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Biomedical subjects

D G MacPhee

Publications and source records attributed to D G MacPhee.

At least 19 recordsLinked to original sources
PubMed2001

Possible role of natural killer cells in negative selection of mutant lymphocytes that fail to express the human leukocyte antigen-A2 allele.

Increased frequencies of cells carrying mutations at several loci have been found in the blood cells of atomic-bomb (A-bomb) survivors upon testing four or five decades after the bombing. Interestingly, though, we have been unable to demonstrate any radiation-associated increases in the frequencies of mutant blood cells in which human leukocyte antigen (HLA)-A expression has been disrupted; this is true both of preliminary tests on the T cells of a small subset of A-bomb survivors and of the much more extensive study reported here in which we screened a much larger group of survivors for HLA-A2 loss mutations in B cells and granulocytes as well as in T cells. In attempting to explain our inability to detect any increases in HLA-A2-negative cell numbers in HLA-A2 heterozygous individuals exposed to A-bomb irradiation, we decided to test the hypothesis that HLA-A mutant lymphocytes might well have been induced by radiation exposure in much the same way as every other type of mutant we encountered, but may subsequently have been eliminated by the strong negative selection associated with their almost inevitable exposure to autologous natural killer (NK) cells in the bloodstream of each of the individuals concerned. We now report that mutant B lymphocyte cell lines that have lost the ability to express the HLA-A2 antigen do indeed appear to be much more readily eliminated than their parental heterozygous counterparts during co-culture in vitro with autologous NK cells. We make this claim first because we have observed that adding autologous NK cells to in vitro cultures of HLA-A2 heterozygous B or T cell lines appeared to cause a dose-dependent decrease in the numbers of HLA-A2-negative mutants that could be detected over a period of 3 days, and second because when we used peripheral blood HLA-A2 heterozygous lymphocyte cultures from which most of the autologous NK cells had been removed we found that we were able to detect newly-arising HLA-A2 mutant T cells in substantial numbers. Taken together, these results strongly support the hypothesis that autologous NK cells are responsible for eliminating mutant lymphocytes that have lost the ability to express self-HLA class I molecules in vivo, and may well therefore explain why we have been unable to detect increased frequencies of HLA-A2 mutants in samples from any of the 164 A-bomb survivors whose HLA-A2 heterozygote status made their lymphocytes suitable for our tests.

Aged↗
PubMed2001

Chromosomal instability in BRCA1- or BRCA2-defective human cancer cells detected by spontaneous micronucleus assay.

The BRCA1 and BRCA2 gene products are believed to play an important part in the onset and/or development of many sporadic mammary cancers. Recently, it has been reported that these two proteins contribute to a centrosome function which is believed to help maintain the integrity of the chromosome segregation process. This may mean a reduced level of the BRCA1 or BRCA2 protein in mammary cells will occasionally lead to nondisjunctional chromosomal loss or gain. We now report that spontaneous micronuclei arising from chromosome(s) which fail to be incorporated into the relevant daughter nuclei during mitosis tend to occur more frequently in BRCA1- or BRCA2-defective human cancer cells than in BRCA-positive cancer cells. Some cases of mammary carcinogenesis may therefore stem from the loss of integrity of chromosome segregation in cells which have a reduced capacity to express either BRCA1 or BRCA2.

BRCA2 Protein↗
PubMed1999

Specific inhibition of the eubacterial DNA ligase by arylamino compounds.

All known DNA ligases catalyze the formation of a phosphodiester linkage between adjacent termini in double-stranded DNA via very similar mechanisms. The ligase family can, however, be divided into two classes: eubacterial ligases, which require NAD(+) as a cofactor, and other ligases, from viruses, archaea, and eukaryotes, which use ATP. Drugs that discriminate between DNA ligases from different sources may have antieubacterial activity. We now report that a group of arylamino compounds, including some commonly used antimalarial and anti-inflammatory drugs and a novel series of bisquinoline compounds, are specific inhibitors of eubacterial DNA ligases. Members of this group of inhibitors have different heterocyclic ring systems with a common amino side chain in which the two nitrogens are separated by four carbon atoms. The potency, but not the specificity of action, is influenced by the DNA-binding characteristics of the inhibitor, and the inhibition is noncompetitive with respect to NAD(+). The arylamino compounds appear to target eubacterial DNA ligase in vivo, since a Salmonella Lig(-) strain that has been rescued with the ATP-dependent T4 DNA ligase is less sensitive than the parental Salmonella strain.

Adenosine Triphosphate↗
PubMed1998

Glucose and related catabolite repressors are powerful inhibitors of pKM101-enhanced UV mutagenesis in Escherichia coli.

When stationary phase Escherichia coli K12 trp (amber) cells were exposed to UV doses ranging from 180-540 J m(-2), we found that we could not recover any induced Trp+ revertants unless the irradiated cultures were first supplied with the Muc+ mutation-enhancing IncP plasmid pKM101 (by conjugation). We also found that the numbers of UV-induced Trp+ revertants recovered from pKM101+ cultures varied quite dramatically depending upon which of several commonly-used carbon sources were present in the post-irradiation plating medium, e.g., there were always significantly fewer revertants on minimal glucose plates than on minimal glycerol plates. More importantly, there were also fewer UV-induced revertants on glycerol + glucose plates than on 'glycerol-only' plates. We then tested two glucose-related compounds which are known to depress intracellular cyclic AMP (cAMP) levels even more effectively than glucose (glucose-6-phosphate and the non-utilisable methyl-alpha-D-glucopyranoside) and found that they too were able to exert powerfully antimutagenic effects in UV-treated pKM101-containing bacteria. Taken together, these results provide strong additional support for our working hypothesis that at least one component of the mutational pathway which operates in UV-irradiated pKM101-containing cells is extremely sensitive to classical cAMP-mediated catabolite repression.

Conjugation, Genetic↗
PubMed1998

Time-dependent mutagenesis and cancer: a new role for antimutagenesis in cancer prevention?

Most attempts to identify potential antimutagens (and/or presumptive anticarcinogens) involve testing individual compounds or mixtures in tandem combinations with specific physical or chemical mutagens and measuring the sought-after reductions in mutation numbers in one or more experimental organisms. Relatively few investigators appear to have set out to identify antimutagens which are efficacious in reducing spontaneous mutation yields (possibly because of the poor mutation yields which tend to be available for downward manipulation in spontaneous mutation assays). The net effect is that we currently know very little about what may well prove to be one of the most interesting and exciting areas of antimutagenesis and anticarcinogenesis research in the future. This paper is primarily concerned with the main features of several interrelated (and often overlapping) pathways which are likely to be involved in the generation of newly-mutant sequences in cellular organisms in the absence of a deliberately-added mutagen. Attempts will be made to highlight some of the cellular processes which may have to be blocked in subtle (or perhaps even unsubtle) ways if we are to achieve our somewhat ambitious goal of discovering antimutagenic anticarcinogens which are both usable and useful in delaying the onset of primarily age-dependent mammalian cancers whose origins may well owe a great deal more to spontaneous mutations than they do to environmentally-provoked ones.

Anticarcinogenic Agents↗
PubMed1998

Epigenetics and epimutagens: some new perspectives on cancer, germ line effects and endocrine disrupters.

It is known that a variety of chemicals, including certain base analogues and reactive oxygen species, can alter the phenotypes of mammalian cells epigenetically, i.e., without changing their DNA sequence information in any way. The implications of such findings are not trivial, but do not seem to have been the focus of a great deal of attention amongst mutation researchers to date. In part this may be a reflection of the confused state of terminology in the chemical carcinogenesis research area and in part may signal a reluctance on the part of many of us to come to terms with the idea of heritable non-sequence changes to DNA molecules. In this review, some of the most obvious outcomes of spontaneous and induced epimutagenic change for human carcinogenesis and germ line inheritance are discussed, and an attempt is made to place the so-called endocrine disrupters in a context in which their modes of action may be more readily analysed and integrated into the broader chemical hazard framework.

Animals↗
PubMed1998

Catabolite repressors are potent antimutagens in Escherichia coli plate incorporation assays: experiments with glucose, glucose-6-phosphate and methyl-alpha-D-glucopyranoside.

Having previously found that the yields of spontaneous valine-resistant (Val(r)) Escherichia coli mutants which appeared on plates containing 40 microg/ml of valine were always much lower when glucose was present in the glycerol-containing defined medium normally used to select them, we now sought to determine whether or not the global regulatory mechanism known as catabolite repression (formerly also called glucose repression) might be involved. We therefore tested glucose (the archetypal catabolite repressor), glycerol (a non catabolite-repressing substrate), glucose-6-phosphate (G6P, an exceptionally powerful catabolite repressor) and methyl-alpha-D-glucopyranoside (alphaMG, a strongly catabolite-repressing but non-utilisable glucose analogue), as potential inhibitors of spontaneous mutagenesis in plate incorporation assays, using three distinct mutation detection systems. We found that the numbers of spontaneous Val(r) and Lac+ mutations appearing on the selective plates tended to be highest when the medium contained only a non-repressing primary carbon source (glycerol in the Val(s) --> Val(r) system, lactose in the Lac- --> Lac+ system) and lowest when it had been supplemented with a strongly catabolite-repressing compound such as alphaMG, G6P or glucose. These results would seem to establish that catabolite repression is an important factor in determining the outcome of the spontaneous mutation generation process in E. coli and hence that the numbers of spontaneous mutations which can be expected to arise in any given set of mutation assay conditions may often be dependent upon the levels of catabolite repression which prevail during the course of the assay. The implications of these results for conventional plate-incorporation mutation assays are discussed.

Antimutagenic Agents↗
PubMed1996

Frameshift mutagenesis by 9-aminoacridine: antimutagenic effects of adenosine compounds.

It has been shown that frameshift mutagenesis by 9-aminoacridine (9AA) in Salmonella typhimurium is significantly inhibited if glucose is present while cells are being treated in liquid defined medium. We suggested that this effect might be a result of glucose-provoked alterations of cAMP levels within the cell. We therefore sought to investigate the effects of exogenous cAMP on mutagenesis by 9-aminoacridine in both Salmonella typhimurium and Escherichia coli. Contrary to expectation, we found that frameshift mutagenesis was significantly depressed when high concentrations of cAMP were added to the defined medium during liquid treatment with 9AA. Other adenosine 5'-phosphates such as adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP) and adenosine 5'-monophosphate (AMP) added to the liquid medium during 9AA treatment also substantially decreased the reversion rate to prototrophy in both S. typhimurium and E. coli, as did adenosine itself. Further experiments showed that neither influx nor efflux of 9-aminoacridine molecules were greatly affected by adenosine compounds, and that although cAMP and adenosine exerted similar antimutagenic effects on 9AA-treated stationary phase cells, their effects on log phase cells were quite different. The antimutagenic effect of a representative adenosine compound (ATP) was found to persist for some time after stationary phase cells had been washed, with maximal mutability being regained only after about 3 h.

Adenine Nucleotides↗
PubMed1996

Mismatch repair as an important source of new mutations in non-dividing cells.

This paper describes a mechanism which permits somatic cells to generate random mutations in the complete absence of cell proliferation. Knowledge of the existence of this mechanism should provide us with the basis for a better understanding of a number of important biological phenomena, and in particular may help to explain the origins of many human cancers.

Cell Division↗
PubMed1996

Mismatch repair as a source of mutations in non-dividing cells.

This paper describes a mechanism which permits somatic cells to generate random mutations in the complete absence of cell proliferation. The mechanism itself is remarkably simple, involving a well-known cellular process (mismatch repair or MMR) which is primarily associated with mutation avoidance, but which is also capable of generating mutations when circumstances are not ideal for avoidance. When MMR operates in its so-called 'methylation-instructed' mode to remove mismatches from newly-replicated portions of genomic DNA, it does so in a way which serves to minimize mutation yields. By contrast, when MMR operates in a non-instructed or 'randomly-templated' way to remove mismatches from DNA molecules, it does so without distinguishing between the two strands of DNA that contain the mismatched bases. Randomly-templated mismatch repair (RT-MMR) therefore generates new and complete mutations whenever it removes the correct bases from either base-pair mismatches or frameshift mispairs and replaces them without incorrect bases or sequences. Wider recognition of the existence of this mechanism--and especially of its proclivity for mutation generation when it is operating in non-dividing cells--should help us to develop a better understanding of a number of important biological phenomena, and may be of particular value in our attempts to explain the origins of many human cancers.

Animals↗
PubMed1996

Spontaneous mutations in bacteria: chance or necessity?

Several investigators have recently reported that significant numbers of appropriately adapted mutants can be induced in bacterial and yeast strains by exposing stationary phase cells to specific environmental challenges. The resulting mutants are said to be both selection-induced and demonstrably non-random in origin; if this interpretation is correct, it is in direct conflict with the conventional neo-Darwinian view, which is that spontaneous mutants are truly random in origin and arise without the intervention of any overtly adaptive forces. We believe that there are alternative ways of accounting for the appearance of many (and probably all) of the additional mutants which proponents of the adaptive mutation theory claim are observed only after they applied the appropriate selective pressure. Having reviewed the available evidence, we consider that most (if not all) of the sorts of mutants which are said to have been induced following exposure of stationary-phase cells to intense selective pressure are equally likely to have been generated during the operation of certain well-known, conventional (and essentially random) cellular DNA repair processes. Evidence in support of our view can be found in the mainstream literature on the origins of spontaneous mutations. We also note that some of the molecular models which have recently been proposed to explain the production of selection-induced mutations preferentially (or even only) in genes of adaptive significance may turn out to be of considerable interest in their own right, even although the mutants whose origins they were intended to explain may turn out to have arisen in a manner which is totally independent of the conditions used for their selection.

Adaptation, Physiological↗
PubMed1995

Mismatch repair, somatic mutations, and the origins of cancer.

This paper outlines the basic properties of a newly recognized pathway that should enable somatic cells to generate double-stranded mutations in the complete absence of cell proliferation. Recognition of the existence of this pathway provides us with the basis for a better understanding of a number of important biological phenomena and, in particular, may help us to understand the origins of cancers in unselected human populations.

Cell Division↗
PubMed1994

Regulatory processes and the origins of spontaneous mutations.

A review of information currently available about the origins of spontaneous mutational events suggests that there may be a role for known cellular control mechanisms in determining the frequencies with which such events can occur. Attention is also directed to recent findings with antimutator (dnaE) mutants of Escherichia coli which indicate that the final step involved in generating a spontaneous mutational event may be different from that involved in generating an SOS-dependent mutational event. Finally, the possible involvement of various sorts of treatments collectively referred to as stress responses (heat shock, cold shock or oxidative damage, etc.) in generating random mutations is discussed; if there is such an involvement, this may represent one way in which organisms are programmed to adapt to a wide variety of environmental challenges.

Escherichia coli↗