncogenes or inactivate tumour suppressor genes in human cancer with the nature of exogenous and endogenous mutagens and the chemistry of their reactions with DNA, in order to deduce whether specific carcinogens can be implicated in the etiology of cancers. Ancillary to these studies are determinations of carcinogen-DNA reaction products in DNA from human sources.

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. This was even before evolution had been interpreted as involving a continuous succession of mutations. Evidence was already to hand before the end of the 18th century that exogenous agents, notably soot, a product of the "industrial revolution," could cause skin cancer. Somewhat over 100 years later, another industrial innovation, the manufacture of synthetic dyestuffs, implicated specific chemical compounds that could act systemically to cause bladder cancer. Meanwhile, the 19th century saw the establishment of the fundamentals of modern medical science; of particular relevance to cancer was the demonstration that it involved abnormalities in the process of cell division. The commencement of the 20th century was marked by a rediscovery of the concept of mutation; and it was proposed that cancer originated through uncontrolled division of somatically mutated cells. At around this time, two further important exogenous causative agents were discovered: X-rays and tumor viruses. In the late 1920s, x-radiation became the first established exogenous cause of mutagenesis. The discoverer of this phenomenon, H. J. Muller, suggested that while mutation in a single cell was the primary causative mechanism in carcinogenesis, its generally observed logarithmic increase in incidence with age reflected a "multihit" process, and that multiple successive mutations were required in the progeny of the original mutants. He also recognized that the rate of proliferation of potentially cancerous cells would markedly influence the probability of their subsequent mutation. These considerations are essentially the foundation of the generally accepted view of carcinogenesis that now seems unlikely to be superseded. However, this acceptance did not come about unopposed. The analogy between carcinogenesis and evolution was disliked by many biologists because it embodied the concept that cancer was an inevitable consequence of our evolutionary origins.(ABSTRACT TRUNCATED AT 400 WORDS)

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., Chem.-Biol. Interact., 73 (1990) 183-194). The reaction of melphalan with 5'-deoxyguanylic acid yielded the deoxyribonucleotide of II and products resulting from reaction with the phosphate group. The initial products, which were formed with a half-life of approximately 40 min at 37 degrees, still had a reactive chloroethyl group; this was displaced more slowly, by reaction with water or with another molecule of dGMP. The products of reaction of melphalan with DNA were released by treatment with acid (0.1 M HCl, 70 degrees, 30 min) and separated from each other on a cation exchange column. They were identified as II, III and an adenine adduct, in a ratio of approximately 3:1:2.

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. Biochem. 136, 461-467], is sensitive, highly reproducible, accurate and is, as described here and relative to previously published methods, well suited for use with a large number of test samples. We identified two problems with the O6-[Me-3H]methylguanine-DNA substrate used in the present work and in other reported assay: firstly, that of progressively higher assay backgrounds with increasing age of substrate, which was nullified by once-only purification of the double-stranded substrate by hydroxyapatite chromatography; secondly, a substrate of high specific radioactivity (30 Ci/mmol), made with freshly prepared tritiated methylnitrosourea, behaved as a substrate of 5 Ci/mmol when referenced against radiolabelled O6-methylguanine-DNA made with either [3H]- or [14C]-methylnitrosourea at the lower specific radioactivities of 1 Ci/mmol and 61 mCi/mmol respectively. This apparently stemmed from the known instability of high-specific-radioactivity [3H]methylnitrosourea and indicated that an expected increase in sensitivity of the assay does not necessarily result from increasing the specific radioactivity of substrates above approx. 1 Ci/mmol. Although O6-MT was stable to preincubation at 25 degrees C, marked losses of activity were observed at 37 degrees C, and more so at 45 degrees C. Enzyme lability at the higher temperatures was not, however, seen during preincubation in the presence of its substrate. O6-[Me-3H]methylguanine-DNA, which apparently protected O6-MT against thermal inactivation. As previously seen with other human cells and tissues, extracts of human spleen in the present study showed wide interindividual differences in O6-MT specific activity (18-fold), which spanned the range 50-900 fmol/mg of protein. Cultured human lymphoblastoid Jurkat cells contained approx. 57,000 enzyme molecules/cell. Substrate-inactivated [Me-3H]methylated O6-MT was analysed by SDS/PAGE and electroblotting. The different but similarly sized forms of this enzyme that we previously detected in human spleen [Major, Gardner, Carne & Lawley (1990) Nucleic Acids Res. 18, 1351-1359] were clearly resolved by fluorography of electroblots, but only at considerable expense of time. As expected, scintillation counting of the protein extracted from gel slices and linear-wire scanning of enzyme-associated radioactivity on electroblots were quicker methods for detecting the [Me-3H]methylated inactivated O6-MT.

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. Several apparent fragments of the methylated form of the protein were purified to homogeneity following reaction of partially-purified extract enzyme with O6-[3H-CH3]methylguanine-DNA substrate. One of these fragments yielded amino acid sequence information spanning fifteen residues, which was identified as probably belonging to human methyltransferase by virtue of both its significant sequence homology to three procaryote forms of O6-MT encoded by the ada, ogt (both from E. coli) and dat (B. subtilis) genes, and sequence position of the radiolabelled methyl group which matched the position of the conserved procaryote methyl acceptor site cysteine residue. Statistical prediction of secondary structure indicated good homologies between the human fragment and corresponding regions of the constitutive form of O6-MT in procaryotes (ogt and dat gene products), but not with the inducible ada protein, indicating the possibility that we had obtained partial amino acid sequence for a non-inducible form of the human enzyme. The identity of the fragment sequence as belonging to human methyltransferase was more recently confirmed by comparison with cDNA-derived amino acid sequence from the cloned human O6-MT gene from HeLa cells (1). The two sequences compared well, with only three out of fifteen amino acids being different (and two of them by only one nucleotide in each codon).

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. When tested in a competitive immunoassay using an antibody specific for melphalan-alkylated DNA it showed an affinity intermediate between that of melphalan-alkylated DNA and melphalan. From this and other assays it seemed possible that the cross-linked moiety in DNA was recognised by the antibody, but that its conformation differed from that of the free base tested, sufficiently to account for the discrepancy. It seemed possible that cross-linked guanine nucleotides would provide a better model, and these were therefore isolated, characterised and tested. Products derived from cross-linking of guanylic acid moieties through N-7 and N-7, and through N-7 and phosphate, had higher affinity than the cross-linked base, approximately the same as for alkylated native DNA, but less than for alkylated denatured DNA or RNA.

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. Both showed rearrangements of c-myc and Pim-1 genes that appeared to result from integration of recombinant mink cytopathic focus-forming (MCF) proviruses but failed to reveal evidence for activation of ras oncogenes, either by DNA transfection experiments or by hybridization of DNA to specific oligonucleotide probes. Some alteration in c-myc and Pim-1 genes were also found in MNU-induced tumors, but, mainly, these involved integration of ecotropic-like rather than recombinant MCF viruses. Furthermore, MNU-induced tumors frequently (in 24% of thymomas) contained G----A transition mutations, activating the Ki-ras oncogene at codon 12 position 2. Another feature that distinguishes the MNU-induced tumors from those occurring in untreated and MMS-treated mice was the consistently high level of c-myc mRNA that occurred in the absence of c-myc gene rearrangement. Taken together, the data indicate that the mechanisms of development of tumors following treatment with MNU and MMS are distinct, and that the effect of MMS is probably to speed up the process of viral leukemogenesis.

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 0.05) and a statistically non-significant deficiency of repair of O6-MeGua in 14/42 first and second degree relatives in comparison with healthy and non-autoimmune disease controls, respectively. A significant correlation of the repair status of O6-MeGua in DNA between individual probands with RA and respective spouses (P less than 0.01) and probands and respective family members (P less than 0.001) supported the idea that an environmental, transmissible agent could influence the expression of the protein, O6-methylguanine-DNA-transferase (O6-MT), involved in the repair of O6-MeGua. The present results, however, cannot entirely exclude an additional hereditary influence.

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. Southern analysis of tumour DNA revealed that 2/30 spontaneous tumours and 2/52 MNU-induced tumours contained alterations in the c-myc gene while 5/29 spontaneous tumours and 6/56 MNU-induced tumours contained alterations in the Pim-1 gene. A more detailed analysis of the Pim-1 gene demonstrated that the alterations observed in most MNU-induced and spontaneous tumours resulted from proviral integration at the 3' end of this gene. Our analyses also demonstrated that the majority of MNU-induced tumours, including those containing rearrangements in the Pim-1 gene, lacked the somatically acquired recombinant MCF proviruses that are present in most spontaneous AKR lymphomas. These results provide evidence that the mechanisms of development of MNU-induced and spontaneous tumours in AKR mice are distinct and the development of thymomas that contain proviral integrations at the Pim-1 locus in the MNU-treated AKR mice involve cooperation between the chemical carcinogen and endogenous murine leukaemia viruses.

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. Thus while proficient lymphocytes always produced proficient cell lines, deficient lymphocytes, in 3/8 cases, gave rise to more proficient cell lines. No evidence was found that groups of individuals exist who lack ability to remove 3-methyladenine from DNA, either from their blood lymphocytes or derived lymphoid cell lines.